Updated Spitzer Emission Spectroscopy of Bright Transiting Hot Jupiter HD189733b

We analyze all existing secondary eclipse time series spectroscopy of hot Jupiter HD189733b acquired with the now defunct Spitzer/IRS instrument. We describe the novel approaches we develop to remove the systematic effects and extract accurate secondary eclipse depths as a function of wavelength in order to construct the emission spectrum of the exoplanet. We compare our results to a previous study by Grillmair et al. that did not examine all data sets available to us. We are able to confirm the detection of a water feature near 6{\mu}m claimed by Grillmair et al. We compare the planetary emission spectrum to three model families -- based on isothermal atmosphere, gray atmosphere, and two realizations of the complex radiative transfer model by Burrows et al., adopted in Grillmair et al.'s study. While we are able to reject the simple isothermal and gray models based on the data at the 97% level just from the IRS data, these rejections hinge on eclipses measured within relatively narrow wavelength range, between 5.5 and 7{\mu}m. This underscores the need for observational studies with broad wavelength coverage and high spectral resolution, in order to obtain robust information on exoplanet atmospheres.Comment: 16 pages, 13 figures and 3 tables. Accepted for publication in Ap

292. Towards Large-Scale Manufacturing of Adeno-Associated Virus by Transient Transfection of HEK293 Suspension Cells in a Stirred Tank Bioreactor Using Serum-Free Medium

Adeno-Associated Virus (AAV) vectors showing safety profile in phase I clinical trials and its ability to transduce gene expression in various tissues have made it a vector of choice for gene delivery. There are different modes of AAV vector production and each has advantages and disadvantages. Here we demonstrated that the production of AAV by transient transfection in a serum-free medium using NRC's patented cGMP compliant human embryonic kidney HEK293 cell line (clone HEK293SF-3F6) adapted for growth in suspension can be readily scaled-up in stirred tank bioreactors. We employed triple-plasmid / polyethylenimine (PEI) based transient transfection technique. As a proof of concept, we demonstrated that nine serotypes of AAV (AAV-1 to AAV-9) encoding GFP can be produced by our cell line HEK293SF with yields of about 1E+13 genome-containing particles per liter (Vg/L). Depending on the serotypes 4-30% of AAV is present in the supernatant of the cell culture at 48hpt. The presence of plasmids and plasmid polyplexes that were not taken up by the cells or were not brought into the cell nucleus were removed by Iodixanol-ultracentrifugation method and Benzonase treatment before analyzing by real-time PCR. About 25% loss in genome containing viral particle counts were observed by Iodixanol purification method based on infectivity assay. Productions of AAV2 and AAV6 encoding GFP were demonstrated in 3L stirred tank bioreactors. Purification scheme was based on column chromatography - a scalable process. Different chromatography media, such as cation exchanger, anion exchanger and hydrophobic interaction chromatography, were tested with each AAV serotypes for their ability to adsorb and elute efficiently. The purification scheme was then adopted by integrating best chromatography medium and sequence dependent upon the AAV serotype in use. We demonstrated the purification scheme for AAV2 based on ion-exchange and hydrophobic interaction chromatography steps. The SDS-PAGE showed the purity of the final product and the presence of three capsid proteins VP1, VP2 and VP3 on Western blot corresponding to the only three bands present in the final product on SDS-PAGE. To extend the storage life of AAV we explored lyophilization technique to study the stability of AAV2 and AAV6 under lyophilized conditions. The AAV2 and AAV6 were stable for over 40 weeks based on infectivity assay. We demonstrated the scalability of the process up to 45L. Productions tested in 20 and 500 mL cultures in shake flasks were scaled up in 2 and 45L cultures (in 3- and 60-L stirred tank bioreactors, respectively). The volumetric yields and purification recoveries were comparable at all of these production scale levels demonstrating scalability of transient transfection at even larger scale is possible to generate material necessary for dosages required for gene therapy application

Advancements in molecular design and bioprocessing of recombinant adeno-associated virus gene delivery vectors using the insect-cell baculovirus expression platform.

AbstractDespite rapid progress in the field, scalable high‐yield production of adeno‐associated virus (AAV) is still one of the critical bottlenecks the manufacturing sector is facing. The insect cell‐baculovirus expression vector system (IC‐BEVS) has emerged as a mainstream platform for the scalable production of recombinant proteins with clinically approved products for human use. In this review, we provide a detailed overview of the advancements in IC‐BEVS for rAAV production. Since the first report of baculovirus‐induced production of rAAV vector in insect cells in 2002, this platform has undergone significant improvements, including enhanced stability of Bac‐vector expression and a reduced number of baculovirus‐coinfections. The latter streamlining strategy led to the eventual development of the Two‐Bac, One‐Bac, and Mono‐Bac systems. The one baculovirus system consisting of an inducible packaging insect cell line was further improved to enhance the AAV vector quality and potency. In parallel, the implementation of advanced manufacturing approaches and control of critical processing parameters have demonstrated promising results with process validation in large‐scale bioreactor runs. Moreover, optimization of the molecular design of vectors to enable higher cell‐specific yields of functional AAV particles combined with bioprocess intensification strategies may also contribute to addressing current and future manufacturing challenges

