The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications.
Since the development of recombinant human insulin, protein-based therapeutics have been the fastest-growing segment of the pharmaceutical industry. Three of the five highest revenue drugs in oncology, Rituxan, Avastin and Herceptin, are monoclonal antibodies . The appearance of hundreds of non-antibody therapeutic proteins, including enzymes, cytokines and growth factors and blood proteins, validates predictions from 2004  that this would be an area of significant growth. Between 2011 and 2016, 62 new therapeutic protein drugs were approved by the US FDA . A further 27 were approved between September 2016 and December 2017 . Although the unique benefits of biopharmaceuticals are clear, the costs to the health care system are becoming prohibitive . Prices charged have traditionally been based upon patient benefit, but the cost of goods is also significant, especially in the transition to generics and biosimilars. Many biopharmaceuticals are produced using bacterial, yeast or mammalian cell culture expression systems. Current mammalian cell-culture based systems are increasingly optimised and industrialized . Each system is constrained in some measure by yield, cost, complex purification procedures and appropriate post-translational modification when required for biological function.
Development of genetic modification systems for livestock offered the possibility of harnessing natural high-volume protein production systems (e.g. milk from farmed livestock and eggs from poultry) for the production of biologics. Transgenic sheep, goats and cows were generated that produced biopharmaceuticals in their milk, with the aim of establishing animal bioreactors for large scale production of high quality proteins, potentially at lower capital and production costs than cell culture systems [7, 8]. The first drug produced in a transgenic animal, antithrombin III in goat milk, was approved for use by the European Medicines Agency in 2006 and the FDA in 2009 . However, there are limitations to the use of large mammals as bioreactors. They have few offspring, long gestation times and require a large amount of space and feed, all of which can make industrial scale-up difficult and expensive. It can also be difficult to purify a recombinant protein from milk due to the high content of lipids and other proteins . Reabsorption of the protein by the animal can have deleterious effects, particularly if the human protein is biologically active in the host .
Interest in developing the laying hen as a bioreactor is based on the high protein synthetic capacity of the hen: an average 60 g egg contains nearly 3.5 g of protein in the egg white  and each hen may lay over 300 eggs per year. Chickens are relatively inexpensive to keep  and large flocks can be built quickly from small numbers of founder birds. In addition, chickens have closely-related glycosylation patterns to humans , which may result in reduced immunogenicity of biologics from egg white, as well as improving functionality of proteins where glycosylation is required for activity [13–15]. We showed previously that lentiviruses can be used for efficient production of transgenic chickens . Transgene expression can be restricted to the oviduct (and thus egg white) using sequences from the ovalbumin gene, encoding the most abundant egg white protein . While this approach allowed successful production of two biologically active proteins in egg white, the expression levels achieved were well below the > 1 g/L likely to be required as the basis of a commercially viable system. In addition, purification of recombinant proteins from egg white, a matrix characterized by high levels of other proteins including albumin and the gel-like glycoconjugate ovomucin, remained to be tackled . Here we demonstrate the improved and optimized production of two new proteins, including a cytokine dimer, and robust purification protocols. Interferon α2a is a legacy, off-patent biologic used to treat certain forms of cancer and hepatitis [18–20] and was selected as a model for biosimilar production in the chicken system. Macrophage colony-stimulating factor (CSF1) is a candidate therapeutic protein in many areas of regenerative medicine . Like interferon, CSF1 is a 4-helix bundle protein, structurally-related to many cytokines, including insulin, somatotrophin, placental lactogen, G-CSF, GM-CSF and erythropoietin (EPO)  that are in widespread clinical use. Immunoglobulin Fc fusion proteins are an emerging class of biopharmaceuticals [23, 24]. Several therapeutic 4 helix bundle proteins have been tested as fusion proteins with the Fc region to improve their circulating half-life and biological activity [24–28]. We recently produced a Fc fusion protein of CSF1 and demonstrated increased in vivo efficacy in rodent and large animal models, and potential as therapeutic in liver regeneration [29–32]. An additional attraction of the Fc tag in generation of therapeutic proteins in bioreactors is that it provides a generic tag for affinity purification. Here we demonstrate the optimal production, downstream processing and biological activity of recombinant proteins produced in the transgenic avian system. The chicken bioreactor clearly has the potential to address the cost and availability of therapeutic proteins for both human and veterinary medicine.
Human interferon α2a is a cytokine commonly used for treatment of hepatitis and various cancers [18–20]. The 0.5 kb coding sequence for interferon α2a was synthesized and cloned into a replication-defective equine infectious anemia virus (EIAV) lentiviral vector with a modified form of the EREOVA promoter previously published . To increase the level of expression, we reintroduced an additional 0.9 kb of the ovalbumin promoter sequence between the estrogen response element (ERE) and the steroid-dependent regulatory element (SDRE) (EREOVA2; Fig. 1a, Additional file 1: Figure S1). Transgenic birds were generated using this lentiviral vector with our established methods . A single G1 male was identified that carried an intact transgene and a line established from this bird. The presence of interferon α2a in egg white in hens from this line was confirmed by western blot analysis with an antibody against human interferon α (Fig. 1b), and concentration measured by a commercial ELISA kit. Biological activity before purification was confirmed by applying diluted whole egg white to cells transfected with a reporter of interferon-stimulated response element (ISRE) driving luciferase expression (Fig. 1c).
A multi-step purification protocol was developed that involves clarification of ovomucin by pH reduction and its removal by centrifugation, and purification of the interferon α2a by HiTrap Blue capture followed by size exclusion chromatography (SEC) polishing; purity was confirmed by SDS-PAGE (Fig. 1d). Recovery of interferon α2a was approximately 15 mg from 100 mL of egg white, which is 60% of the original 25 mg/100 mL estimate determined by ELISA, with most of the loss occurring in the wash of the capture step; there is no observable protein loss in the ovomucin precipitation step or polishing step (Fig. 1e). Purity of > 95% was determined by reversed phase chromatography and SEC-multi-angle light scattering analyzes (Additional file 2: Figure S2).
The anti-viral properties of the purified interferon α2a were examined using a dose-inhibition assay against an H1N1 influenza A virus (A/Puerto Rico/8/1934 PR8) in A549 cells pre-treated with interferon α2a. The assay was performed as described previously  with commercially-available interferon α2a (produced in E. coli) as a control. The egg white interferon showed improved activity compared to the commercial protein (Fig. 1f), calculated to be as high as 1 × 109 U/mg, an order of magnitude greater than the bacterially-produced protein.
Colony stimulating factor 1 (CSF1) is required for differentiation, proliferation and function of macrophages, and has shown promise as a therapeutic protein for a variety of indications, particularly in regenerative medicine [21, 29, 30]. Porcine CSF1 is active across mammalian species , and an Fc fusion protein of CSF1 was shown to have increased half-life and efficacy in vivo . The coding sequence for porcine CSF1 fused to an antibody Fc region (pCSF1-Fc)  was synthesized and cloned into a pLenti6 (Invitrogen) lentiviral vector derived from human immunodeficiency virus (HIV), downstream of the EREOVA2 promoter and upstream of optimized woodchuck hepatitis virus posttranscriptional regulatory element  (oPRE) (Fig. 2a). Founder (G0) cockerels were generated  and bred to identify G1 transgenic birds. The presence of a single insertion of the intact transgene was confirmed in two hens and one cockerel by Southern blot analysis (Additional file 3: Figure S3). The two hens had the same insertion site (Line 1), while the cockerel had a different insertion (Line 2); all three were bred with stock birds to generate G2 hens. Expression of pCSF1-Fc in egg white was confirmed by SDS-PAGE and western blot with an antibody against porcine Fc (Fig. 2b), and expression was equivalent between two generations (Additional file 4: Figure S4). To test the functionality of pCSF1-Fc egg white, porcine CSF1 receptor was expressed in factor-dependent Ba/F3 cells  as previously described , making them dependent on functional CSF1-Fc for survival. Whole egg white from both Line 1 hens promoted survival and proliferation at similar concentrations to control pCSF1-Fc produced in CHO cells , while wild type egg white alone was unable to promote survival (Fig. 2c).
Egg white from pCSF1-Fc eggs was clarified of ovomucin as described for interferon purification. All of the protein came through in the supernatants (Fig. 2d). The active dimer form of pCSF1-Fc was purified by MabSelect SuRe capture followed by size exclusion chromatography (SEC) polishing, and purity confirmed by SDS-PAGE (Fig. 2e). Due to the difficulty in quantifying pCSF1-Fc in whole egg white by western blot and the lack of an ELISA test, concentration of protein was calculated based on the final recovery and estimates of protein loss through the purification process based on western blots of samples collected throughout the process (Fig. 2f). Purity was > 97%, as determined by reversed phase chromatography and SEC-multi-angle light scattering. The molecular mass average across the elution profile is 95.1 kDa with good protein mono-dispersity (Mw/Mn = 1.011) and no significant aggregation. Co-polymer model analysis indicated that peak mass was comprised of 85.6 kDa of protein and 9.5 kDa of oligosaccharide (~ 10% glycosylation). (Additional file 5: Figure S5). Mass spectrometry analysis confirmed the identity of pCSF1-Fc; presence of contaminating egg white proteins was below the limit of detection (Additional file 6: Table S1). The Ba/F3 cell survival assay was repeated with pCSF1-Fc purified from fresh egg white, egg white that had been stored at -20 °C for at least 1 month, and egg white that had been stored at -80 °C for at least 1 month. The control pCSF1-Fc and all purified egg pCSF1-Fc samples were able to promote cell survival at comparable levels (Fig. 2g), demonstrating that egg white can be collected and frozen for a significant period of time prior to purification without loss of recovery or activity. The ability of the purified protein to interact with the native CSF1 receptor was confirmed using mouse bone marrow cells. Both control and egg-derived pCSF1-Fc promoted cell differentiation at comparable levels (Fig. 2h).
To determine the activity of egg-derived pCSF1-Fc in vivo, mice were treated with 1 μg/g of either control or purified egg pCSF1-Fc, following which the livers, spleens and blood were analyzed for weight and histology as previously described . Consistent with previous reports, both forms of pCSF1-Fc caused an increase in F4/80+CD11b+ cells in the blood (Fig. 3a) and F4/80+ tissue macrophages in the liver (Fig. 3b). There were also significant increases in the weights of the liver and spleen (Fig. 3c-d).
Although pCSF1-Fc is active on human cells and might have short-term therapeutic applications in human patients and in veterinary medicine, it is likely to be immunogenic. Indeed, we have immunized mice with the CSF1-Fc protein to produce monoclonal antibodies that cross-react with human CSF1 (hCSF1; L.Waddell, DAH, manuscript in preparation). As a prelude to testing human clinical applications, we have generated additional chicken lines expressing a human CSF1-Fc (hCSF1-Fc) protein using the same vector system and employing the optimized purification protocol. The presence of a single insertion of the intact transgene was confirmed in two hens by Southern blot analysis (Additional file 7: Figure S6), and both were calculated to have correct protein expression (Fig. 4a) at levels of approximately 1 mg/mL, calculated by the same method as for pCSF1-Fc. Purification was identical to that of pCSF1-Fc, including recovery efficiency (Fig. 4b). Since hCSF1 has previously been shown to interact with pig bone marrow , the bone marrow differentiation assay was repeated to test activity of purified hCSF1-Fc, and protein from both lines was shown to promote survival and differentiation at an equivalent level to each other and pCSF1-Fc control, even after vacuum drying and reconstitution (Fig. 4c).
These results indicate that both interferon α2a and CSF1-Fc are efficiently synthesized as a component of egg white, readily recoverable using common chromatography methods, and biologically active in vitro and in vivo at levels equal to control cytokines produced in different systems.
Lentiviruses are efficient vectors for the generation of transgenic chickens with stable germline transmission and consistent, oviduct-specific expression of correctly folded and functional protein . Here we have demonstrated an improved promoter design that generated levels of expression compatible with cost-effective scale-up. There were no negative effects on the health of the hens despite the substantial increase in expression of exogenous protein. The method can be applied to untagged and Fc-tagged proteins, and common chromatographic techniques can be applied to purify the proteins from egg white. Initial characterization of egg-derived proteins showed high levels of purity and activity as good as or better than the equivalent proteins produced in traditional cell culture or E. coli systems. Contamination by egg proteins was below levels detectable by mass spectrometry and in vivo testing showed no immunogenic reaction in mice. It is likely that similarly-structured proteins, including the many therapeutic Fc fusion proteins currently under clinical evaluation [23, 24]. will be amenable to production in the egg white of transgenic chickens and purification as we have described at commercially relevant scale.
The infrastructure and upstream costs of a transgenic chicken bioreactor production system are predicated to be substantially lower than those for mammalian cell culture, perhaps by as much as two orders of magnitude, due largely to the comparably lower cost of chicken facilities and lack of requirement for GMP conditions until egg crack. Initial analysis provided by the Centre for Process Innovation National Biologics Manufacturing Centre (Darlington, UK) indicates that downstream processing costs should be equivalent to current methods, leading to an overall lower manufacturing cost. Relative to mammalian transgenic animal bioreactors, a flock of bioreactor chickens is faster and cheaper to produce, and egg white is a simpler mixture of proteins with a significantly lower lipid content than milk. In addition, the favorable glycosylation patterns and oxidative environment of the chicken system make it appealing for production of proteins with specific requirements for these characteristics that are currently difficult to recapitulate in cell culture.
The lentivirus system was the best available at the time of generation of these chicken lines, but since then, significant advances have taken place in chicken primordial germ cell culture and genome editing. Combining these techniques means precise control over the insertion site of the transgene and uniformity of the resulting chicken population. It also means substantially increased efficiency, reduced need for genotype screening, no biosafety concerns around lentiviral particles, and overall lower upstream line generation costs, making the transgenic chicken bioreactor an even more appealing option.
