Life Sciences Commons^™

Transition State Interactions In A Promiscuous Enzyme: Sulfate And Phosphate Monoester Hydrolysis By Pseudomonas Aeruginosa Arylsulfatase, Bert Van Loo, Ryan Berry, Usa Boonyuen, Mark F. Mohamed, Marko Golicnik, Alvan C. Hengge, Florian Hollfelder

Chemistry and Biochemistry Faculty Publications

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PAS^_WT has a much less negative Brønsted coefficient (ß_{leaving group} ^obs-Enz=-0.33) than the uncatalyzed reaction (ß_{leaving group} ^obs=-1.81). This situation is diminished when cationic active site groups are exchanged for alanine. …

Influence Of Loss Of Function Of The Fatty Acid Desaturase 7 Gene On Photosynthetic Activity And Foliar Redox Status, Janithri Savindya Wickramanayake

Graduate Theses and Dissertations

Fatty Acid Desaturase7 (FAD7) is a chloroplast-localized enzyme that converts 16 and 18 carbon dienoic fatty acids to trienoic fatty acids. The suppressor of prosystemin-mediated response2 (spr2) mutant in tomato (Solanum lycopersicum) and the fad7-1 mutant in Arabidopsis (Arabidopsis thaliana) result in the loss of function of FAD7, which alter the fatty acid profiles of chloroplast membranes and enhance resistance against aphids. This research contributes toward the long-term goal of identifying factors that determine aphid resistance in FAD7 mutants. Previous data suggested constitutive differences between spr2 and WT including increased expression of genes associated with photosynthesis and differences in redox …

Isolation And Characterization Of Natural Protease Producers Of Bacillus Spp. From Soil Samples, Ylberinë Baliu, Bahtir Hyseni, Shkëlqim Hyseni, Aida Rushiti, Flora Ferati, Fatos Rexhepi, Rifat Morina, Sabri Hajdini, Emrah Nikerel

UBT International Conference

Proteases are group of enzymes that catalyze the hydrolysis of proteins, resulting in peptide and amino acid production. These are considered as commercially most significant among industrial enzymes, consisting about 60% of the total enzyme market. Proteases have wide areas of application in several industries like food, detergent, textile, pulp and paper, and pharmaceutical industries, as these offer environmentally benign, sustainable solutions to several challenges in the corresponding industry.Enzymes can be produced by plants, animals or microorganisms, yet the majority is produced by (recombinant) microorganisms, typically with modified features for high enzyme production. However, for e.g. food industry, the use …

Theobromine And Related Methylxanthines As Inhibitors Of Primary Amine Oxidase, Padraig Shanahan, Jeffrey O’Sullivan, Keith Tipton, Gemma Kinsella, Barry Ryan, Gary Henehan

Articles

Methylxanthines are the most widely consumed drugs in the world and evidence of their health benefits has been growing in recent years. Primary Amine Oxidase (PrAO) has been recognised as a therapeutic target for amelioration of inflammatory, vascular and neurodegenerative diseases. Previous work in our laboratories showed that caffeine inhibited Bovine PrAO with a Ki of 1.0mM using benzylamine as substrate.

This study aimed to extend our previous work and explore the possibility that related methylxanthines might influence PrAO activity. While paraxanthine, theophylline and 7-methylxanthine had little effect on PrAO, theobromine was a noncompetitive inhibitor with a Ki of 276±44µM. …

The Distinctive Regulatory Mechanisms Of Bacterial Acetyl-Coa Carboxylase, Alexandra Leigh Evans

LSU Doctoral Dissertations

Metabolic Regulation is a complex system used to control cellular metabolism in response to conditions in the cell’s environment. For most enzymes, the cell can rely upon a minimal amount of regulation; however, critical enzymes, such as acetyl-CoA carboxylase, must be regulated at multiple levels. Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In bacteria, acetyl-CoA carboxylase forms a complex of three subunits–biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase–which catalyze the carboxylation of acetyl-CoA to form malonyl-CoA via two half-reactions. In the first half-reaction, biotin covalently linked to biotin carboxyl carrier protein is carboxylated by biotin …

The N-Terminal Methyltransferase Homologs Nrmt1 And Nrmt2 Exhibit Novel Regulation Of Activity Through Heterotrimer Formation., Jon David Faughn

