Discovery, Characterization and Mechanistic Study of a Novel L-Tyrosine Hydroxylase in the Biosynthesis of Anthramycin

A tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster is proposed to catalyze the ortho-hydroxylation of L-tyrosine to L-DOPA as the initial step of a unique transformation to the hydropyrrole moiety found in anthramycin. The sequence of Orf13 is not similar to any known characterized proteins, nor does it contain conserved domains or motifs characteristic of enzymes performing aromatic hydroxylation. The lack of information for this common enzymatic reaction suggests the identification of a new class of tyrosine hydroxylases which may have novel cofactor requirements, novel folds and/or chemical mechanisms. Heme B has been identified in purified Orf13 and full heme B occupancy is achieved during expression with iron (III) citrate in E. coli. Maximal L-tyrosine to L-DOPA conversion is observed in the presence of hydrogen peroxide (H2O2). This confirmed heme B as the required catalytic cofactor and the putative function of Orf13 as a tyrosine hydroxylase. This information also classified Orf13 as a heme peroxidase. Spectroscopic data from a reduced-CO (g) spectrum of Orf13 and electron paramagnetic resonance of ferric-heme Orf13 are consistent with histidyl-ligated heme peroxidases. The steady-state kinetics of L-tyrosine hydroxylation show similar catalytic efficiency for L-tyrosine and H2O2. Orf13 has a secondary tyrosine hydroxylation activity in the presence of molecular oxygen (O2) and dihydroxyfumaric acid (DHFA), which is also found with histidyl-heme peroxidases. Orf13 is substrate specific and stereoselective for L-tyrosine. Turnover is only observed with para-substituted phenols but not with D-tyrosine, implicating the para-phenol substituent is required for hydroxylation. Although the catalytic requirements of heme B and H2O2 are in agreement with heme peroxidases, the resulting hydroxylated product (L-DOPA) by a H2O2 dependent pathway is unprecedented. Heme dependent aromatic hydroxylation is typically catalyzed by cytochrome P450s through an O2 dependent pathway. Mechanistic investigation of Orf13 revealed H2O2 as the oxygen source in a labeling study using H218O2. A proposed mechanism of L-tyrosine hydroxylation is suggested to proceed through an oxygen rebound mechanism similar to cytochrome P450 aromatic hydroxylation. Therefore, Orf13 represents a new class of heme-histidyl ligated H2O2 dependent hydroxylases and is the first identified bacterial tyrosine hydroxylase

A sociometric analysis of the Pierce Elementary School

Thesis (M.A.)--Boston University, 1947. This item was digitized by the Internet Archive

Genetic interactions between PXY and the ERECTA and PLETHORA gene families drive cell size and cell proliferation during vascular development in Arabidopsis thaliana.

A significant proportion of terrestrial biomass is constituted of xylem cells that make up woody plant tissue (Etchells., 2015). This thesis further explores the genetic interactions that underpin the vascular developmental process in Arabidopsis thaliana stems, and the transition from primary to secondary growth in roots. To date, progress in the understanding of the underlying molecular mechanisms and genetic controls that underpin vascular development during secondary growth is incomplete. The PXY gene is one element in a ligand receptor pair that is a known central factor important for vascular development regulation and control. PXY signalling is known to be a part of a complex signalling system and it is clear that other interacting factors are also extremely important for the development of the vasculature. The research described as part of this thesis has shown that there exists a genetic interaction between the PXY gene and those of the PLETHORA3 and PLETHORA5 genes in Arabidopsis thaliana. This thesis has shown that PLETHORA3 and PLETHORA5 genes act redundantly and are in part responsible for the process of cellular proliferation within the vasculature during secondary growth. The loss of function mutations in PLT3 and PLT5 result in enhanced phenotypes in a pxy background for increased loss of cell proliferation. Further, they result in the suppression of hallmark characteristics and cellular proliferation in transgenic 35S::CLE41 over-expression backgrounds. Additionally, the interactions of PXY with the ERECTA family of genes has shown that ERECTA-LIKE genes interact with PXY to regulate the cell size in vascular cell types. Together PXY, ERECTA and PLETHORA interact to drive cell size and cell proliferation within the vascular cambium in regard to secondary growth

