A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

Background: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand ( hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.Results: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIF alpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFa peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.Conclusion: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes

Silk physico-chemical variability and mechanical robustness facilitates intercontinental invasibility of a spider.

There are substantive problems associated with invasive species, including threats to endemic organisms and biodiversity. Understanding the mechanisms driving invasions is thus critical. Variable extended phenotypes may enable animals to invade into novel environments. We explored here the proposition that silk variability is a facilitator of invasive success for the highly invasive Australian house spider, Badumna longinqua. We compared the physico-chemical and mechanical properties and underlying gene expressions of its major ampullate (MA) silk between a native Sydney population and an invasive counterpart from Montevideo, Uruguay. We found that while differential gene expressions might explain the differences in silk amino acid compositions and protein nanostructures, we did not find any significant differences in silk mechanical properties across the populations. Our results accordingly suggest that B. longinqua's silk remains functionally robust despite underlying physico-chemical and genetic variability as the spider expands its range across continents. They also imply that a combination of silk physico-chemical plasticity combined with mechanical robustness might contribute more broadly to spider invasibilities

Keggin heteropolyacid H3PW12O40 supported on different oxides for catalytic and catalytic photo-assisted propene hydration

Catalytic and catalytic photo-assisted hydration of propene to form 2-propanol in gas–solid regime at atmospheric pressure and 85 1C were carried out by using a heteropolyacid (POM) supported on different oxides. Binary materials were prepared by impregnation of H3PW12O40 on different commercial and home prepared supports (TiO2, SiO2, WO3, ZrO2, ZnO, Al2O3). Some of the composites were active both for catalytic and catalytic photo-assisted reactions. The Keggin type POM was completely and partially degraded, when supported on ZnO and Al2O3, respectively, and these binary solids always resulted as inactive for both catalytic and catalytic photo-assisted reactions. The supported Keggin POM species played a key role both for the catalytic and the photo-assisted catalytic reactions, due to their strong acidity and ability to form strong oxidant species under UV irradiation, respectively. The contemporary presence of heat and UV light improved the activity of almost all POM supported materials. All materials were characterized by X-ray diffraction (XRD), scanning electron microscopy observations (SEM), diffuse reflectance spectroscopy (DRS), determination of the conduction and valence band energy by photovoltage measurements, Fourier transform infrared spectroscopy (FTIR), NH3-TPD experiments and time resolved microwave conductivity (TRMC). Introduction Propene hydration to obtain 2-propanol is a reaction carried out at moderate temperatures (ca. 150–200 1C) and pressure (2MPa) in the presence of an acid catalyst;1 however the realization of this reaction at ambient conditions is of great interest. The us

Hierarchies of Susy Splittings and Invisible Photinos as Dark Matter

We explore how to generate hierarchies in the splittings between superpartners. Some of the consequences are the existence of invisible components of dark matter, new inflaton candidates, invisible monopoles and a number of invisible particles that might dominate during various eras, in particular between BBN and recombination and decay subsequently.Comment: 16 pages. v3: Ref. 27 has been modified. v4: Published versio

Helical Chirality: a Link between Local Interactions and Global Topology in DNA

DNA supercoiling plays a major role in many cellular functions. The global DNA conformation is however intimately linked to local DNA-DNA interactions influencing both the physical properties and the biological functions of the supercoiled molecule. Juxtaposition of DNA double helices in ubiquitous crossover arrangements participates in multiple functions such as recombination, gene regulation and DNA packaging. However, little is currently known about how the structure and stability of direct DNA-DNA interactions influence the topological state of DNA. Here, a crystallographic analysis shows that due to the intrinsic helical chirality of DNA, crossovers of opposite handedness exhibit markedly different geometries. While right-handed crossovers are self-fitted by sequence-specific groove-backbone interaction and bridging Mg2+ sites, left-handed crossovers are juxtaposed by groove-groove interaction. Our previous calculations have shown that the different geometries result in differential stabilisation in solution, in the presence of divalent cations. The present study reveals that the various topological states of the cell are associated with different inter-segmental interactions. While the unstable left-handed crossovers are exclusively formed in negatively supercoiled DNA, stable right-handed crossovers constitute the local signature of an unusual topological state in the cell, such as the positively supercoiled or relaxed DNA. These findings not only provide a simple mechanism for locally sensing the DNA topology but also lead to the prediction that, due to their different tertiary intra-molecular interactions, supercoiled molecules of opposite signs must display markedly different physical properties. Sticky inter-segmental interactions in positively supercoiled or relaxed DNA are expected to greatly slow down the slithering dynamics of DNA. We therefore suggest that the intrinsic helical chirality of DNA may have oriented the early evolutionary choices for DNA topology

A socio-environmental geodatabase for integrative research in the transboundary Rio Grande/Río Bravo basin

Integrative research on water resources requires a wide range of socio-environmental datasets to better understand human-water interactions and inform decision-making. However, in transboundary watersheds, integrating cross-disciplinary and multinational datasets is a daunting task due to the disparity of data sources and the inconsistencies in data format, content, resolution, and language. This paper introduces a socio-environmental geodatabase that transcends political and disciplinary boundaries in the Rio Grande/Río Bravo basin (RGB). The geodatabase aggregates 145 GIS data layers on five main themes: (i) Water & Land Governance, (ii) Hydrology, (iii) Water Use & Hydraulic Infrastructures, (iv) Socio-Economics, and (v) Biophysical Environment. Datasets were primarily collected from public open-access data sources, processed with ArcGIS, and documented through the FGCD metadata standard. By synthesizing a broad array of datasets and mapping public and private water governance, we expect to advance interdisciplinary research in the RGB, provide a replicable approach to dataset compilation for transboundary watersheds, and ultimately foster transboundary collaboration for sustainable resource management.The project was funded by Grant No. G15AP00132 from the United States Geological Survey. Open Access fees paid for in whole or in part by the University of Oklahoma Libraries.Ye

From presence to consciousness through virtual reality

Immersive virtual environments can break the deep, everyday connection between where our senses tell us we are and where we are actually located and whom we are with. The concept of 'presence' refers to the phenomenon of behaving and feeling as if we are in the virtual world created by computer displays. In this article, we argue that presence is worthy of study by neuroscientists, and that it might aid the study of perception and consciousness

Role of thrombin receptor in breast cancer invasiveness

Invasion, the ability of an epithelial cancer cell to detach from and move through a basement membrane, is a central process in tumour metastasis. Two components of invasion are proteolysis of extracellular matrix and cellular movement through it. A potential promoter of these two processes is thrombin, the serine proteinase derived from the ubiquitous plasma protein prothrombin. Thrombin promotes the invasion of MDA-MB231 breast tumour cells (a highly aggressive cell line) in an in vitro assay. Invasion by MDA-MB436 and MCF-7 cells, less aggressive cell lines, is not promoted by thrombin. Thrombin, added to the cells, is a stimulator of cellular movement; fibroblast-conditioned medium is the chemotaxin. Thrombin-promoted invasion is inhibited by hirudin. Stimulation of invasion is a receptor-mediated process that is mimicked by a thrombin receptor-activating peptide. Thrombin has no effect on chemotaxis in vitro. Thrombin receptor is detectable on the surface of MDA-MB231 cells, but not on the other two cell lines. Introduction of oestrogen receptors into MDA-MB231 cells by transfection with pHEO had no effect on thrombin receptor expression, in the presence or absence of oestradiol. This paper demonstrates that thrombin increases invasion by the aggressive breast cancer cell line MDA-MB231 by a thrombin receptor-dependent mechanism. © 1999 Cancer Research Campaig