Human interferons were amongst the first patented biologicals. The extension of many of the therapeutic applications of human interferon α2a (RoferonA) and other isoforms to companion animal veterinary medicine have been reviewed recently . Interferon also has increased bioavailability as a Fc fusion protein , and this form would be even more readily produced in a chicken bioreactor using the purification approach described here. Production in chickens may bring the cost down to levels that would support wider applications, and extension to containment of viral diseases in valuable livestock. For the CSF1-Fc fusion protein, a dose of 1 mg/kg twice a week was required to produce a maximal elevation of blood monocyte and tissue macrophage numbers when tested in pigs . Presuming similar efficacy in humans, a single dose could be obtained from 2 to 3 eggs. One of the therapeutic applications of CSF1-Fc already validated in mouse models is in the treatment of bone fractures , an application of relevance to both human and companion animal medicine. Even if production of proteins such as CSF1-Fc in chickens faces hurdles in translation to the human clinic, the bioreactor will enable cost-effective production of the amounts required to test clinical applications in informative large animal models.
We have demonstrated that a human cytokine, interferon α2a, can be recovered at a level of 15 mg/100 ml egg white (the approximate volume from 3 eggs) with biological activity estimated as 1 × 109 IU/mg. In addition, we have generated two transgenic lines in which porcine and human CSF1 have been synthesized as FC fusion proteins. Very high recovery (80–90%) from ~ 100 mg/100 ml of highly pure proteins with biological activity comparable to equivalent proteins produced in cell culture was demonstrated. We have validated the transgenic chicken system for the cost-effective production of pure, high quality, biologically active proteins for therapeutics and other applications.
The human interferon α2a coding sequence was codon-optimized for chicken and synthesized with a Kozak consensus sequence, start codon and chicken lysozyme signal peptide at the 5′ end. The synthetic gene was ligated into a replication defective vector derived from equine infectious anemia virus (EIAV) containing a 4.4 kb modified form of the EREOVA promoter and regulatory elements (EREOVA2) previously shown to drive protein expression restricted to the hen oviduct . The original EREOVA promoter had 0.9 kb deleted between the estrogen-responsive enhancer element (ERE) and the steroid-dependent regulatory element (SDRE) that was preserved in EREOVA2. The new promoter was found to enhance protein expression in egg white at least 10-fold compared to EREOVA. The synthesized, codon-optimized gene for pCSF1-Fc (Entelechon, Germany) consisted of a Kozak sequence, start codon, lysozyme signal peptide and 154 amino acid active form of porcine CSF1 joined to the hinge-CH3 region of porcine IgG1a as previously described (13). This transgene was cloned using designed restriction sites AatII and NheI into a modified pLenti6/DEST Gateway vector, with the V5 tag removed and downstream oPRE added. This vector was derived from human immunodeficiency virus (HIV) and contained the EREOVA2 promoter.
The birds were derived from a flock of Novagen Brown commercial egg layer strain. Lentiviral vectors were packaged and injected into embryos from new-laid eggs to generate G0 transgenic birds . Cockerels estimated by PCR analysis to have vector sequence present in the germ line at frequency of > 1% were bred with wild-type hens to generate G1 birds, which were screened by PCR and Southern transfer analysis to confirm that the complete gene was present as a single copy. Egg white from G1 hens or female G2 offspring of G1 cockerels was tested for presence of transgene-derived protein. Lines were established from validated G1/G2 birds for each transgene.
Protein expression was demonstrated by SDS-PAGE and western blot. Samples were prepared by addition of 4x NuPAGE LDS sample buffer, and 10x sample reducing agent for gels that were run under reducing conditions and heated at 70 °C for 10 min before gel electrophoresis using Novex NuPAGE 12% Bis-Tris/MOPS gels (Life Technologies). Proteins were transferred onto nitrocellulose membranes by use of an XCell II Blot Module (Life Technologies). Transfer was verified by Ponceau S staining and membranes were blocked overnight at 4 °C. For westerns imaged using the LI-COR scanner, blocking was performed with Odyssey Blocking Buffer, while for westerns visualized using HRP, blocking was performed with 5% (w/v) powdered milk in TBS. Interferon α2a was detected with monoclonal anti-interferon α (Sigma, UK, catalogue number SAB1409236) at 1:1000 dilution and sheep anti-mouse IgG conjugated HRP (GE Healthcare NXA931-1ML) or LI-COR IRDye 680RD Donkey anti-Rabbit IgG (H + L) at 1:20,000 dilution and imaged with a LI-COR Odyssey Imager according to manufacturer's instructions. Concentration was estimated with a commercial ELISA kit according to manufacturer's instructions (PBL Biomedical Laboratories 41,100). Due to a lack of antibody against porcine CSF1, CSF1-Fc was immunoblotted with anti-porcine Fc conjugated to HRP (Bethyl Laboratories A100-104P) at a 1:10,000 dilution in 3% (w/v) powdered milk in TBS 0.1% (v/v) Tween-20. Interferon concentration was determined by quantitative Western blot using LI-COR Image Studio software.
Whole egg white from transgenic hens' eggs was tested for activity prior to purification. For interferon, the ability to stimulate interferon-stimulated response element (ISRE)-driven transcription of luciferase was used to determine initial whole egg white activity. The pGL4.45[luc2P/ISRE/Hygro] vector (Promega) was transfected in HEK293T cells seeded in white-walled 96-well plates at 1 × 104 cells/well using FuGENE HD (Promega) according to manufacturer's instructions. 24 h post-transfection, the medium was replaced with antibiotic-free medium and either egg white from transgenic hens or wild type egg white with control interferon protein (Sigma) added at an estimated final concentration of 10 international units (IU), 102 IU and 104 IU, with diluted wild type egg white used as a negative control. After a further 24 h, the One Glo Luciferase assay kit (Promega) was used to measure luminescence according to manufacturer's instructions. For pCSF1-Fc, a cell survival assay based upon the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed as previously described . Briefly, stable Ba/F3 cells expressing porcine CSF1 receptor were grown in complete RPMI (10% (v/v) fetal calf serum, penicillin, streptomycin, and GlutaMAX) and supplemented with 5% (v/v) X63 cell supernatant from X63 Ag8–653 myeloma cells carrying an expression vector for IL-3 (22). Cells were seeded in 96-well plates at a density of 2 × 104 cells/well in triplicate and treated with serial dilutions of egg or CHO cell-produced pCSF1-Fc instead of IL-3, with negative controls containing complete RPMI with no X63 or pCSF1-Fc. Plates were incubated at 37 °C, 5% (v/v) CO2 for 48 h and MTT (Sigma) at a final concentration of 0.5 mg/mL was added for a further 3-h incubation, followed by solubilization overnight with acidified isopropanol. Absorbance was measured at 570 nm and results normalized to the highest dose positive control.
For pCSF1-Fc and hCSF1-Fc purification, egg white was diluted 1:4 in 50 mM phosphate buffer pH 3, adjusted to pH 4, and stirred at room temperature for 10 min to precipitate ovomucin. The diluted egg white was centrifuged at 25,000 g for 45 min at 10 °C, the supernatant adjusted to pH 7 and centrifuged again at 25,000 g for 45 min at 10 °C to remove any further precipitates. The clarified egg white was diluted to a final volume of 10 times the original egg white volume and pCSF1-Fc was bound to a 5 mL HiTrap MabSelect SuRe column (GE Healthcare) pre-equilibrated with 50 mM phosphate buffer, pH 7, (PB) at 5 mL/min by use of an Äkta Prime Plus liquid chromatography system (GE Healthcare). The column was washed with 10 column volumes of PB and the protein was eluted with 3–5 column volumes of 100 mM citrate buffer (CB) pH 3, or until the UV peak had returned to baseline, into tubes containing 1 M Tris pH 9 at 30% of the final fraction volume. The column was regenerated with 3 column volumes of CB and 5 column volumes of PB, cleaned in place with 5 column volumes of 0.5 M NaOH, pH adjusted with 5 column volumes of PB and finally cleaned with 10 column volumes of 20% (v/v) ethanol for storage. The fractions containing eluted protein, as determined by SDS-PAGE, were pooled and concentrated to < 13 mL in an Amicon stirred cell with 10,000 Da ultrafiltration regenerated cellulose membrane and injected onto a Superdex 200 26/600 column (GE Healthcare) equilibrated with PBS at 2.5 mL/min by use of the Äkta Prime Plus. Fractions were collected manually to ensure that only the main portion of the correct peak was isolated. After use the column was cleaned with 1–2 column volumes of 20% (v/v) ethanol and stored.
For Interferon α2a, egg white was diluted 1:4 in 20 mM phosphate buffer pH 3, adjusted to pH 4, and stirred at room temperature for 10 min to precipitate ovomucin. The diluted egg white was then centrifuged at 25,000 g for 45 min at 10 °C, the supernatant adjusted to pH 7 and centrifuged again at 25,000 g for 35 min at 10 °C to remove any further precipitates. The clarified egg white was diluted to a final volume of 10 times the original egg white volume and interferon α2a was bound onto a 5 mL HiTrap Blue column (GE Healthcare) equilibrated with 20 mM PB pH 7 at 5 mL/min by use of an Äkta Prime Plus liquid chromatography system (GE Healthcare). The column was washed with 10 column volumes of PB and protein eluted with 20 mM PB containing 1 M NaCl. The fractions containing interferon α2a, as determined by SDS-PAGE, were pooled, diluted to 10 times the pooled volume in ddH2O to prevent salt-induced association of interferon and ovalbumin, concentrated to < 13 mL in an Amicon stirred cell with 10,000 Da ultrafiltration regenerated cellulose membrane and injected onto a Superdex 200 26/600 column (GE Healthcare) equilibrated with PBS at 2.5 mL/min by use of the Äkta Prime Plus. After use the column was cleaned with 1–2 column volumes of 20% (v/v) ethanol and stored. Interferon-containing fractions, as determined by SDS-PAGE, were concentrated by Amicon filtration. Protein concentration was determined by UV280. Identity was confirmed by SDS-PAGE and mass spectrometric analyzes (Roslin Proteomics and Metabolomics Facility) and quality was assessed by reversed phase chromatography and SEC-multi-angle light scattering at the Edinburgh Protein Production Facility (EPPF), Kings Buildings, Edinburgh.
A dose-inhibition assay was performed to determine interferon activity compared to a commercially available control (Sigma SRP4594). HEK 293 T cells were used to produce influenza A/PR/8/34 (PR8) virus as described previously (17). A549 cells were pre-treated with varying concentrations of interferon for 24 h, then infected at low multiplicity (MOI 0.001). After 1 h of adsorption at 37 °C, virus-containing medium was replaced with virus growth medium (DMEM, 1 μg/mL TPCK-treated trypsin, 0.14% (w/v) BSA). After 48 h, virus was harvested and titer determined by plaque assay on MDCK cells with an Avicel overlay (virus growth medium supplemented with 1.2% (v/v) Avicel) followed by toluidine blue staining (17, 23). The virus titer was normalized to a no-interferon control to determine dose-inhibition.
In vitro activity of purified egg pCSF1-Fc was tested by the MTT assay in Ba/F3 cells as described for whole egg white, and in pig bone marrow cells as previously described . Briefly, pig bone marrow cells were derived from pigs culled by captive bolt at Dryden farm as previously described [31, 38]. Cells were plated at 5 × 104 cells/well in a 96-well plate directly from frozen stocks and grown in complete RPMI supplemented with either CHO-derived or egg purified pCSF1-Fc at 10 ng/mL, 100 ng/mL or 1000 ng/mL. After 7 days, the culture medium was replaced with 50 μl of 1 mg/ml MTT solution and incubated for 1 h at 37 °C. The MTT solution was removed and tetrazolium salt solubilized with 100 μl of solubilization agent (0.1 M HCL, 10% Triton X-100 and isopropanol) followed by incubation at 37 °C with 5% CO2 for 10 min. Plates were read at 570 nm.
In vivo activity of pCSF1-Fc was tested in mice as previously described . Approval was obtained from The Roslin Institute's and The University of Edinburgh's Protocols and Ethics Committees. The experiments were carried out under the authority of a UK Home Office Project License under the regulations of the Animals (Scientific Procedures) Act 1986. Twelve male C57BL/6 mice aged 6–8 weeks were purchased from Charles River Laboratories (UK) and injected subcutaneously with either PBS, 1 mg/kg pCSF1-Fc from CHO cells or 1 mg/kg pCSF1-Fc from eggs (n = 4 per group) for 4 days and sacrificed on day 5 by rising levels of carbon dioxide. Liver and spleen were removed, weighed and fixed in 10% neutral buffered formalin for histological analysis. Blood was collected by cardiac puncture and transferred into EDTA-coated tubes, then prepared for flow cytometry using the Uti-Lyse kit (Dako, Ely, UK) and stained with PE anti-mouse F4/80 (Biolegend, CA, USA, catalogue number 122616, 1:200) and APC anti-mouse Mac1/CD11b (Abcam, Cambridge, UK, catalogue number ab25482, 1:2000) to identify the macrophage population. Results were analyzed with FlowJo software.
Activity of purified egg hCSF1-Fc was tested by the bone marrow MTT assay as described for pCSF1-Fc. To test whether the protein could withstand drying for long-term storage and easier shipping, aliquots of protein were brought to dryness using a Savant SPD2010 Speedvac (Thermo) at a constant vacuum pressure of 5.1 Torr over 1.5-h without heating. The protein was reconstituted in the same volume of ddH2O and used as described for pCSF1-Fc in the bone marrow differentiation assay.
Analysis was performed with GraphPad Prism Version 6.04 and statistical significance was assessed using Student's two-tailed t-test. Resulting values were considered statistically significant at p < 0.05.
David A. Hume, Andrew C. Gill and Helen M. Sang contributed equally to this work.
We thank Dr. E. Eliot, Dr. C. Robertson and colleagues at the former Viragen Inc. for their contributions to the early stages of developing the ovalbumin promoter-human interferon α2a transgenic line. We thank the Centre for Process Innovation National Biologics Manufacturing Centre for cost of goods analysis and assistance with protein downstream processing. We thank the staff of the Transgenic Chicken Facility and Greenwood Poultry Unit for the breeding and husbandry of the transgenic lines. We thank the staff of the Biological Resource Facility for assistance in carrying out the experiments on mice.
This work was supported by BBSRC strategic funding to The Roslin Institute BBS/E/D/20221658, BBSRC grants BB/I013113/1 (in collaboration with Zoetis, Inc.), BB/M021920/1, and BBSRC-University of Edinburgh IAA award; the EPPF is supported by the Wellcome Trust Multi-User Equipment grants 081287/Z/06/Z, 101527/Z/13/Z and the Wellcome-UoE ISSF award; M.L.T. and N.S. were supported by BBSRC grant BB/J01446X/1 and MRC grant MR/K000276/1 (respectively) to P.D. The funding bodies were not involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
LRH, DAH, HMS and ACG conceived the study. LRH wrote the manuscript with contributions for the different portions of the study as follows: CSF1-Fc protein design, MTT assays and mouse experiments: CP and DAH. Interferon viral assays: MLT, NS and PD. Preparative chromatography: ACG and GP. Analytical chromatography: MW. Promoter and lentiviral construct design and transgenic chicken generation: HMS, SL, AS, and HJG. Mass spectrometry: DK. LRH generated the CSF1-Fc chicken lines with assistance from AS and HJG. SL generated the interferon chicken line with assistance from AS and HJG in collaboration with Viragen Inc. LRH and CP performed mouse experiments. LRH performed protein purification experiments with advice and assistance from ACG and GP. MLT, NS and PD designed, performed and analyzed influenza virus assays; LRH and GP performed CSF1-Fc activity assays with advice from CP. MW and DK performed chromatographic and mass spectrometry (respectively) analysis of purified protein. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Approval for animal experiments was obtained from The Roslin Institute's and The University of Edinburgh's Protocols and Ethics Committees. The experiments were carried out under the authority of a UK Home Office Project License under the regulations of the Animals (Scientific Procedures) Act 1986.
D.A.H. holds a patent position on the applications of CSF1-Fc in liver disease, and of CSF1 in kidney regeneration. Part of this work was performed on a collaborative grant part-funded by Zoetis, Inc. L.R.H. is now employed by Roslin Technologies, who will be commercializing the chicken platform, as well as the current proteins for use as research reagents.


