Focused directed evolution of aryl-alcohol oxidase in Saccharomyces cerevisiae by using chimeric signal peptides

Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating a-factor and the K1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: preproa-AAO> preaproK-AAO>preKproa-AAO>preproK-AAO. The chimeric preaproK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Meta1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preaproK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability

Detection of the ISW effect and corresponding dark energy constraints made with directional spherical wavelets

Using a directional spherical wavelet analysis we detect the integrated Sachs-Wolfe (ISW) effect, indicated by a positive correlation between the first-year Wilkinson Microwave Anisotropy Probe (WMAP) and NRAO VLA Sky Survey (NVSS) data. Detections are made using both a directional extension of the spherical Mexican hat wavelet and the spherical butterfly wavelet. We examine the possibility of foreground contamination and systematics in the WMAP data and conclude that these factors are not responsible for the signal that we detect. The wavelet analysis inherently enables us to localise on the sky those regions that contribute most strongly to the correlation. On removing these localised regions the correlation that we detect is reduced in significance, as expected, but it is not eliminated, suggesting that these regions are not the sole source of correlation between the data. This finding is consistent with predictions made using the ISW effect, where one would expect weak correlations over the entire sky. In a flat universe the detection of the ISW effect provides direct and independent evidence for dark energy. We use our detection to constrain dark energy parameters by deriving a theoretical prediction for the directional wavelet covariance statistic for a given cosmological model. Comparing these predictions with the data we place constraints on the equation-of-state parameter $w$ and the vacuum energy density $\Omega_\Lambda$. We also consider the case of a pure cosmological constant, i.e. $w=-1$. For this case we rule out a zero cosmological constant at greater than the 99.9% significance level. All parameter estimates that we obtain are consistent with the standand cosmological concordance model values.Comment: 16 pages, 13 figures; replaced to match version accepted by MNRA

Filtering techniques for the detection of Sunyaev-Zel'dovich clusters in multifrequency CMB maps

The problem of detecting Sunyaev-Zel'dovich (SZ) clusters in multifrequency CMB observations is investigated using a number of filtering techniques. A multifilter approach is introduced, which optimizes the detection of SZ clusters on microwave maps. An alternative method is also investigated, in which maps at different frequencies are combined in an optimal manner so that existing filtering techniques can be applied to the single combined map. The SZ profiles are approximated by the circularly-symmetric template $\tau (x) = [1 +(x/r_c)^2]^{-\lambda}$, with $\lambda \simeq \tfrac{1}{2}$ and $x\equiv |\vec{x}|$, where the core radius $r_c$ and the overall amplitude of the effect are not fixed a priori, but are determined from the data. The background emission is modelled by a homogeneous and isotropic random field, characterized by a cross-power spectrum $P_{\nu_1 \nu_2}(q)$ with $q\equiv |\vec{q}|$. The filtering methods are illustrated by application to simulated Planck observations of a $12.8^\circ \times 12.8^\circ$ patch of sky in 10 frequency channels. Our simulations suggest that the Planck instrument should detect $\approx 10000$ SZ clusters in 2/3 of the sky. Moreover, we find the catalogue to be complete for fluxes $S > 170$ mJy at 300 GHz.Comment: 12 pages, 7 figures; Corrected figures. Submitted to MNRA

Proper motions of the HH1 jet

We describe a new method for determining proper motions of extended objects, and a pipeline developed for the application of this method. We then apply this method to an analysis of four epochs of [S~II] HST images of the HH~1 jet (covering a period of $\sim 20$~yr). We determine the proper motions of the knots along the jet, and make a reconstruction of the past ejection velocity time-variability (assuming ballistic knot motions). This reconstruction shows an "acceleration" of the ejection velocities of the jet knots, with higher velocities at more recent times. This acceleration will result in an eventual merging of the knots in $\sim 450$~yr and at a distance of $\sim 80"$ from the outflow source, close to the present-day position of HH~1.Comment: 12 pages, 8 figure