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

Equilibrium Isotherm, Kinetic and Thermodynamic Studies of the Adsorption of Erythrosine Dye onto Activated Carbon from Coconut Fibre

Equilibrium isotherm, kinetic and thermodynamic studies of the adsorption of erythrosine dye onto activation carbon from coconut fire was carried out. The coconut fibre obtain from Elele, Rivers State Nigeria, was washed, dried, carbonized at 400oC, crushed, sieved and activated at 800oC, before it was washed and dried at 110oC. Variable influencing factors, such as contact time, temperature and initial concentration were studied through single-factor experiment, while other factors are kept constant (at 30min, 30oC and 50mg/L) in each adsorption experiment. The Freundlich isotherm fits adsorption compare to others used, the adsorption kinetic followed pseudo-second order reaction, while the thermodynamic parameters, (∆H) = 28.73KJ/mol, (∆G) = 94.45J/mol.K and (∆S) = -0.10, -0.27, -0.82, -1.05, -1.77, -2.49KJ/mol. From the results obtained, activated carbon from coconut fibre, will be an excellent low-cost adsorbent for the removal of Erythrosine from industrial waste water

Development of suspension adapted Vero cell culture process technology for production of viral vaccines

Abstract Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 10 6 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10 6 cells/mL

A novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient siRNA delivery in breast cancer therapy: preparation and in vitro analysis

Wei Shao1, Arghya Paul1, Sana Abbasi1, Parminder S Chahal2, Jimmy A Mena2, Johnny Montes2, Amine Kamen2, Satya Prakash11Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering and Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, 2Animal Cell Technology, Bioprocess Centre, Biotechnology Research Institute, National Research Council, Montreal, Quebec, CanadaBackground: Systemic delivery of small interfering RNA (siRNA) is limited by its poor stability and limited cell-penetrating properties. To overcome these limitations, we designed an efficient siRNA delivery system using polyethyleneimine-coated virus-like particles derived from adeno-associated virus type 2 (PEI-AAV2-VLPs).Methods: AAV2-VLPs were produced in insect cells by infection with a baculovirus vector containing three AAV2 capsid genes. Using this method, we generated well dispersed AAV2-VLPs with an average diameter of 20 nm, similar to that of the wild-type AAV2 capsid. The nanoparticles were subsequently purified by chromatography and three viral capsid proteins were confirmed by Western blot. The negatively charged AAV2-VLPs were surface-coated with PEI to develop cationic nanoparticles, and the formulation was used for efficient siRNA delivery under optimized transfection conditions.Results: PEI-AAV2-VLPs were able to condense siRNA and to protect it from degradation by nucleases, as confirmed by gel electrophoresis. siRNA delivery mediated by PEI-AAV2-VLPs resulted in a high transfection rate in MCF-7 breast cancer cells with no significant cytotoxicity. A cell death assay also confirmed the efficacy and functionality of this novel siRNA formulation towards MCF-7 cancer cells, in which more than 60% of cell death was induced within 72 hours of transfection.Conclusion: The present study explores the potential of virus-like particles as a new approach for gene delivery and confirms its potential for breast cancer therapy.Keywords: adeno-associated virus type 2, virus-like particles, small interfering RNA delivery, breast cancer therapy, nanomedicin

Statistical Modeling and Optimization of Biodiesel Production from Azadirachta Indica (Neem) Using Co-Solvent Technique

In this work, statistical modeling and optimization of biodiesel production from Azadirachta Indica(neem) using co-solvent technique via a two-step transesterification process was carried out. Neem oil was extracted from neem seeds and properties such as moisture content, specific gravity, acid value, saponification value and iodine value were determined. The experimental design used was Central Composite Design. The range of factor levels used for the Central Composite Design were reaction temperature (30°C to 46°C), catalyst amount (0.8% to 1.2%, w/w), reaction time (20 to 40min) and methanol-to-oil molar ratio (5:1 to 9:1). The co-solvents used were methanol and diethyl ether. The co-solvent-to-methanol volume ratio for all the experimental runs was kept constant at 1:1. Also the biodiesel produced was characterized for some important properties including acid value, specific gravity, saponification value, iodine value, cetane number, ester value, kinematic viscosity, flash point, pour point and cloud point. Optimized biodiesel yield of 84.77% was obtained for reaction time of 35 min, catalyst amount of 1.10g, reaction temperature of 34°C, and oil-to-methanol molar ratio of 6:1. The cetane number (51.733), specific gravity (0.8881g/cm3), flash point (134oC) and kinematic viscosity (5.86mm2/s) of the produced biodiesel met the ASTM specifications. The results of characterization of the biodiesel revealed that biodiesel can be produced at lower reaction conditions and with comparable fuel property with biodiesel produced using conventional methods