Functional factor VIII made with von Willebrand factor at high levels in transgenic milk

Background—Current manufacturing methods for recombinant human Factor VIII (rFVIII) within mammalian cell cultures are inefficient which limit the abundance needed for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary gland of mice, rabbits, sheep and pigs. Unfortunately, it is secreted into milk with low specific activity due in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives—To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods—Transgenic mice were made with a mammary specific FVIII gene (226/N6) bioengineered for efficient expression and stability containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand Factor (VWF). 226/N6 was evaluated by ELISA, SDS-PAGE, Western blot, and one- and two-stage clotting assays. Hemostatic activity of immunoaffinity enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions—With or without co-expression of VWF, 226/N6 was secreted into milk as a biologically active single chain molecule that retained high specific activity similar to therapeutic-grade FVIII. 226/N6 had \u3e450-fold higher IU/ml than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma-derived VWF as therapeutic-grade rFVIII and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof-of-principle to study expression of 226/N6 and perhaps other single chain bioengineered rFVIII in the milk of transgenic livestock

Bovine Follicular Dynamics, Oocyte Recovery,and Development of Oocytes Microinjected with a Green Fluorescent Protein Construct

The present study was carried out to 1) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA constructs. A potential predictor of DNA integration frequency at d 10 was assessment of the incidence of green fluorescing embryos. The proportion of cleaved embryos that developed to morulae or blastocysts was not different between groups with 1 pronucleus injected (45%), 1 pronucleus uninjected (64%), or 2 pronuclei injected (49%). However, the development of morulae or blastocysts was higher in the group with 2 pronuclei uninjected (69%). The overall developmental score of green fluorescent protein-positive embryos was higher for grade A oocytes (1.3 &#;&#;0.1) than for grade B (0.8 &#; 0.1), C (0.6 &#;&#;0.1), or D (0.3 &#;&#;0.1) oocytes. The results show that production of transgenic bovine blastocysts can occur from the microinjection of a presumptive zygote having only one visible pronucleus. Initial oocyte quality is an important factor in selection of oocytes suitable for microinjection of DNA and for preimplantation development to produce bovine transgenic embryos

3D Cosmic Shear: Cosmology from CFHTLenS

This paper presents the first application of 3D cosmic shear to a wide-field weak lensing survey. 3D cosmic shear is a technique that analyses weak lensing in three dimensions using a spherical harmonic approach, and does not bin data in the redshift direction. This is applied to CFHTLenS, a 154 square degree imaging survey with a median redshift of 0.7 and an effective number density of 11 galaxies per square arcminute usable for weak lensing. To account for survey masks we apply a 3D pseudo-Cl approach on weak lensing data, and to avoid uncertainties in the highly non-linear regime, we separately analyse radial wave numbers k<=1.5h/Mpc and k<=5.0h/Mpc, and angular wavenumbers l~400-5000. We show how one can recover 2D and tomographic power spectra from the full 3D cosmic shear power spectra and present a measurement of the 2D cosmic shear power spectrum, and measurements of a set of 2-bin and 6-bin cosmic shear tomographic power spectra; in doing so we find that using the 3D power in the calculation of such 2D and tomographic power spectra from data naturally accounts for a minimum scale in the matter power spectrum. We use 3D cosmic shear to constrain cosmologies with parameters OmegaM, OmegaB, sigma8, h, ns, w0, wa. For a non-evolving dark energy equation of state, and assuming a flat cosmology, lensing combined with WMAP7 results in h=0.78+/-0.12, OmegaM=0.252+/-0.079, sigma8=0.88+/-0.23 and w=-1.16+/-0.38 using only scales k<=1.5h/Mpc. We also present results of lensing combined with first year Planck results, where we find no tension with the results from this analysis, but we also find no significant improvement over the Planck results alone. We find evidence of a suppression of power compared to LCDM on small scales 1.5 < k < 5.0 h/Mpc in the lensing data, which is consistent with predictions of the effect of baryonic feedback on the matter power spectrum.Comment: Full journal article here http://mnras.oxfordjournals.org/content/442/2/1326.full.pdf+htm

Haemophilic factors produced by transgenic livestock: abundance that can enable alternative therapies worldwide