text:
St Bernadette's Primary School was established in 1960, only two years after the suburb of Lalor Park began.
Father Leonard, the founding Parish Priest of Lalor Park Parish approached the Sisters of St Joseph to establish a school. The Sisters of St Joseph came from Baulkham Hills Novitiate to teach at St Bernadette's Parish School. Originally the school was set up in the church with all furniture and classroom partitions packed away to enable weekend mass.
In December 1963 a convent was built for the Sisters to live on site. With the population expanding, a parish and school assembly hall was built in 1971 and two kindergarten classrooms were added in 1980.
St Bernadette's School has grown from its humble beginning sharing a space with the Parish Church. The convent was converted to a dedicated administration area for the school and the original Church was remodeled to be a modern library and multipurpose room. The school has a functioning parent run canteen and four state of the art contemporary learning spaces that are fully resourced to ensure the best personalised teaching and learning is delivered to all our students.
The school has evolved throughout its long history of serving the local community. It now includes an extensive modern playground including infants play equipment, synthetic turf areas, netball court, basketball court, covered deck area, vegetable garden, shade structures and access to a parish hall. The school also has easy access to the playing fields adjoining the school.
The school maintains a strong partnership with the Parish. Together we ensure a Catholic education is available for all students. We remain committed to ensuring students have a positive encounter with God.