The Limits on Cosmological Anisotropies and Inhomogeneities from COBE Data

Assuming that the cosmological principle holds, Maartens, Ellis and Stoeger (MES) recently constructed a detailed scheme linking anisotropies in the cosmic background radiation (CMB) with anisotropies and inhomogeneities in the large scale structure of the universe and showed how to place limits on those anisotropies and inhomogeneities simply by using CMB quadrupole and octupole limits. First we indicate and discuss the connection between the covariant multipole moments of the temperature anisotropy used in the MES scheme and the quadrupole and octupole results from COBE. Then we introduce those results into the MES limit equations to obtain definite quantitative limits on the complete set of cosmological measures of anisotropy and inhomogeneity. We find that all the anisotropy measures are less than 10^{-4} in the case of those not affected by the expansion rate H, and less than 10^{-6} Mpc^{-1} in the case of those which are. These results quantitatively demonstrate that the observable universe is indeed close to Friedmann-Lemaitre-Robertson-Walker (FLRW) on the largest scales, and can be adequately modelled by an almost-FLRW model -- that is, the anisotropies and inhomogeneities characterizing the observable universe on the largest scales are not too large to be considered perturbations to FLRW.Comment: Original paper with corrections. ApJ 476 435 (1997) erratum to appear ApJ Sept 199

Comparing filters for the detection of point sources

This paper considers filters (the Mexican hat wavelet, the matched and the scale-adaptive filters) that optimize the detection/separation of point sources on a background. We make a one-dimensional treatment, we assume that the sources have a Gaussian profile, i. e. $\tau (x) = e^{- x^2/2R^2}$, and a background modelled by an homogeneous and isotropic Gaussian random field, characterised by a power spectrum $P(q)\propto q^{-\gamma}, \gamma \geq 0$. Local peak detection is used after filtering. Then, the Neyman-Pearson criterion is used to define the confidence level for detections and a comparison of filters is done based on the number of spurious and true detections. We have performed numerical simulations to test theoretical ideas and conclude that the results of the simulations agree with the analytical results.Comment: 17 pages, 17 figures, accepted for publication in MNRA

Cosmological applications of a wavelet analysis on the sphere

The cosmic microwave background (CMB) is a relic radiation of the Big Bang and as such it contains a wealth of cosmological information. Statistical analyses of the CMB, in conjunction with other cosmological observables, represent some of the most powerful techniques available to cosmologists for placing strong constraints on the cosmological parameters that describe the origin, content and evolution of the Universe. The last decade has witnessed the introduction of wavelet analyses in cosmology and, in particular, their application to the CMB. We review here spherical wavelet analyses of the CMB that test the standard cosmological concordance model. The assumption that the temperature anisotropies of the CMB are a realisation of a statistically isotropic Gaussian random field on the sphere is questioned. Deviations from both statistical isotropy and Gaussianity are detected in the reviewed works, suggesting more exotic cosmological models may be required to explain our Universe. We also review spherical wavelet analyses that independently provide evidence for dark energy, an exotic component of our Universe of which we know very little currently. The effectiveness of accounting correctly for the geometry of the sphere in the wavelet analysis of full-sky CMB data is demonstrated by the highly significant detections of physical processes and effects that are made in these reviewed works.Comment: 17 pages, 8 figures; JFAA invited review, in pres

Seeking the Ultraviolet Ionizing Background at z~3 with the Keck Telescope

We describe the initial results of a deep long-slit emission line search for redshifted (2.7<z<4.1) Lyman-alpha. These observations are used to constrain the fluorescent Ly-alpha emission from the population of clouds whose absorption produces the higher-column-density component of the Ly-alpha forest in quasar spectra. We use the results to set an upper limit on the ultraviolet ionizing background. Our spectroscopic data obtained with the Keck II telescope at lambda/(Delta lambda FWHM)~2000 reveals no candidate Ly-alpha emission over the wavelength range of 4500-6200 Ang along a 3 arcmin slit in a 5400 s integration. Our 3 sigma upper bound on the mean intensity of the ionizing background at the Lyman limit is J(nu 0) < 2E-21 erg/s/cm**2/Hz/sr for 2.7<z<3.1 (where we are most sensitive), assuming Lyman limit systems have typical radii of 70 kpc (q_0=0.5, H_0=50 km/s/Mpc). This constraint is more than an order of magnitude more stringent than any previously published direct limit. However, it is still a factor of three above the ultraviolet background level expected due to the integrated light of known quasars at z~3. This pilot study confirms the conclusion of Gould \& Weinberg (1996) that integrations of several hours on a 10-m class telescope should be capable of measuring J(nu 0) at high redshift.Comment: 22 pages, 2 postscipt figures. Latex requires aaspp4.sty and epsf.sty (included). Accepted for publication in the Astronomical Journal (Nov 1998