Haemophilia replacement factors, both plasma-derived and recombinant, are in relatively short supply and are high-cost products. This has stymied the study and development of alternative methods of administration of haemophilia therapy even in the most economically advanced countries, owing to the large amounts of material needed because bioabsorption and bioavailability of haemophilic factors can be less than 10% when using non-intravenous routes of delivery. There is therefore a need to increase access to therapy worldwide by decreasing the cost and increasing the abundance so that therapy can be achieved through simplified, alternative delivery methods. Transgenic livestock have been used to produce haemophilic factors in milk. Only the pig mammary gland has been shown to carry out the post-translational processing necessary to enable both the biological activity and long circulation half-life needed for therapeutic glycoproteins. Furthermore, the large amounts of recombinant protein that can be produced from pig milk make feasible the use of alternative delivery methods such as oral, intratracheal, subcutaneous, and intramuscular administration

The galaxy-halo connection from a joint lensing, clustering and abundance analysis in the CFHTLenS/VIPERS field

We present new constraints on the relationship between galaxies and their host dark matter halos, measured from the location of the peak of the stellar-to-halo mass ratio (SHMR), up to the most massive galaxy clusters at redshift $z\sim0.8$ and over a volume of nearly 0.1~Gpc$^3$. We use a unique combination of deep observations in the CFHTLenS/VIPERS field from the near-UV to the near-IR, supplemented by $\sim60\,000$ secure spectroscopic redshifts, analysing galaxy clustering, galaxy-galaxy lensing and the stellar mass function. We interpret our measurements within the halo occupation distribution (HOD) framework, separating the contributions from central and satellite galaxies. We find that the SHMR for the central galaxies peaks at $M_{\rm h, peak} = 1.9^{+0.2}_{-0.1}\times10^{12} M_{\odot}$ with an amplitude of $0.025$, which decreases to $\sim0.001$ for massive halos ($M_{\rm h} > 10^{14} M_{\odot}$). Compared to central galaxies only, the total SHMR (including satellites) is boosted by a factor 10 in the high-mass regime (cluster-size halos), a result consistent with cluster analyses from the literature based on fully independent methods. After properly accounting for differences in modelling, we have compared our results with a large number of results from the literature up to $z=1$: we find good general agreement, independently of the method used, within the typical stellar-mass systematic errors at low to intermediate mass (${M}_{\star} < 10^{11} M_{\odot}$) and the statistical errors above. We have also compared our SHMR results to semi-analytic simulations and found that the SHMR is tilted compared to our measurements in such a way that they over- (under-) predict star formation efficiency in central (satellite) galaxies.Comment: 31 pages, 18 figures, 4 table. Accepted for publication in MNRAS. Online material available at http://www.cfhtlens.or

Shear-flexion cross-talk in weak-lensing measurements

Gravitational flexion, caused by derivatives of the gravitational tidal field, is potentially important for the analysis of the dark-matter distribution in gravitational lenses, such as galaxy clusters or the dark-matter haloes of galaxies. Flexion estimates rely on measurements of galaxy-shape distortions with spin-1 and spin-3 symmetry. We show in this paper that and how such distortions are generally caused not only by the flexion itself, but also by coupling terms of the form (shear $\times$ flexion), which have hitherto been neglected. Similar coupling terms occur between intrinsic galaxy ellipticities and the flexion. We show, by means of numerical tests, that neglecting these terms can introduce biases of up to 85% on the $F$ flexion and 150% on the $G$ flexion for galaxies with an intrinsic ellipticity dispersion of $\sigma_{\epsilon}=0.3$. In general, this bias depends on the strength of the lensing fields, the ellipticity dispersion, and the concentration of the lensed galaxies. We derive a new set of equations relating the measured spin-1 and spin-3 distortions to the lensing fields up to first order in the shear, the flexion, the product of shear and flexion, and the morphological properties of the galaxy sample. We show that this new description is accurate with a bias $\leq 7$ (spin-1 distortion) and $\leq 3$ (spin-3 distortion) even close to points where the flexion approach breaks down due to merging of multiple images. We propose an explanation why a spin-3 signal could not be measured yet and comment on the difficulties in using a model-fitting approach to measure the flexion signal.Comment: 11 pages, MNRAS accepte

