Biology subjects

Simpson, J.

Publications and source records attributed to Simpson, J..

28 records · Page 2Linked to original sources

bioRxiv2019

Discrete virus factories form in the cytoplasm of cells co-infected with two strains of the segmented dsRNA virus, infectious bursal disease virus (IBDV), that subsequently coalesce

The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, are non-enveloped viruses with a segmented double-stranded (ds)RNA genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common, however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus co-infection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11- or Tetracysteine (TC)- tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98-VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection, or stained with ReAsH following PBG98-VP1-TC infection, had green or red foci in the cytoplasm respectively that co-localised with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 hours post infection (hpi) (p<0.001), while the average area increased from 1.24 m2 to 45.01m2 (p<0.001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57m/s at 16 hpi compared to 0.22 m/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During co-infection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10-16 hours post-infection. We speculate that dsRNA virus reassortment requires VF coalescence, and the potential for reassortment increases at later time points in infection. ImportanceReassortment is common in viruses with segmented double stranded (ds)RNA genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs from two different strains of dsRNA virus during a co-infection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 hours pi. We hypothesise that VF coalescence is required for the reassortment of dsRNA viruses and the potential for reassortment increases later in the replication cycle. This study provides new information that adds to our understanding of dsRNA virus VF trafficking.

microbiology↗

bioRxiv2019

Genome-wide association and functional analyses identify CASC20 and KIF26B as target loci in heterotopic ossification

Heterotopic ossification (HO) is bone formation that occurs after trauma within tissues that do not normally have the property of ossification, resulting in pain and disability. The genetic architecture of HO remains unclear. In the first genome-wide association studies of this disease, we identify the human-only long non-coding RNA-encoding gene CASC20 as a robust, replicating susceptibility locus for HO and KIF26B as a potential severity locus. We find that both CASC20 and KIF26B are expressed in human bone. Both CASC20 and KIF26B expression is upregulated upon BMP2 induced osteogenic differentiation in primary human mesenchymal stem cells, followed by RUNX2 and OSTERIX upregulation and mineralised nodule formation. A CRISPR-Cas9 mediated knockout of Kif26b inhibits BMP2-induced Runx2, Sp7/Osterix, Col1A1, Alp, and Bglap/Osteocalcin expression in a murine myocyte model of osteogenic trans-differentiation, and prevents mineralised nodule formation. These studies provide the first insights into the heritable biology of common, complex HO.

genetics↗

bioRxiv2019

The porcine deltacoronavirus replication organelle comprises double membrane vesicles and zippered endoplasmic reticulum with double membrane spherules

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea and the virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to begin to address questions regarding virus-host cell interactions for this genera of coronavirus. Here we present a detailed study of PDCoV induced replication organelles. All positive strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta- and Gammacoronavirus genera have been characterized. However the structures induced by deltacoronaviruses, in particular the presence of convoluted membranes or double membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis and progeny particle release determined. Subsequently, virus induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double membrane vesicles. Significantly however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum and small associated tethered vesicles, double membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

microbiology↗

bioRxiv2018

Biomarkers of collagen synthesis predict progression in the PROFILE idiopathic pulmonary fibrosis cohort

Idiopathic pulmonary fibrosis (IPF) is characterised by excessive extracellular matrix (ECM) deposition and remodelling. Measuring this activity provides an opportunity to develop tools capable of identifying individuals at-risk of progression. Longitudinal change in markers of ECM synthesis was assessed in 145 newly-diagnosed individuals with IPF. Serum levels of collagen synthesis neoepitopes, PRO-C3 and PRO-C6 (collagen type 3 and 6), were elevated in IPF compared with controls at baseline, and progressive disease versus stable disease during follow up, (PRO-C3 p<0.001; PRO-C6 p=0.029). Assessment of rate of change in neoepitope levels from baseline to 3 months (defined as slope to month 3: HIGH slope, slope > 0 vs. LOW slope, slope <=0) demonstrated no relationship with mortality for these markers (PRO-C3 (HR 1.62, p=0.080); PINP (HR 0.76, p=0.309); PRO-C6 (HR 1.14, p=0.628)). As previously reported, rising concentrations of collagen degradation markers C1M, C3M, C6M and CRPM were associated with an increased risk of overall mortality (HR=1.84, CI 1.03 - 3.27, p=0.038, HR=2.44, CI 1.39-4.31, p=0.002; HR= 2.19, CI 1.25-3.82, p=0.006; HR= 2.13 CI 1.21-3.75, p=0.009 respectively). Elevated levels of PRO-C3 and PRO-C6 associate with IPF disease progression. Collagen synthesis and degradation biomarkers have the potential to enhance clinical trials in IPF and may inform prognostic assessment and therapeutic decision making in the clinic.

clinical trials↗

bioRxiv2018

Acute effects of systemic inflammation upon neurovascular unit and cerebrovascular function.

