A genome-wide CRISPR screen to generate high-yield cell lines for pandemic influenza vaccine production

All influenza vaccines currently sold in Canada require one fertilized chicken egg to produce roughly one dose of vaccine. During pandemic influenza outbreaks, the limited availability of eggs stresses the ability of this method to deliver vaccine in a timely manner (1). Unlike eggs, cell lines grow exponentially, resulting in virtually limitless substrate for cultivating influenza vaccines. This ability to rapidly scale production during periods of increased demand is ideal for effectively responding to pandemic influenza outbreaks. While promising, cell-based influenza vaccine production suffers from low volumetric yield (~10-fold lower) compared to egg-based methods (2). In this study, a genome-wide screen was used to identify gene knockouts that increase influenza yield in the HEK-293SF cell line. Viral replication is dependent upon a myriad of cellular factors, for example an estimated 9.5% of human protein-coding genes affect HIV-1 replication (3). Many of these cellular factors are components of the innate immune system and actively inhibit virus replication. Identifying these factors will allow the generation of cell lines where they have been knocked out, enhancing influenza yield. To carry out the screen, knockouts were induced with a lentivirus-vectored, pooled CRISPR/Cas9 library. Cells were then infected with Green Fluorescence Protein (GFP)-tagged influenza. Cells with a favorable environment for influenza replication expressed high amounts of GFP, allowing them to be collected using Fluorescence Assisted Cell Sorting. Next Generation Sequencing was then used to determine which knockouts enhanced influenza replication. The results of the screen will inform the generation of high-yield vaccine production cell lines based on the HEK-293SF parent line, advancing efforts towards cell-based vaccine production methods that are able to effectively address pandemic outbreaks. The results also offer insights into the host determinants of influenza infection within the unique environment of bioreactor culture. Krammer F, Palese P. Advances in the development of influenza virus vaccines. Nat Rev Drug Discov. 2015;14(3):167-82. Genzel Y. Designing cell lines for viral vaccine production: Where do we stand? Biotechnol J. 2015;10(5):728-40. Bushman FD, Malani N, Fernandes J, D\u27Orso I, Cagney G, Diamond TL, Zhou H, Hazuda DJ, Espeseth AS, Konig R, Bandyopadhyay S, Ideker T, Goff SP, Krogan NJ, Frankel AD, Young JA, Chanda SK. 2009. Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathog 5:e1000437

Ground-based optical transmission spectrum of the hot Jupiter HAT-P-1b

Time-series spectrophotometric studies of exoplanets during transit using ground-based facilities are a promising approach to characterize their atmospheric compositions. We aim to investigate the transit spectrum of the hot Jupiter HAT-P-1b. We compare our results to those obtained at similar wavelengths by previous space-based observations. We observed two transits of HAT-P-1b with the Gemini Multi-Object Spectrograph (GMOS) instrument on the Gemini North telescope using two instrument modes covering the 320 - 800 nm and 520 - 950 nm wavelength ranges. We used time-series spectrophotometry to construct transit light curves in individual wavelength bins and measure the transit depths in each bin. We accounted for systematic effects. We addressed potential photometric variability due to magnetic spots in the planet's host star with long-term photometric monitoring. We find that the resulting transit spectrum is consistent with previous Hubble Space Telescope (HST) observations. We compare our observations to transit spectroscopy models that marginally favor a clear atmosphere. However, the observations are also consistent with a flat spectrum, indicating high-altitude clouds. We do not detect the Na resonance absorption line (589 nm), and our observations do not have sufficient precision to study the resonance line of K at 770 nm. We show that even a single Gemini/GMOS transit can provide constraining power on the properties of the atmosphere of HAT-P-1b to a level comparable to that of HST transit studies in the optical when the observing conditions and target and reference star combination are suitable. Our 520 - 950 nm observations reach a precision comparable to that of HST transit spectra in a similar wavelength range of the same hot Jupiter, HAT-P-1b. However, our GMOS transit between 320 - 800 nm suffers from strong systematic effects and yields larger uncertainties.Comment: A&A, accepted, 16 pages, 8 figures, 5 table

THE MORTALITY OF BACTERIOPHAGE CONTAINING ASSIMILATED RADIOACTIVE PHOSPHORUS

The bacteriophage T4 containing assimilated radioactive phosphorus is inactivated at a rate proportional to the specific radioactivity of the constituent phosphorus. The beta radiation from the phosphorus makes a negligible contribution to this effect. The inactivation is therefore a direct consequence of the nuclear reaction, which kills the phage with an efficiency of about 1/12. Several phages related to T4 behave similarly. When radioactive phage is grown from a seed of non-radioactive phage, all of the phage progeny are subject to killing by radioactive decay. The phage is killed by beta radiation from P32 with an efficiency of about 1/100 per ionization within the particle volume. Bacteriophage T4 and its relatives contain about 500,000 atoms of phosphorus per infective particle. Virtually all this phosphorus is adsorbed to bacteria with the specificity characteristic of the infective particles, and none of it can be removed from the particles by the enzyme desoxyribonuclease. The phosphorus content per particle, together with the published data on analytical composition, indicates a particle diameter close to 110 mµ for the varieties of phage studied

Effect of Vitamin D on serum markers of bone turnover in SLE in a randomised controlled trial

© 2019 Author(s). Objective Bone health in SLE is adversely affected by vitamin D deficiency, inflammatory cytokines and glucocorticoid use. We hypothesised that vitamin D supplementation would increase markers of bone formation and decrease markers of bone resorption in SLE subjects. Methods We studied 43 vitamin D-deficient SLE subjects who participated in a 12-week randomised controlled trial of 2000-4000 IU/day vitamin D supplementation versus placebo. Subjects had inactive SLE (SLE Disease Activity Index ≤4) and were taking D, N-terminal propeptide of type 1 collagen (P1NP) and C-telopeptide (CTX). We tested the effect of vitamin D versus placebo on change (δ) in P1NP and δCTX in an intention-to-treat analysis. Secondary analyses evaluated whether vitamin D affected bone turnover among subjects achieving vitamin D repletion (≥30 ng/mL) or currently taking glucocorticoids. Results 28 subjects were randomised to vitamin D and 15 to placebo. Mean age was 39 years and 40% were using glucocorticoids at enrolment. Repletion was achieved by 46% in the vitamin D group versus none in the placebo group. Changes in bone turnover markers were not significantly different in the vitamin D group versus placebo group (median δP1NP -0.2 vitamin D group vs -1.1 placebo group (p=0.83); median δCTX +3.5 vitamin D group vs -37.0 placebo group (p=0.50)). The effect of vitamin D did not differ based on achieving vitamin D repletion or baseline glucocorticoid use. Conclusion Vitamin D supplementation did not affect the 12-week change in bone turnover markers among SLE subjects in this trial