redpajama_set_name: RedPajamaCommonCrawl


On Saturday, April 7, starting at 3 PM Central Daylight Time, you will be able to see this year's Hugo Award Nominations announced live in person at five conventions in the United States and Europe, with the announcements simultaneously carried live over the internet on UStream. Special guests will be present at some of the live announcements as well.
Norwescon 35, one of the Pacific Northwest's premier science fiction and fantasy conventions, at Seatac, WA (1 PM PDT). With Special Guest John Picacio. Ustream channel www.ustream.tv/channel/hugo-nominee-announcement-live-from-norwescon.
Leprecon 38, Arizona's annual science fiction and fantasy convention, at Tempe, AZ (1 PM MST). With Special Guest Joe Haldeman. Ustream channel www.ustream.tv/channel/hugo-nominee-announcement-live-from-leprecon.
Minicon 47, Minnesota's longest-running science fiction convention, at Bloomington, MN (3 PM CDT). With guest Chris Garcia. Ustream channel www.ustream.tv/channel/hugo-nominee-announcement-live-from-minicon.
Marcon 47, the Midwest's largest annual convention, at Columbus, OH (4 PM EDT). Ustream channel www.ustream.tv/channel/hugo-nominee-announcement-live-from-marcon.
Olympus 2012, the British National Science Fiction Convention (Eastercon) (9 PM BST). With Chicon 7 Chair Dave McCarty and Special Guest George R.R. Martin. Ustream channel www.ustream.tv/channel/hugo-nominee-announcement-live-from-eastercon.
The 2012 Hugo Awards will be presented at Chicon 7, the 2012 World Science Fiction Convention, August 30 to September 3, 2012 at the Hyatt Regency Chicago. All members of Chicon 7 (both attending and supporting) will be eligible to vote on the 2012 Hugo Awards.