Background: Brain health relies on a tightly regulated system known as neurovascular function whereby the cellular constituents of the neurovascular unit (NVU) regulate cerebral haemodynamics in accordance with neuronal metabolic demand. Disruption of neurovascular function impairs brain health and is associated with the development of disease, including Alzheimer's disease (AD). The NVU is the site of action of neuroinflammatory responses and contributes to the transition of systemic inflammation to neuroinflammatory processes. Thus, systemic inflammatory challenges may cause a shift in the NVU focus, prioritising neuroimmune over neurovascular actions leading to altered neurovascular function. Methods: Rats were injected with lipopolysaccharide (LPS) (2mg/kg) or vehicle and haemodynamic responses to sensory and non-sensory (hypercapnia) stimuli were assessed in vivo. Following imaging, animals were perfused and their brain extracted to histologically characterise components of the NVU to determine the association between underlying pathology to altered blood flow regulation in vivo. Results: LPS-treated animals showed altered haemodynamic function and cerebrovascular dynamics 6 hours after LPS administration. Histological assessment identified a significant increase in astrogliosis, microgliosis and endothelial activation in LPS-treated animals. Conclusions: Our data shows that an acutely induced systemic inflammatory response is able to rapidly alter in-vivo haemodynamic function and is associated with significant changes in the cellular constituents of the NVU. We suggest that these effects are initially mediated by endothelial cells, which are directly exposed to the circulating inflammatory stimulus and have been implicated in regulating functional hyperaemia.

neuroscience↗

bioRxiv2018

Abnormal microstructural development of the cerebral cortex in neonates with congenital heart disease is associated with impaired cerebral oxygen delivery

BackgroundAbnormal macrostructural development of the cerebral cortex has been associated with hypoxia in infants with congenital heart disease (CHD). Animal studies have suggested that hypoxia results in cortical dysmaturation at the cellular level. New magnetic resonance imaging (MRI) techniques offer the potential to investigate the relationship between cerebral oxygen delivery and microstructural development of the cortex in newborn infants with CHD.\n\nMethodsWe measured macrostructural and microstructural properties of the cortex in 48 newborn infants with complex CHD and 48 age-matched healthy controls. Cortical volume and gyrification index were calculated from high resolution structural MRI. Neurite density and orientation dispersion indices were modelled using high angular resolution diffusion MRI. Cerebral oxygen delivery was estimated in infants with CHD using phase contrast MRI and pre-ductal pulse oximetry. We used tract-based spatial statistics to examine voxel-wise group differences in cortical microstructure.\n\nResultsMicrostructural development of the cortex was abnormal in 48 infants with CHD, with regions of increased fractional anisotropy (FA) and reduced orientation dispersion index (ODI) compared to 48 healthy controls, correcting for gestational age at birth and scan (FWE-corrected for multiple comparisons at P<0.05). Regions of reduced cortical ODI in infants with CHD were related to impaired cerebral oxygen delivery (R2=0.637, n=39). Cortical ODI was associated with gyrification index (R2=0.589, P<0.0001, n=48).\n\nConclusionsThis study suggests that the primary component of cerebral cortex dysmaturation in CHD is impaired dendritic arborisation, which may underlie abnormal macrostructural findings reported in this population. The degree of impairment was related to cerebral oxygen delivery, supporting the hypothesis that maternal oxygen therapy may be beneficial in this population.

neuroscience↗

bioRxiv2017

Allele specific expression analysis identifies regulatory variation associated with stress-related genes in the Mexican highland maize landrace Palomero Toluqueno