Novel fibrin-fibronectin matrix accelerates mice skin wound healing

Plasma fibrinogen (F1) and fibronectin (pFN) polymerize to form a fibrin clot that is both a hemostatic and provisional matrix for wound healing. About 90% of plasma F1 has a homodimeric pair of γ chains (γγF1), and 10% has a heterodimeric pair of γ and more acidic γ′ chains (γγ′F1). We have synthesized a novel fibrin matrix exclusively from a 1:1 (molar ratio) complex of γγ′F1 and pFN in the presence of highly active thrombin and recombinant Factor XIII (rFXIIIa). In this matrix, the fibrin nanofibers were decorated with pFN nanoclusters (termed γγ′F1:pFN fibrin). In contrast, fibrin made from 1:1 mixture of γγF1 and pFN formed a sporadic dis- tribution of “pFN droplets” (termed γγF1+pFN fibrin). The γγ′F1:pFN fibrin enhanced the adhesion of primary human umbilical vein endothelium cells (HUVECs) relative to the γγF1+FN fibrin. Three dimensional (3D) culturing showed that the γγ′F1:pFN complex fibrin matrix enhanced the proliferation of both HUVECs and primary human fibroblasts. HUVECs in the 3D γγ′F1:pFN fibrin exhibited a starkly enhanced vascular mor- phogenesis while an apoptotic growth profile was observed in the γγF1+pFN fibrin. Relative to γγF1+pFN fibrin, mouse dermal wounds that were sealed by γγ′F1:pFN fibrin exhibited accelerated and enhanced healing. This study suggests that a 3D pFN presentation on a fibrin matrix promotes wound healing

CFHTLenS: higher order galaxy–mass correlations probed by galaxy–galaxy–galaxy lensing

We present the first direct measurement of the galaxy–matter bispectrum as a function of galaxy luminosity, stellar mass and type of spectral energy distribution (SED). Our analysis uses a galaxy–galaxy–galaxy lensing technique (G3L), on angular scales between 9 arcsec and 50 arcmin, to quantify (i) the excess surface mass density around galaxy pairs (excess mass hereafter) and (ii) the excess shear–shear correlations around single galaxies, both of which yield a measure of two types of galaxy–matter bispectra. We apply our method to the state-of-the-art Canada–France–Hawaii Telescope Lensing Survey (CFHTLenS), spanning 154 square degrees. This survey allows us to detect a significant change of the bispectra with lens properties. Measurements for lens populations with distinct redshift distributions become comparable by a newly devised normalization technique. That will also aid future comparisons to other surveys or simulations. A significant dependence of the normalized G3L statistics on luminosity within −23 ≤ Mr ≤ −18 and stellar mass within 5 × 10^9 M_⊙ ≤ M* ≤ 2 × 10^(11) M_⊙ is found (h = 0.73). Both bispectra exhibit a stronger signal for more luminous lenses or those with higher stellar mass (up to a factor of 2–3). This is accompanied by a steeper equilateral bispectrum for more luminous or higher stellar mass lenses for the excess mass. Importantly, we find the excess mass to be very sensitive to galaxy type as recently predicted with semi-analytic galaxy models: luminous (M_r < −21) late-type galaxies show no detectable signal, while all excess mass detected for luminous galaxies seems to be associated with early-type galaxies. We also present the first observational constraints on third-order stochastic galaxy biasing parameters

Bayesian galaxy shape measurement for weak lensing surveys – III. Application to the Canada–France–Hawaii Telescope Lensing Survey

A likelihood-based method for measuring weak gravitational lensing shear in deep galaxy surveys is described and applied to the Canada–France–Hawaii Telescope (CFHT) Lensing Survey (CFHTLenS). CFHTLenS comprises 154 deg^2 of multi-colour optical data from the CFHT Legacy Survey, with lensing measurements being made in the i′ band to a depth i′_(AB) < 24.7, for galaxies with signal-to-noise ratio ν_(SN) ≳ 10. The method is based on the lensfit algorithm described in earlier papers, but here we describe a full analysis pipeline that takes into account the properties of real surveys. The method creates pixel-based models of the varying point spread function (PSF) in individual image exposures. It fits PSF-convolved two-component (disc plus bulge) models to measure the ellipticity of each galaxy, with Bayesian marginalization over model nuisance parameters of galaxy position, size, brightness and bulge fraction. The method allows optimal joint measurement of multiple, dithered image exposures, taking into account imaging distortion and the alignment of the multiple measurements. We discuss the effects of noise bias on the likelihood distribution of galaxy ellipticity. Two sets of image simulations that mirror the observed properties of CFHTLenS have been created to establish the method's accuracy and to derive an empirical correction for the effects of noise bias