text:
Quentin Tarantino's Star Trek
by TheButcher on Mon Dec 04, 2017 11:38 pm
Deadline December 4, 2017:
Quentin Tarantino Hatches 'Star Trek' Movie Idea; Paramount, JJ Abrams To Assemble Writers Room
Mike Fleming Jr wrote: EXCLUSIVE: Already busy prepping to direct the film he just set up at Sony Pictures, Quentin Tarantino is also planning to boldly go where he has not gone before. Sources said that Tarantino has come up with a great idea for a Star Trek movie at Paramount. After sharing his idea with JJ Abrams (who himself is busy prepping Star Wars Episode IX), I've heard the plan is to assemble a writers room of scribes who'll hear Tarantino's take and begin to put together a movie. If it all works out, Tarantino might direct it, with Abrams producing.
While Tarantino has always come up with his own original films, many have wondered what he might do if he took the reins of an existing franchise. He has only done that on television, twice directing episodes of CSI and once an episode of ER. He has spoken about the appeal of taking on one of the James Bond movies, but the hard part of something like that is getting the rights holders to give him a wide creative swath that comes along with a final cut auteur like Tarantino. This would give a remarkable boost to the venerable franchise for Paramount, which is looking to build them under studio chief Jim Gianopulos.
As Deadline revealed last month, Tarantino agreed to make his next film for Sony Pictures. The untitled film is an ensemble that deals with a period in Los Angeles around the time of the Manson murder spree around 1969. He has asked Margot Robbie to play Sharon Tate, and has been discussing two great male lead roles with Tom Cruise, Leonardo DiCaprio and Brad Pitt, though he hasn't yet set final casting. Release date is August 9, 2019. That is the 50th anniversary of the death of Sharon Tate, but the description of the picture as a Manson Family pic isn't really accurate, kind of like describing Inglorious Basterds as a movie about Hitler.
Paramount declined comment, and attempts to reach Tarantino's camp were unavailing. Stay tuned.
NERDIST PODCAST DECEMBER 18, 2015:
EPISODE 774: QUENTIN TARANTINO
Quentin Tarantino (director, Pulp Fiction, Kill Bill) chats with Chris and Matt about going to Comic-Con back in the day, what movies inspired him as a kid, and how he turns everything he reads and sees into a movie in his head. He also talks about his process while directing and writing, things that almost made it into his movies, and what to expect with The Hateful Eight!
Re: Quentin Tarantino's Star Trek
by Ribbons on Wed Dec 06, 2017 8:45 pm
I don't believe for a second that Tarantino is going to end up making a Star Trek movie. This is the wild hair he gets up his ass and speculates about in interviews every now and then, like those James Bond and Godzilla entries he used to bang on about.
Although I'd be first in line if he did.
by TheButcher on Sat Dec 09, 2017 5:47 am
Quentin Tarantino's 'Star Trek' Will Be R-Rated: 'The Revenant's Mark L. Smith Frontrunner Scribe
Mike Fleming Jr wrote: EXCLUSIVE: After Deadline this week revealed that Quentin Tarantino pitched a Star Trek film to JJ Abrams and Paramount, the whole thing is moving at warp speed. Tarantino met for hours in a writers room with Mark L. Smith, Lindsey Beer, Drew Pearce and Megan Amram. They kicked around ideas and one of them will get the job. I'm hearing the frontrunner is Smith, who wrote The Revenant. The film will most certainly go where no Star Trek has gone before: Tarantino has required it to be R rated, and Paramount and Abrams agreed to that condition. Most mega budget tent poles restrict the film to a PG-13 rating in an effort to maximize the audience. That was the reason that Guillermo Del Toro's $150 million At The Mountains of Madness didn't go forward at Universal, even though Tom Cruise was ready to star. The exception to this rule was Fox's Deadpool, but that film started out with modest ambitions before it caught on and became the biggest R rated film ever.
That rating was crucially important to Tarantino, who hopes to direct this Star Trek and who has helmed R rated films his entire career. Imagine how this could open storytelling lanes, or even what the banter on the bridge of the Starship Enterprise might be, if you conjure up memories of the conversations between Samuel L. Jackson and John Travolta in Pulp Fiction, or the banter at the diner between robbers before the heist gone wrong that triggered the action in Reservoir Dogs.
Smith is best known for writing the Alejandro Gonzalez Inarritu-directed The Revenant and subsequently overhauled Overlord, the WWII thriller that Abrams' Bad Robot is producing for Paramount. Pearce's script credits include Iron Man 3, Sherlock Holmes 3, Mission Impossible: Rogue Nation and the TV series Runaway TV; he just directed his script Hotel Artemis; Beer's credits are mostly upcoming, and include the Doug Liman-directed Chaos Walking, as well as Godzilla Vs. Kong, Masters of the Universe, Barbarella and Dungeons and Dragons, all big scale stuff.
They will lock one of the three quickly (if there is a front runner, it might be Smith), and the film will be scripted based on Tarantino's idea while Tarantino is filming his next film about the Manson summer of 1969, which got set at Sony and has I, Tonya's Margot Robbie poised to play Sharon Tate, and Cruise, Leonardo DiCaprio and Brad Pitt all having met with the filmmaker about roles.
BMD:
Here's Quentin Tarantino On What He Wants To Do With STAR TREK
ANDREW TODD wrote: We're still reeling over today's news that Quentin Tarantino has pitched a new Star Trek movie to Paramount, is working on it with JJ Abrams, and may even direct the damn thing. That's news so improbable it had to come either from April 1st of any year, or any day of 2017. But Deadline's story didn't feature any information about what Tarantino's pitch is, exactly. Luckily, thanks to a December 2015 interview on the Nerdist Podcast, we might have a little glimmer of an idea.
I'll let you listen first - it's pretty short, though as dense as you'd imagine:
Tarantino expresses distaste for Star Trek Into Darkness (check), but appreciation for the reboot cast (also check); he also notes that in the reboot universe (or the Kelvin timeline, for true fans), the events of the original series very well may still happen, even if they're slightly altered. He then goes on to discuss the notion of remaking classic episodes with full casts and bigger budgets, namechecking The Original Series' "City on the Edge of Forever" (as well as the fact that it's obvious to namecheck "City on the Edge of Forever"). That's when things get real interesting.
Tarantino's pitch - if it's anything like what he describes in the interview - would seem to revolve around the Next Generation Season 3 episode "Yesterday's Enterprise." One of the best episodes in the franchise's history, it deals with time travel and alternate realities, forcing Picard and his crew to choose between altering the timeline to save the mysteriously reappeared USS Enterprise-C, throwing the quadrant into war, or saving the timeline and dooming that ship and her crew. A classic trolley-problem episode, only with significantly higher stakes.
While the interview cuts off before Tarantino can explain his precise take on the idea, my suspicion/speculation is that, rather than straight-up remaking "Yesterday's Enterprise," his pitch could rework its premise to unify the two parallel realities into which Trek canon is now split. Perhaps the reboot's Enterprise could be put into the Enterprise-C's role; perhaps Kirk, Spock, and company would have to make a similar decision about a completely different vessel. Like, for example, the USS Kelvin, destroyed at the opening of 2009's Star Trek. There's already been talk of bringing Chris Hemsworth's George Kirk back for the fourth film in the reboot series; maybe that idea can be combined with Tarantino's to seal off the Kelvin timeline once and for all.
Whatever happens, it'll be completely surreal seeing Quentin Tarantino credited on a Star Trek movie - even if it's only a "Story By" nod.
THR DECEMBER 08, 2017:
Patrick Stewart Might Reprise Jean-Luc Picard Role if Tarantino Directs 'Star Trek'
by Peven on Sat Dec 09, 2017 11:24 am
TheButcher wrote: BMD:
Make it so, motherfuckers!
by wonkabar on Wed Mar 14, 2018 5:55 pm
Does anybody remember me posting this idea years ago on AICN? I remember getting SO much shit lol
by Peven on Wed Mar 14, 2018 8:44 pm
I remember you posting your head shots and talking about reading for a bad guy part for the new TJ Hooker tv show except some guy named Dick Ritchie got the part instead.....
by Ribbons on Wed Mar 14, 2018 11:42 pm
I'm pretty sure I came up with the idea


text:
All notable changes to this project will be documented in this file.

