Identification of Amino Acids within Nonstructural Proteins 10 and 14 of the Avian Coronavirus Infectious Bronchitis Virus That Result in Attenuation In Vivo and In Ovo

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens' eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo. Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens' eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo. Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application

Multiple novel non-canonically transcribed sub-genomic mRNAs produced by avian coronavirus infectious bronchitis virus

Funding: This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/L003988/1 and 1645891, and strategic funding to The Pirbright Institute, BBS/E/I/00007035, BBS/E/I/00007034, BBS/E/I/00007037 and BBS/E/I/00007039.Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.Publisher PDFPeer reviewe

Multi-channel whole-head OPM-MEG: Helmet design and a comparison with a conventional system

© 2020 The Authors Magnetoencephalography (MEG) is a powerful technique for functional neuroimaging, offering a non-invasive window on brain electrophysiology. MEG systems have traditionally been based on cryogenic sensors which detect the small extracranial magnetic fields generated by synchronised current in neuronal assemblies, however, such systems have fundamental limitations. In recent years, non-cryogenic quantum-enabled sensors, called optically-pumped magnetometers (OPMs), in combination with novel techniques for accurate background magnetic field control, have promised to lift those restrictions offering an adaptable, motion-robust MEG system, with improved data quality, at reduced cost. However, OPM-MEG remains a nascent technology, and whilst viable systems exist, most employ small numbers of sensors sited above targeted brain regions. Here, building on previous work, we construct a wearable OPM-MEG system with ‘whole-head’ coverage based upon commercially available OPMs, and test its capabilities to measure alpha, beta and gamma oscillations. We design two methods for OPM mounting; a flexible (EEG-like) cap and rigid (additively-manufactured) helmet. Whilst both designs allow for high quality data to be collected, we argue that the rigid helmet offers a more robust option with significant advantages for reconstruction of field data into 3D images of changes in neuronal current. Using repeat measurements in two participants, we show signal detection for our device to be highly robust. Moreover, via application of source-space modelling, we show that, despite having 5 times fewer sensors, our system exhibits comparable performance to an established cryogenic MEG device. While significant challenges still remain, these developments provide further evidence that OPM-MEG is likely to facilitate a step change for functional neuroimaging

Science and Technology Issues in the 115th Congress

Science and technology (S&T) have a pervasive influence over a wide range of issues confronting the nation. Public and private research and development spur scientific and technological advancement. Such advances can drive economic growth, help address national priorities, and improve health and quality of life. The constantly changing nature and ubiquity of science and technology frequently create public policy issues of congressional interest. The federal government supports scientific and technological advancement directly by funding and performing research and development and indirectly by creating and maintaining policies that encourage private sector efforts. Additionally, the federal government establishes and enforces regulatory frameworks governing many aspects of S&T activities. This report briefly outlines an array of science and technology policy issues that may come before the 115th Congress. Given the rapid pace of S&T advancement and its importance in many diverse public policy issues, S&T-related issues not discussed in this report may come before the 115th Congress. The selected issues are grouped into 9 categories: - Overarching S&T Policy Issues, - Agriculture, - Biomedical Research and Development, - Defense, - Energy, - Environment and Natural Resources, - Homeland Security, - Information Technology, - Physical and Material Sciences, and - Space. Each of these categories includes concise analysis of multiple policy issues. The material presented in this report should be viewed as illustrative rather than comprehensive. Each section identifies CRS reports, when available, and the appropriate CRS experts to contact for further information and analysis

SnoN expression is differently regulated in microsatellite unstable compared with microsatellite stable colorectal cancers

BACKGROUND: SnoN is an important regulator of the transforming growth factor beta (TGFβ) signalling pathway and has been shown to exhibit both tumour promotion and suppression activity. METHODS: To further explore the role of this complex molecule in colorectal tumorigenesis, we examined 52 paired normal and tumour colorectal specimens stratified by level of microsatellite instability; 18 with high-level microsatellite instability (MSI-H) and 34 microsatellite stable (MSS). SnoN transcript expression was quantitated by real-time PCR and analysed with respect to clinical indicators of prognosis. RESULTS: Within the MSI-H subgroup, SnoN was commonly either up-regulated (6/18, 33%) or down-regulated (7/18, 39%). A significantly different distribution of SnoN expression was observed in MSS cancers compared with MSI-H (P ≤ 0.001). Whilst 17/34 (50%) of MSS tumours demonstrated up-regulation, none showed down-regulated expression. Within the MSI-H subgroup, up-regulation was significantly correlated with lack of repeat tract mutation in the TGFβRII gene (P ≤ 0.025), suggesting that SnoN is more frequently up-regulated in the presence of functional TGFβ signalling. CONCLUSION: Together these data support the notion that SnoN has both oncogenic and tumour suppressive properties depending on other genetic changes within the tumour, and that the MSI-H pathway of colorectal tumorigenesis presents an excellent model for the study of these opposing functions

