An enzyme-based screening system for the rapid assessment of protein N-glycosylation efficiency in yeast

N-Glycosylation efficiency is a key parameter when studying components of the protein N-glycosylation pathway, but was recently also recognized as an important factor in the production of glycosylated proteins. We have developed a novel assay to quantify N-glycosylation efficiency of proteins. This assay is based on the secreted activity of yeast acid phosphatase, the proper folding and hence secretion of which is strongly dependent on its N-glycosylation status. The results show that the reporter yields a quantitative measure for protein N-glycosylation in yeast, which is in good agreement with classically used assay based on protein migration patterns on SDS-PAGE. However, the assay is less laborious and is adaptable to high-throughput screening approaches as exemplifie

Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth propertie

Vitreoscilla hemoglobin promoter is not responsive to nitrosative and oxidative stress in Escherichia coli

The Vitreoscilla hemoglobin gene (vhb) is expressed under oxygen-limited conditions via an FNR-dependent mechanism. Furthermore, cAMP-CRP has been implicated in its regulation. Recently, VHb protein has been reported to protect a heterologous host from nitrosative stress. In this study we analyzed the regulation of the Vitreoscilla hemoglobin promoter (Pvhb) in Escherichia coli under nitrosative and oxidative stress conditions. Our results show unambiguously that expression of neither VHb nor chloramphenicol acetyltransferase under the control of Pvhb is induced under the experimental conditions used. Thus, a clear discrepancy between in vivo function, i.e. protection against nitrosative stress, and regulation of gene expression is obvious. The regulation of Pvhb reported here is in clear contrast to the expression pattern of flavohemoglobins from various microorganisms, which are generally induced by nitrosative stress. However, the length of Pvhb is only 146 bp and therefore, we cannot rule out that additional regulatory sequences may be located in the upstream region of Pvh

Intrinsic non-symbiotic and truncated haemoglobins and heterologous Vitreoscilla haemoglobin expression in plants

To date, haemoglobins (Hbs) have been shown to exist in all kingdoms of life. The least studied and understood groups are plant non-symbiotic haemoglobins (nsHbs) and the recently found plant truncated Hbs (trHbs). From a biotechnological point of view, the best characterized and almost exclusively applied Hb is the bacterial Vitreoscilla haemoglobin (VHb). In this review, the present state of knowledge of structural features and ligand binding kinetics of plant nsHbs and trHbs and their proposed roles as oxygen carriers, oxygen sensors, and for oxygen storage, in nitric oxide (NO) detoxification, and in peroxidase activity are described. Furthermore, in order to predict the functioning of plant Hbs, their characteristics will be compared with those of the better known bacterial globins. In this context, the effects of heterologous applications of VHb on plants are reviewed. Finally, the challenging future of plant Hb research is discusse

Morphing Ensemble Kalman Filters

A new type of ensemble filter is proposed, which combines an ensemble Kalman filter (EnKF) with the ideas of morphing and registration from image processing. This results in filters suitable for nonlinear problems whose solutions exhibit moving coherent features, such as thin interfaces in wildfire modeling. The ensemble members are represented as the composition of one common state with a spatial transformation, called registration mapping, plus a residual. A fully automatic registration method is used that requires only gridded data, so the features in the model state do not need to be identified by the user. The morphing EnKF operates on a transformed state consisting of the registration mapping and the residual. Essentially, the morphing EnKF uses intermediate states obtained by morphing instead of linear combinations of the states.Comment: 17 pages, 7 figures. Added DDDAS references to the introductio

Similarity Index for the Fat Fraction between Breast Milk and Infant Formulas

The similarity of the fat fraction in infant formulas rich in either bovine milk fat (MF) or vegetable oil (VO) to breastmilk was evaluated by analyzing their lipid composition. Milk fat-rich formulas were highly similar (average similarity index 0.68) tobreast milk compared to the VO-rich formulas (average similarity index 0.56). The highest difference in the indices was found in thecontents of cholesterol (0.66 vs 0.28 in MF- and VO-rich formulas, respectively, on average) and polar lipids (0.84 vs 0.53), thepositional distribution of fatty acids in thesn-2 position of triacylglycerols (0.53 vs 0.28), and fatty acid composition (0.72 vs 0.54).The VO-based formulas were superior in similarity inn-6 PUFA. Thus, the addition of bovine MF fractions is an effective way toincrease the similarity between the lipid composition of infant formulas and human mil

Light Induced Melting of Colloidal Crystals in Two Dimensions

We demonstrate that particles confined to two dimensions (2d) and subjected to a one-dimensional (1d) periodic potential exhibit a rich phase diagram, with both ``locked floating solids'' and smectic phases. The resulting phases and phase transitions are studied as a function of temperature and potential strength. We find reentrant melting as a function of the potential strength. Our results lead to universal predictions consistent with recent experiments on 2d colloids in the presence of a laser-induced 1d periodic potential.Comment: 4 pages, 3 figures, also available at http://cmtw.harvard.edu/~fre

Novel Phases and Reentrant Melting of Two Dimensional Colloidal Crystals

We investigate two-dimensional (2d) melting in the presence of a one-dimensional (1d) periodic potential as, for example, realized in recent experiments on 2d colloids subjected to two interfering laser beams. The topology of the phase diagram is found to depend primarily on two factors: the relative orientation of the 2d crystal and the periodic potential troughs, which select a set of Bragg planes running parallel to the troughs, and the commensurability ratio p= a'/d of the spacing a' between these Bragg planes to the period d of the periodic potential. The complexity of the phase diagram increases with the magnitude of the commensurabilty ratio p. Rich phase diagram, with ``modulated liquid'', ``floating'' and ``locked floating'' solid and smectic phases are found. Phase transitions between these phases fall into two broad universality classes, roughening and melting, driven by the proliferation of discommensuration walls and dislocations, respectively. We discuss correlation functions and the static structure factor in these phases and make detailed predictions of the universal features close to the phase boundaries. We predict that for charged systems with highly screened short-range interactions these melting transitions are generically reentrant as a function of the strength of the periodic potential, prediction that is in accord with recent 2d colloid experiments. Implications of our results for future experiments are also discussed.Comment: 37 pages, 24 figure

Democratic cultural policy : democratic forms and policy consequences

The forms that are adopted to give practical meaning to democracy are assessed to identify what their implications are for the production of public policies in general and cultural policies in particular. A comparison of direct, representative, democratic elitist and deliberative versions of democracy identifies clear differences between them in terms of policy form and democratic practice. Further elaboration of these differences and their consequences are identified as areas for further research

Nearly K\"ahler heterotic compactifications with fermion condensates

We revisit AdS_4 heterotic compactifications on nearly K\"ahler manifolds in the presence of H-flux and certain fermion condensates. Unlike previous studies, we do not assume the vanishing of the supersymmetry variations. Instead we determine the full equations of motion originating from the ten-dimensional action, and subsequently we provide explicit solutions to them on nearly K\"ahler manifolds at first order in alpha'. The Bianchi identity is also taken into account in order to guarantee the absence of all anomalies. In the presence of H-flux, which is identified with the torsion of the internal space, as well as of fermion condensates in the gaugino and dilatino sectors, new solutions are determined. These solutions provide a full classification of consistent backgrounds of heterotic supergravity under our assumptions. All the new solutions are non-supersymmetric, while previously known supersymmetric ones are recovered too. Our results indicate that fully consistent (supersymmetric or not) heterotic vacua on nearly K\"ahler manifolds are scarce, even on AdS_4, and they can be completely classified.Comment: 1+17 pages, 1 figure; v2: remark and two references added, published versio