The format is based on [Keep a Changelog](http://keepachangelog.com/en/1.0.0/)
and this project adheres to [Semantic Versioning](http://semver.org/spec/v2.0.0.html).

Filterrific major versions match the Ruby on Rails major versions they work with.

## [5.2.3] - Mar. 18, 2022

* Added support for Rails 7

## [5.2.2] - Jul. 11, 2021

* Fixed Ruby 2.7 deprecated warning when trying to regex match an Integer.
* Added ability to pass custom url parameter instead of default :filterrific.
* Added basic Aria attrs to spinner for accessibility.
* Improved concurrent ajax requests: Abort prior request when new ones are triggered.

## [5.2.1] - Aug. 5, 2019

* Fixed issue where uncommitted code was pushed to rubygems and broke array filters.

## [5.2.0] - Jul. 21, 2019

* Make Filterrific master branch compatible with Rails 5 and 6.
* Trigger synthetic JS events before and after form submission ajax requests.

## [5.1.0] - Aug. 3, 2018

* Breaking change: all Filterrific params are sanitized by default to prevent XSS attacks. You can disable sanitization (you really shouldn't!) by setting the :sanitize_params option to false when calling #initialize_filterrific in the controller.

## [5.0.1] - Jan. 2, 2018

* Changed all instances of #deep_stringify_keys back to #stringify_keys. This was changed in 5.0.0, but it shouldn't have been changed.

## [5.0.0] - Dec. 31, 2017

* We're switching to a new versioning strategy for Filterrific: Filterrific major releases (the first number in the version) will be matched with the supported major version of Rails. Minor and path versions may diverge from Rails. That means for any version of Rails 5.x you will use the most current version of Filterrific 5.x.

## [4.0.0] - Dec. 31, 2017

* This is the first release of Filterrific specifically geared towards Rails 4.x. No functional changes, just new versioning system and removal of unused code.

## [3.0.0] - Dec. 31, 2017

* This is the first release of Filterrific specifically geared towards Rails 3.x. No functional changes, just new versioning system and removal of unused code.

## [2.1.2] - Nov. 11, 2016

* Removed older way of initializing assets, relying on config/initializers/assets.rb for all versions of Rails.

## [2.1.1] - Nov. 11, 2016

* Reverted asset initialization back to working state for versions prior to Rails5.

## [2.1.0] - Nov. 6, 2016

* Updated filterrific for Rails 5 compatibility:
    * Turbolinks compatibility (thanks @olegantonyan)
    * Updated to Ruby 2.2.2 in CI test matrix
    * Added Ruby 2.3.1 to CI test matrix (thanks @rodrigoargumedo)
    * Fixed #stringify_keys deprecation warning (thanks @olegantonyan)
* When generating sorting headers, allow usage of HTML (e.g., for icons) using #safe_join (thanks @ScottKolo)
* Improved param casting to Int: hyphens and zeros (thanks @grit96)

## [2.0.5] - May 4, 2015

* Feature: Allow disabling of session persistence by passing `false` as
  `:persistence_id` option.
* Fix: Direction indicators on sortable headers are now correct.
* Fix: Make JS event observers work with TurboLinks.
* Fix: Make reset_filterrific_url available in controller.

## [2.0.4] - Mar. 10, 2015

* Objectify nested params so that they render correctly on the form when
  restored from session or URL params.
* Don't convert strings that almost look like integers to integers. Example:
  `"0123"` as part of a phone number should not be converted to `123`.

## [2.0.3] - Jan. 30, 2015

* Cleaned up obsolete option names

## [2.0.2] - Jan. 28, 2015

* Fixed bugs in ActionControllerExtension
* Improved test coverage

## [2.0.1] - Jan. 28, 2015

* Fixed regression with Rails 3.2 (doesn't support `#deep_stringify_keys`)

## [2.0.0] - Jan. 28, 2015

API changes:

* Filterrific model API option names have changed.
* Better initialization of Filterrific via `initialize_filterrific` method:
    * It resets the filter params, so the `reset_filterrific` action is not required any more.
    * It persists filter params in session.
* Simplified `@filterrific.find` method to load collection from DB.
  Replaces `Student.filterrific_find(@filterrific)`
* The `form_for_filterrific` form builder doesn't override the standard
  `form_for` method any more.
* Dropped support for Ruby 1.8.7 (because of 1.9 Hash syntax)
* Dropped support for Rails <= 3.0.0 (because of ActiveRecord
  bug fixes in 3.1, and use of asset pipeline)

## [1.4.3] - Jan. 12, 2015

* Handle case where Filterrific filter params are empty.

## [1.4.2] - Oct. 18, 2014

* Updated initialization of ActiveRecord and ActionView extensions again

## [1.4.1] - Oct. 15, 2014

* Updated initialization of ActiveRecord and ActionView extensions

## [1.4.0] - Oct. 13, 2014

* Better support for new versions of Rails (integration tests are done in filterrific_demo)
* Fixed asset pipeline for filterrific-spinner.gif.
* Isolate_namespace
* Update Gem dependencies
* Switch from Rspec to Minitest
* Require Rails 3.1 or greater

## [1.3.1] - Jun. 18, 2014

* Changed ParamSet#select_options so that a complete hash can be assigned

## [1.3.0] - Jun. 3, 2014

* Added ParamSet#select_options (thanks @pnomolos).
* Added ParamSet#signature to quickly test two param_sets for equality.
* Bugfix: When using with Rails 4.0: Fixed 'wrong number of arguments 0 for 1' exception (thanks @sebboh).
* Bugfix: jQuery bug in `form_for` helper (thanks @sebboh).
* Bugfix: removed duplicate dependency in .gemspec.

* Updated documentation.
* Updated Gemfile source
* Refactored ParamSet initialization.
* Refactored ActiveRecordExtension initialization.

## [1.2.0] - May 16, 2013

* Added simple wrapper for Filterrific::ParamSet.new so that it can be
  instantiated with Filterrific.new instead of Filterrific::ParamSet.new.
* Overrode ActionView's form_for to add filterrific magic when applied to a
  Filterrific object.
* Fixed bug with javascript periodic observer, changed css selector class to
  avoid conflicts.
* Moved observe_form_field jquery plugin into filterrific namespace to avoid
  conflicts.

## [1.1.0] - May 8, 2013

* Major refactor.
* Added specs.
* Tied in Rails asset pipeline.
* Added gh-pages branch for documentation.

## [1.0.1] - Nov. 9, 2011

* Bug fix: Replaced stringify_keys with map.to_s (filter_names is an Array, not a Hash!).

## [1.0.0] - Nov. 9, 2011

* Support for Rails 3.1.
* New model api.

## [0.1.0] - Aug. 1, 2010

* Replicate functionality of Rails 2.3 version.

