Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

Transpeptidation catalyzed by ​sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of ​sortase A on a solid support (Sepharose beads). Immobilization of ​sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca²⁺-independent variant of ​sortase A with increased catalytic activity. This heptamutant variant of ​sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized ​sortase A takes 1–2 d. Batch reactions take 3–12 h and flow reactions proceed at 0.5 ml h⁻¹, depending on the geometry of the reactor used.United States. National Institutes of Health (RO1 AI087879

Combined Forward-Backward Asymmetry Measurements in Top-Antitop Quark Production at the Tevatron

The CDF and D0 experiments at the Fermilab Tevatron have measured the asymmetry between yields of forward- and backward-produced top and antitop quarks based on their rapidity difference and the asymmetry between their decay leptons. These measurements use the full data sets collected in proton-antiproton collisions at a center-of-mass energy of $\sqrt s =1.96$ TeV. We report the results of combinations of the inclusive asymmetries and their differential dependencies on relevant kinematic quantities. The combined inclusive asymmetry is $A_{\mathrm{FB}}^{t\bar{t}} = 0.128 \pm 0.025$. The combined inclusive and differential asymmetries are consistent with recent standard model predictions

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Simplet/Fam53b is required for Wnt signal transduction by regulating beta-catenin nuclear localization

Canonical beta-catenin-dependent Wnt signal transduction is important for several biological phenomena, such as cell fate determination, cell proliferation, stem cell maintenance and anterior-posterior axis formation. The hallmark of canonical Wnt signaling is the translocation of beta-catenin into the nucleus where it activates gene transcription. However, the mechanisms regulating beta-catenin nuclear localization are poorly understood. We show that Simplet/Fam53B (Smp) is required forWnt signaling by positively regulating beta-catenin nuclear localization. In the zebrafish embryo, the loss of smp blocks the activity of two beta-catenin-dependent reporters and the expression of Wnt target genes, and prevents nuclear accumulation of beta-catenin. Conversely, overexpression of smp increases beta-catenin nuclear localization and transcriptional activity in vitro and in vivo. Expression of mutant Smp proteins lacking either the nuclear localization signal or the beta-catenin interaction domain reveal that the translocation of Smp into the nucleus is essential for beta-catenin nuclear localization and Wnt signaling in vivo. We also provide evidence that mammalian Smp is involved in regulating beta-catenin nuclear localization: the protein colocalizes with beta-catenin-dependent gene expression in mouse intestinal crypts; siRNA knockdown of Smp reduces beta-catenin nuclear localization and transcriptional activity; human SMP mediates beta-catenin transcriptional activity in a dose-dependent manner; and the human SMP protein interacts with human beta-catenin primarily in the nucleus. Thus, our findings identify the evolutionary conserved SMP protein as a regulator of beta-catenin-dependent Wnt signal transduction

CTPS forms the cytoophidium in zebrafish

Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)2017/20745-