BackgroundGene regulatory variation has been proposed to play an important role in the adaptation of plants to environmental stress. In the central highlands of Mexico, farmer selection has generated a unique group of maize landraces adapted the challenges of the highland niche. In this study, gene expression in Mexican highland maize and a reference maize breeding line were compared to identify evidence of regulatory variation in stress-related genes. It was hypothesised that local adaptation in Mexican highland maize would be associated with a transcriptional signature observable even under benign conditions.\n\nMethodsAllele specific expression analysis was performed using the seedling-leaf transcriptome of an F1 individual generated from the cross between the highland adapted Mexican landrace Palomero Toluqueno and the reference line B73, grown under benign conditions. Results were compared with a published dataset describing the transcriptional response of B73 seedlings to cold, heat, salt and UV treatments.\n\nResultsA total of 2386 genes were identified to show allele specific expression. Of these, 277 showed an expression difference between Palomero Toluqueno and B73 alleles that mirrored the response of B73 cold, heat, salt and/or UV treatments, and, as such, were considered to display a constitutive stress response. Constitutive stress response candidates included genes associated with plant hormone signaling and a number of transcription factors. Construction of a gene co-expression network revealed further signaling and stress-related genes to be among the potential targets of the transcription factors candidates.\n\nDiscussionConstitutive activation of responses may represent the best strategy when stresses are severe but predictable. Expression differences observed here between PT and B73 alleles indicate the presence of cis-acting regulatory variation linked to stress-related genes in PT. Considered alongside gene annotation and population data, allele specific expression analysis of plants grown under benign conditions provides an attractive strategy to identify functional variation potentially linked to local adaptation.

plant biology↗

bioRxiv2017

A Systematic Nomenclature for the Drosophila Ventral Nervous System

The fruit fly, Drosophila melanogaster, is an established and powerful model system for neuroscience research with wide relevance in biology and medicine. Until recently, research on the Drosophila brain was hindered by the lack of a complete and uniform nomenclature. Recognising this problem, the Insect Brain Name Working Group produced an authoritative hierarchical nomenclature system for the adult insect brain, using Drosophila melanogaster as the reference framework, with other taxa considered to ensure greater consistency and expandability (Ito et al., 2014). Here, we extend this nomenclature system to the sub-gnathal regions of the adult Drosophila nervous system, thus providing a systematic anatomical description of the ventral nervous system (VNS). This portion of the nervous system includes the thoracic and abdominal neuromeres that were not included in the original work and contains the motor circuits that play essential roles in most fly behaviours.

neuroscience↗

bioRxiv2017

Epidemic establishment and cryptic transmission of Zika virus in Brazil and the Americas

Zika virus (ZIKV) transmission in the Americas was first confirmed in May 2015 in Northeast Brazil1. Brazil has the highest number of reported ZIKV cases worldwide (>200,000 by 24 Dec 20162) as well as the greatest number of cases associated with microcephaly and other birth defects (2,366 confirmed cases by 31 Dec 20162). Following the initial detection of ZIKV in Brazil, 47 countries and territories in the Americas have reported local ZIKV transmission, with 22 of these reporting ZIKV-associated severe disease3. Yet the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of such information for interpreting past trends in reported microcephaly. To address this we generated 53 complete or partial ZIKV genomes, mostly from Brazil, including data generated by the ZiBRA project - a mobile genomics lab that travelled across Northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Joint analyses of viral genomes with ecological and epidemiological data estimate that the ZIKV epidemic first became established in NE Brazil by March 2014 and likely disseminated from there, both nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates of the international spread of ZIKV from Brazil coincide with periods of high vector suitability in recipient regions and indicate the duration of pre-detection cryptic transmission in those regions. NE Brazils role in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the virus basic reproduction number.\n\nOne Sentence SummaryVirus genomes reveal the establishment of Zika virus in Northeast Brazil and the Americas, and provide an appropriate timeframe for baseline (pre-Zika) microcephaly in different regions.

genomics↗

bioRxiv2017

Activation of NF-κB by a novel, CDK4-regulated, nucleolar stress response pathway

p53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-{kappa}B transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-{kappa}B also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-{kappa}B pathway is as yet unknown. Here we demonstrate that siRNA silencing of PolI complex components stimulates NF-{kappa}B signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-{kappa}B induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of the NF-{kappa}B pathway and is associated with an atypical nucleolar architecture. It is mimicked by CDK4 inhibition and is dependent upon upstream binding factor (UBF) and p14ARF. We show that blocking stress effects on TIF-IA blocks their ability to activate the NF-{kappa}B pathway. Finally, using ex vivo culture, we show a strong correlation between degradation of TIF-IA and activation of NF-{kappa}B in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, NF-{kappa}B nucleolar stress response pathway that has in vivo relevance and therapeutic implications.

cell biology↗