## [0.0.1] - Jul. 30, 2010

* Initial setup.

redpajama_set_name: RedPajamaGithub


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Browse: Home / 2017 / February / 06 / Posts from ExecutiveBiz for 02/06/2017
Posted by Nicholas Hoffman on February 6, 2017 in News | 33 Views
Chris Kubasik, L3 Technologies COO, Inducted Into 2017 Wash100 for Acquisition Leadership
Executive Mosaic is honored to announce Chris Kubasik, president and chief operating officer of L3 Technologies, as an inductee into the 2017 edition of the Wash100 — Executive Mosaic's annual selection of influential leaders in the government contracting industry…
PAE CEO John Heller Named to 2017 Wash100 for Post-Acquisition & Govt Services Leadership
Executive Mosaic is pleased to recognize John Heller, CEO of PAE as an inductee into the 2017 edition of the Wash100 — Executive Mosaic's annual selection of influential leaders in the government contracting arena…
Raytheon Awarded Navy Environmental Satellite Receiver Processor Support Contract
The U.S. Navy has awarded Raytheon a $50 million contract to provide hardware and support services to the military branch's Environmental Satellite Receiver Processor program. The indefinite-delivery/indefinite-quantity hybrid contract covers production, engineering, procurement and in-service engineering agent to support the program executive office for command, control, communications, computers and intelligence ESRP, the Defense Department said Friday…
Former Army Acquisitions Chief Heidi Shyu Named Roboteam Chairman; Shahar Abuhazira Comments
Heidi Shyu, former assistant secretary of the U.S. Army for acquisition, logistics and technology, has joined Roboteam as chairman of the board of directors. The tactical ground robotic systems provider said Monday Shyu will mentor the management team and lead company strategic efforts to grow its unmanned ground vehicle business…
GSA Tech Transformation Service Looks to Establish 'Bug Bounty' Program
The General Services Administration's Technology Transformation Service plans to establish a "bug bounty" program that will engage ethical hackers in efforts to discover and address vulnerabilities in TTS web applications, Federal News Radio reported Monday…
Doug Davis: Intel Seeks Collaboration With DOT Secretary Elaine Chao to Spur Transport Innovation
Doug Davis, senior vice president and general manager of Intel's automated driving group, has said Elaine Chao's appointment as transportation secretary presents an opportunity for the government and industry to collaborate to maintain U…
OmniEarth to Develop Soil Moisture Mapping Tech Under DARPA Contract
OmniEarth has received a contract from the Defense Advanced Research Projects Agency to develop a mapping technology for military personnel to assess, manage and predict soil conditions. The company said Monday it will fuse information from commercially-available synthetic aperture radar and multispectral imagery in a push to develop a precision soil moisture mapping model to aid tactical decisionmaking…
Lockheed, UK Nat'l Cybersecurity Center Partner for Talent Development Initiative
Lockheed Martin and the U.K.-based National Cyber Security Center have partnered to facilitate a program that seeks to develop the cybersecurity skills of students who are currently in their first year or about to start university…
Report: DISA CTO David Mihelcic to Retire Before March
David Mihelcic, chief technology officer at the Defense Information Systems Agency, will retire later this month after a three-decade career in government, C4ISR and Networks reported Friday. Mark Pomerleau writes Mihelcic has advocated for efforts to reform the Defense Department's acquisition process and said he believes DoD should replace its current requirements-driven procurement system with a needs-based approach…
Michael Isman: Booz Allen Opens DC Innovation Hub to Facilitate Partnerships With Entrepreneurs, Startups
Michael Isman, a vice president and strategic innovation group executive at Booz Allen Hamilton, has said the company unveiled a new center in downtown Washington in May 2016 as part of the firm's investment efforts to advance innovation, Federal News Radio reported Friday…
NASA Eyes March 19 Launch for Orbital ATK's 7th ISS Cargo Resupply Mission
NASA aims to launch on March 19 an Orbital ATK-built Cygnus spacecraft to perform the seventh cargo delivery mission to the International Space Station. Orbital ATK's Cygnus will have a 30-minute launch window that will start at approximately 10:56 p…


Meeting in Kelso on the weekend of September 9-11, the Synod of the Diocese of Bathurst has cleared the way for Bishop Ian Palmer to continue as Bishop of Bathurst for an extra two years, until he reaches the age of 70.
This will mean that Bishop Ian may now stay in office until January 2020.
The decision involved amending the Bishop Election Ordinance, which stipulates a retirement age of 68, to allow Synod to authorise a two-year extension.
Archdeacon Frank Hetherington, who moved the motion, had earlier told Synod in his report that he believed the extra two years should be added to the Bishop's episcopate.
"It would provide space for the resolution of any unresolved matters arising from the CBA case, the development of future budgets so that the Episcopal stipend and emoluments are met from the Synod Management Fund and further, to initiate the type of change and restructuring that will enable the Anglican Church in the region to move into a different future," the Archdeacon said.
Bishop Ian vacated the room for the debate, handing the chair to the Primate, Archbishop Philip Freier.
The motion produced some keen debate, with two speakers opposing on the grounds of concern for Bishop Ian's health while others pointed out that the motion merely cleared the way for an extension – the Bishop was not obliged to stay on if he felt in necessary to stand down.
Many speakers took the opportunity to express strong support for Bishop Ian and the manner in which he has guided the diocese through the financial and legal minefield of the past three years.
The final vote was an overwhelming expression of confidence in the Bishop.
Synod also debated a motion brought by Dr Ken Phillis and The Rev'd Canon Brett Watterson (Dubbo Parish) to abolish the mandatory retirement age for Licensed Lay Ministers.
As with the motion on the Bishop's retirement age, this motion also involved changing the relevant ordinance so that "age shall not, of itself, be a determinant in the consideration of a person to appointment as a Licensed Lay Minister in the Diocese."
A further amendment to the ordinance states that "the renewal of any Lay Minister's License upon expiry of its term be subject to a written review by the Parish Priest to the Bishop".
In a further reflection on the ageing of the church, Synod also resolved on a motion by The Reverend Geoff McAuliffe (Forbes) and Sue West (Anglicare) that "this Synod commission BIC to consider the issue of age limits in Ordained Parish Ministry and report back to Synod in 2017".
Acknowledging that a review of the administrative procedures of the Diocese over the next few weeks has meant a delay in preparing the Synod Management Budget for 2017, Synod has resolved to "task the Bishop in Council with receiving and approving the 2017 Synod Management Fund Budget and immediately circulates the budget to Synod members and parishes".


text:
Q: Unable to serialize datetime.datetime object in dictionary to JSON using python and trac I have a list of dictionaries (each dictionary representing 1 record of an SQL query) which I am trying to serialize as JSON. There are several datetime.date and datetime.datetime objects in these dictionaries which are proving difficult to serialize. Initially, the error message is:
TypeError: datetime.date(2012, 5, 23) is not JSON serializable

After adding a handler to the json.dumps call the error message is:
TypeError: date_handler() takes exactly 1 argument (2 given)

date_handler looks like this:
    def date_handler(obj):
        return obj.isoformat() if hasattr(obj, 'isoformat') else obj

I'm running this as part of a Trac plugin however I think this is just an isolated python issue but have no idea what to do to sort it - does anyone have any ideas?

A: I solved this by applying .isoformat() fields that were dates or timestamps in the dictionary as I'm importing the data from a database and so know what the field types are beforehand, thanks for your help!

