We report our initial efforts in the analysis of endogenous nuclear receptor coactivator complexes as a research bridging strand of the Nuclear Receptor Signaling Atlas (NURSA) (www.NURSA.org). A proteomic approach is used to systematically isolate a variety of coactivator complexes using HeLa cells as a model cell line and to identify the coactivator-associated proteins with mass spectrometry. We have isolated and identified seven coactivator complexes including the p160 steroid receptor coactivator family, cAMP response element binding protein-binding protein, p300, coactivator of activating protein-1 and estrogen receptors, and E6 papillomavirus-associated protein. The newly identified coactivator-associated proteins provide unbiased clues and links for understanding of the endogenous hormone receptor coregulator network and its regulation. We hope that the electronic availability of these data to the general scientific community will facilitate generation and testing of new hypotheses to further our understanding of nuclear receptor signaling and coactivator functions.

The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

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The stem cell niche comprises soluble molecules and extracellular matrix components which provide chemical and mechanical cues that determine the differentiation of stem cells. Here, the effect of polyelectrolyte multilayer (PEM) composition and terminal layer fabricated with layer-by-layer technique (LBL) pairing either hyaluronan [in its native (nHA) and oxidized form (oHA)] or chondroitin sulfate (CS) with type I collagen (Col I) is investigated on chondrogenic differentiation of human umbilical mesenchymal stem cells (hUC-MSCs). Physical studies performed to investigate the establishment and structure of the surface coatings show that PEM composed of HA and Col I show a dominance of nHA or oHA with considerably lesser organization of Col I fibrils. In contrast, distinguished fibrilized Col I is found in nCS-containing PEM. Generally, Col I-terminated PEM promote the adhesion, migration, and growth of hUC-MSCs more than GAG-terminated surfaces due to the presence of fibrillar Col I but show a lower degree of differentiation towards the chondrogenic lineage. Notably, the Col I/nHA PEM not only supports adhesion and growth of hUC-MSCs but also significantly promotes cartilage-associated gene and protein expression as found by histochemical and molecular biology studies, which is not seen on the Col I/oHA PEM. This is related to ligation of HA to the cell receptor CD44 followed by activation of ERK/Sox9 and noncanonical TGF-β signaling-p38 pathways that depends on the molecular weight of HA as found by immune histochemical and western blotting. Hence, surface coatings on scaffolds and other implants by PEM composed of nHA and Col I may be useful for programming MSC towards cartilage regeneration.