The Dangers of Dengue Fever

Dengue Fever or also known as breakbone fever is a viral infection that is spread from mosquitoes to people. The first isolation of Dengue Fever was in 1943 Japan and 1945 in Hawaii, the first two dengue viruses were isolated and named DENV1 and DENV2. There are 4 variations of Dengue, DENV 1-4. Each year there are about 50 million dengue infections and of those about 500,000 individuals are hospitalized with hemorrhagic dengue fever. It is estimated that 2.5 billion people are at risk of contracting dengue, Brazil is among the countries most affected by this terrible viral disease, with 13.6 million cases. Common symptoms of Dengue include Nausea/Vomiting, Rashes, Aches and Pains, and Thrombocytopenia. Some cases can become severe and cause hemorrhagic fever. Dengue occurs in hot climates, Temperature is an important determinant of biting rate, egg and immature mosquito development, development time of virus in the mosquito, and survival at all stages of the mosquito life cycle. Studies show that if climate change continues it can lead to high human exposure to dengue fever. It can affect Australia, Europe and North America. Dengue has expanded due to declining vector-control and increasing global trade and travel. Mosquitoes are vector-borne (living organisms) that transmit the disease, in this case Dengue via contact. Mosquitoes release viral particles covered in salivary glands in order to deliver pathogens to humans while they pass through the dermis of the skin. Dengue is transmitted primarily through a mosquito called Aedes aegypti

Deep Radio Interferometric Imaging with POLISH: DSA-2000 and weak lensing

Radio interferometry allows astronomers to probe small spatial scales that are often inaccessible with single-dish instruments. However, recovering the radio sky from an interferometer is an ill-posed deconvolution problem that astronomers have worked on for half a century. More challenging still is achieving resolution below the array's diffraction limit, known as super-resolution imaging. To this end, we have developed a new learning-based approach for radio interferometric imaging, leveraging recent advances in the classical computer vision problems of single-image super-resolution (SISR) and deconvolution. We have developed and trained a high dynamic range residual neural network to learn the mapping between the dirty image and the true radio sky. We call this procedure POLISH, in contrast to the traditional CLEAN algorithm. The feed forward nature of learning-based approaches like POLISH is critical for analyzing data from the upcoming Deep Synoptic Array (DSA-2000). We show that POLISH achieves super-resolution, and we demonstrate its ability to deconvolve real observations from the Very Large Array (VLA). Super-resolution on DSA-2000 will allow us to measure the shapes and orientations of several hundred million star forming radio galaxies (SFGs), making it a powerful cosmological weak lensing survey and probe of dark energy. We forecast its ability to constrain the lensing power spectrum, finding that it will be complementary to next-generation optical surveys such as Euclid

Single View Refractive Index Tomography with Neural Fields

Refractive Index Tomography is an inverse problem in which we seek to reconstruct a scene's 3D refractive field from 2D projected image measurements. The refractive field is not visible itself, but instead affects how the path of a light ray is continuously curved as it travels through space. Refractive fields appear across a wide variety of scientific applications, from translucent cell samples in microscopy to fields of dark matter bending light from faraway galaxies. This problem poses a unique challenge because the refractive field directly affects the path that light takes, making its recovery a non-linear problem. In addition, in contrast with traditional tomography, we seek to recover the refractive field using a projected image from only a single viewpoint by leveraging knowledge of light sources scattered throughout the medium. In this work, we introduce a method that uses a coordinate-based neural network to model the underlying continuous refractive field in a scene. We then use explicit modeling of rays' 3D spatial curvature to optimize the parameters of this network, reconstructing refractive fields with an analysis-by-synthesis approach. The efficacy of our approach is demonstrated by recovering refractive fields in simulation, and analyzing how recovery is affected by the light source distribution. We then test our method on a simulated dark matter mapping problem, where we recover the refractive field underlying a realistic simulated dark matter distribution