A: See a solution from the Django domain.

redpajama_set_name: RedPajamaStackExchange


text:
Pulford pupil aiming for the top
Nine-year-old Elliot Morris of Leighton Buzzard who is starring in Chitty Chitty Bang Bang
Bev Creagh
Seeing the West End show Charlie and the Chocolate Factory two years ago was a light bulb moment for Leighton schoolboy Elliot Morris.
"I was only seven when I went and I knew I wanted to be Charlie," he says simply.
In fact, so enamoured was the Year 4 Pulford School student of Raold Dahl's deliciously dark tale that he persuaded his mum to enrol him in a Charlie's school workshop at the Umbrella Rooms in the heart of London's theatreland on Shaftesbury Avenue.
Elliot – who's about to go on tour as Jeremy in Chitty Chitty Bang Bang – put his name down for all seven of the classes.
He said: "There were about 20 of us to start with and we all had to do some singing, dancing and acting.
"I got down to the last six and then I was cut."
He admits he was really disappointed but adds philosophically: "I knew there were lots of other things I could do because mum gets messages from casting directors about what's going on and tells me where I need to be."
He was absolutely thrilled to land the role of Jeremy in one of the world's favourite musicals, Chitty Chitty Bang Bang – mainly because he'll be appearing in the Newscastle production.
Elliot confesses: "I've always wanted to stay away from home and when mum told me it was a tour I really wanted to do it. I'm on for three days and then I'll have six days off."
Considering he's still only nine, this talented young performer already has quite an impressive theatrical CV.
He said proudly: "I was Gavroche in Les Mis at Stantonbury Campus in Milton Keynes and I also took part in Fagin's Gang at the Grove Theatre in Dunstable."
He loves everything about the experience, save one.
"I really don't like make-up," he admits sheepishly, wrinkling his nose.
He's currently taking modern dance classes and his dual ambitions are to appear on TV and star in Billy Elliott.
Elliot's first brush with fame came on the Chris Evans Breakfast Show in the Kids Get a Fanfare secion.
"It was when I was younger and having my first recorder lesson ever," he explains.
"I'm really good at it now."
Catch Elliot in Chitty Chitty Bang Bang at Milton Keynes Theatre from tonight (May 10) until Saturday Call the Box Office on 0844 871 7652.


text:
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Lend-Lease Settlements
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Present Scope of Lend-Lease Operations
Lend-Lease will round out its third year of service in defense of the United States on March 11, 1944. The original Lend-Lease Act, adopted nine months before the United States entered the war, has been extended by Congress to June 30 of this year. A further extension will be necessary at the present session of Congress and a further appropriation will later be requested by the President to finance lend-lease operations daring the fiscal year 1945.
The lend-lease program is subject to termination at any time by adoption of a concurrent resolution by the two houses of Congress "which declares that the powers conferred [upon the President] are no longer necessary to the defense of the United States." While there is no thought of terminating the program while the war is still in progress, members of Congress have pressed increasingly during recent months for more detailed information on lend-lease operations and on plans for settlement of obligations outstanding at the close of the war.
At the beginning of 1944, Congress by successive measures had appropriated to the President roughly $25 billion for lend-lease expenditure and had authorized transfers from various departments of the government for lend-lease purposes to a total of $36 billion. Of the total funds made available, $18.6 billion had been expended up to November 30, 1943. Lend-lease aid in the form of war materials, industrial machinery and commodities, and foodstuffs amounted to $16.1 billion; services provided by the United States to others of the United Nations were valued at $2.5 billion.
ISSUE TRACKER for Related Reports
World War II Demobilization
Nov. 18, 1950 Conservation of War Materials
Jan. 04, 1946 Future of Light Metals
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Dec. 06, 1944 War Veterans and Employment
Nov. 11, 1944 Reconversion of Agriculture
Jul. 28, 1944 Priorities in Demobilization
May 16, 1944 Termination of War Contracts
Jan. 17, 1944 Lend-Lease Settlements
Nov. 30, 1943 Disposal of Surplus War Materials
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text:
Q: How do I write the scores of my game into the leaderboard.txt and display the top 5 scores only with each player's name I'm currently doing a dice game for my school programming project, and it includes the rules: 'Stores the winner's score, and their name, in an external file' and 'Displays the score and player name of the top 5 winning scores from the external file.' I've done everything in my game apart from the leaderboard. I am able to write the name and score of the user into the txt file, but I am unsure of how to then sort it, and would also like when people first start the program can use the menu to go to the leaderboard where it would then read the txt file and print the top 5 scores in order including the names.
I've checked loads of over questions similar to mine, but none of them exactly worked for my code as I kept on getting errors implementing other people's code into mine which just weren't compatible with my layout.
(deleted)

Thanks in advance, I've never used stack overflow to ask a question so I apologize if there's anything I've done wrong in my post.

A: You did good on the question. You stated the problem clearly and, most importantly, you added enough code to run the code so we can have a look at how the program behaves and what's going wrong. In this case nothing is going wrong which is good :)
Considering you mention that this is a school project I will not give you a fully copy/paste solution but will explain hopefully enough details on how to solve this on your own.
Now according to the question, you don't know how to sort your leaderboard. I ran the program a few times myself (after removing the sleeps because I am impatient ) and see that your leaderboard file looks like this:
90 - somename
38 - anothername
48 - yetanothername

To display this you must do two things:

*

*Open the file and read the data

*Convert the data from the file into something usable by the program

The first step seems to be something you already know as you already use open() to write into the file. Reading is very similar.
The next step is not so obvious if you are new to programming. The file is read as text-data, and you need to sort it by numbers. For a computer the text "10" is not the same a the number 10 (note the quotes). You can try this by opening a Python shell:
>>> 10 == 10
True
>>> 10 == "10"
False
>>> "10" == 10
False

And text sorts differently to numbers. So you one piece of the solution is to convert the text into numbers.
You will also get the data as lines (either using readlines() or splitlines() depending on how you use it. These line need to be split into score and name. The pattern in the file is this:
<score> - <name>

It is important to notice that you have the text " - " as separator between the two (including spaces). Have a look at the Python functions str.split() and str.partition(). These functions can be applied to any text value:
>>> "hello.world".split(".")
['hello', 'world']
>>> "hello.world".partition(".")
('hello', '.', 'world')

You can use this to "cut" the line into multiple pieces.
After doing that you have to remember the previous point about converting text to numbers.
As a last step you will need to sort the values.
When reading from the file, you can load the converted data into a Python list, which can then be sorted.
A convenient solution is to create a list where each element of that list is a tuple with the fields (score, name). Like that you can directly sort the list without any arcane tricks.
And finally, after sorting it, you can print it to the screen.
In summary

*

*Open the file

*Read the data from the file as "lines"

*Create a new, empty list.

*Loop over each line and...

*

*... split the line into multiple parts to get at the score and name separately

*... convert the score into a number

*... append the two values to the new list from point 3

*Sort the list from point 3

*Print out the list.

Some general thoughts
You can improve and simplify the code by using more functions
You already show that you know how to use functions. But look at the comments #THIS IS ROUND1 to #THIS IS ROUND5. The lines of code for each round are the same. By moving those lines into a function you will save a lot of code. And that has two benefits: You will only need to make a code-change (improvement or fix) in one place. And secondly, you guarantee that all blocks behave the same.
To do this, you need to think about what variables that block needs (those will be the new function arguments) and what the result will be (that will be the function return value).
A simple example with duplication:
print("round 1")
outcomes = []
value1 = random(1, 100)
value2 = random(1, 100)
if value1 > value2:
    outcomes.append("A")
else:
    outcomes.append("B")

print("round 2")
outcome = ""
value1 = random(1, 100)
value2 = random(1, 100)
if value1 > value2:
    outcomes.append("A")
else:
    outcomes.append("B")

Rewritten with functions
def run_round(round_name):
    print(round_name)
    value1 = random(1, 100)
    value2 = random(1, 100)
    if value1 > value2:
        return "A"
    else:
        return "B"

outcomes = []
result_1 = run_round("round 1")
outcomes.append(result_1)
result_2 = run_round("round 2")
outcomes.append(result_2)

As you can see, the second code is much shorter and has no more duplication. Your code will have more function arguments. It is generally a challenge in programming to organise your code in such a way that functions have few arguments and no complex return values. Although, as long as it works nobody will look too closely ;)
Safe way to ask for a password
You can use getpass() from the module getpass() to prompt for a password in a secure manner:
from getpass import getpass
password = getpass()

Note however, if you are using PyCharm, this causes some issues which are out of scope of this post. In that case, stick with input().
Sleeps
The "sleep()" calls are nice and give you the chance to follow the program, but make it slow to test the program. Consider to use smaller values (comma-values are possible), or, even better, write your own function that you can "short-circuit" for testing. Something like this:
import time

ENABLE_SLEEP = True

def sleep(s):
    if ENABLE_SLEEP:
        time.sleep(s)

print("some code")
sleep(1)
print("more code")
sleep(4)

You will then use your own sleep() function anytime you want to wait. That way, you can simply set the variable ENABLE_SLEEP to False and your code will run fast (for testing).


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