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation

<p>Abstract</p> <p>Background</p> <p><it>Thrombospondin-4 </it>(<it>THBS4</it>) is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of <it>THBS4 </it>expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter.</p> <p>Methods</p> <p>Fifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP) were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of <it>THBS4 </it>transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon.</p> <p>Results</p> <p><it>THBS4 </it>expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and <it>THBS4 </it>methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high <it>THBS4 </it>methylation, but the correlation was not significant. <it>THBS4 </it>methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and negatively with the occurrence of adenomas elsewhere in the colon.</p> <p>Conclusions</p> <p><it>THBS4 </it>shows increased methylation in colorectal cancer, but this is not strongly associated with altered gene expression, either because methylation has not always reached a critical level or because other factors influence <it>THBS4 </it>expression. <it>THBS4 </it>may act as a tumour suppressor gene, demonstrated by its suppression of tumour colony formation <it>in vitro</it>. <it>THBS4 </it>methylation is detectable in normal colonic mucosa and its level may be a biomarker for the occurrence of adenomas and carcinoma.</p

Large Scale Searches for Brown Dwarfs and Free-Floating Planets

Searches of large scale surveys have resulted in the discovery of over 1000 brown dwarfs in the Solar neighbourhood. In this chapter we review the progress in finding brown dwarfs in large datasets, highlighting the key science goals, and summarising the surveys that have contributed most significantly to the current sample.Comment: Accepted to appear in the Handbook of Exoplanets (Springer); Editors: Hans J. Deeg & Juan Antonio Belmont

A Multicenter Study of the Validity and Reliability of Responses to Hand Cold Challenge as Measured by Laser Speckle Contrast Imaging and Thermography:outcome measures for systemic sclerosis-related Raynaud's phenomenon

Objective: Reliable and objective outcome measures to facilitate clinical trials of novel treatments for systemic sclerosis (SSc)–related Raynaud's phenomenon (RP) are badly needed. Laser speckle contrast imaging (LSCI) and thermography are noninvasive measures of perfusion that have shown excellent potential. This multicenter study was undertaken to determine the reliability and validity of a hand cold challenge protocol using LSCI, standard thermography, and low‐cost cell phone/mobile phone thermography (henceforth referred to as mobile thermography) in patients with SSc‐related RP. Methods: Patients with RP secondary to SSc were recruited from 6 UK tertiary care centers. The patients underwent cold challenge on 2 consecutive days. Changes in cutaneous blood flow/skin temperature at each visit were imaged simultaneously using LSCI, standard thermography, and mobile thermography. Measurements included area under the curve (AUC) for reperfusion/rewarming and maximum blood flow rate/skin temperature after rewarming (MAX). Test–retest reliability was assessed using intraclass correlation coefficients (ICCs). Estimated latent correlations (estimated from multilevel models, taking values between −1 and 1; denoted as rho values) were used to assess the convergent validity of LSCI and thermography. Results: In total, 159 patients (77% with limited cutaneous SSc) were recruited (84% female, median age 63.3 years). LSCI and standard thermography both had substantial reliability, with ICCs for the reperfusion/rewarming AUC of 0.67 (95% confidence interval [95% CI] 0.54, 0.76) and 0.68 (95% CI 0.58, 0.80), respectively, and ICCs for the MAX of 0.64 (95% CI 0.52, 0.75) and 0.72 (95% CI 0.64, 0.81), respectively. Very high latent correlations were present for the AUCs of LSCI and thermography (ρ = 0.94; 95% CI 0.87, 1.00) and for the AUCs of standard and mobile thermography (ρ = 0.98; 95% CI 0.94, 1.00). Conclusion: This is the first multicenter study to examine the reliability and validity of cold challenge using LSCI and thermography in patients with SSc‐related RP. LSCI and thermography both demonstrated good potential as outcome measures. LSCI, standard thermography, and mobile thermography had very high convergent validity