Electronic Theses and Dissertations

Protein, DNA, and RNA methyltransferases have an ever-expanding list of novel substrates and catalytic activities. Even within families and between homologs, it is becoming clear the intricacies of methyltransferase specificity and regulation are far more diverse than originally thought. In addition to specific substrates and distinct methylation levels, methyltransferase activity can be altered through formation of complexes with close homologs. This work involves the N-terminal methyltransferase homologs NRMT1 and NRMT2. NRMT1 is a ubiquitously expressed distributive trimethylase. NRMT2 is a monomethylase expressed at low levels and in a tissue-specific manner. They are both nuclear methyltransferases with overlapping target consensus sequences …

Site-Directed Mutagenesis Of Malate Dehydrogenase: A Class Project, Bruce J. Heyen, Chesley Rowlett, Jon Zatorski, Ryan Burch, Emily Veach, Andy Gemmaka

Scholar Week 2016 - present

Malate dehydrogenase (MDH) is an important enzyme in an organism’s metabolic pathways. MDH is found in almost all living cells and catalyzes the conversion of malate to oxaloacetate which also involves nicotinamide dehydrogenase (NAD) as a coenzyme. A method to study how an enzyme operates is to alter one of its amino acids and compare the activity of the enzyme before and after the mutation. As a class project in Advanced Biochemistry during the spring semester of 2018, we are working as a team to propose and carry out a point-based mutation on MDH.

Secondary Functions And Novel Inhibitors Of Aminoacyl-Trna Synthetases, Patrick Wiencek

Graduate College Dissertations and Theses

The aminoacyl-tRNA synthetases are a family of enzymes involved in the process of translation, more specifically, ligating amino acids to their cognate tRNA molecules. Recent evidence suggests that aminoacyl-tRNA synthetases are capable of aminoacylating proteins, some of which are involved in the autophagy pathway. Here, we test the conditions under which E. coli and human threonyl-tRNA synthetases, as well as hisidyl-tRNA synthetase aminoacylate themselves. These reactions are ATP dependent, stimulated by Mg2+, and are inhibited by increasing cognate tRNA concentrations. These data represent the foundation for future aminoacylation experiments, specifically delving into the relationship between the autophagy pathway and the …

The Influences Of Feeding Broilers On Graded Inclusion Of Sunflower Meal With Or Without Avizyme On Growth, Protein And Energy Efficiency, Carcass Traits, And Nutrient Digestibility, Mahmoud Alagwany, Adel Attia, Zenat Ibrahim, Mohamed Abd El-Hack1, Muhammed Arif, Mohamed Emam

Turkish Journal of Veterinary & Animal Sciences

The present study aimed to investigate the substituting effect of sunflower meal (SFM) for soybean meal (SBM) with or without supplemental Avizyme on growth performance, protein and energy efficiency ratios, carcass traits, and nutrient digestibility of broiler chickens. A 4 × 2 factorial design experiment was conducted with four levels of SFM replacing SBM (0%, 25%, 50%, 75%) and two levels of supplemented Avizyme (0 or 0.1 g/kg diet). Results revealed that increasing SFM substitution for SBM up to 50% was associated with an increase in body weight (P < 0.01). Daily weight gain was statistically (P < 0.01) enhanced with increasing SFM up to 50% through 22 to 42 and 7 to 42 days of age. Feed conversion ratio improved (P < 0.01) in groups given SFM at 25% and 50% compared to the control. No carcass values were affected by SFM inclusion or Avizyme. The inclusion of 25% or 50% SFM increased (P < 0.01) protein efficiency at periods of 7?21 and 22?42 days of age. Energy efficiency ratio improved (P < 0.01) due to SFM incorporation at levels of 25% or 50% in all periods. The interaction effect between SFM and Avizyme was not significant on a majority of studied parameters. The digestion coefficients of crude fiber, N-free extract, and organic matter increased (P < 0.05 or P < 0.01) with increased levels of SFM. In conclusion, the incorporation of SFM as a substitute for SBM in broiler diets improved the growth performance of broilers. The use of SFM up to 50% without Avizyme supplementation could be recommended in broiler diets from 7 to 42 days of age.

Lonicera Maackii Alters Decay Dynamics Of Coarse Woody Debris, Michaela J. Woods

Browse all Theses and Dissertations

Since industrialization, anthropogenic carbon emissions have led to excess atmospheric carbon dioxide that may alter the stability of ecosystem processes. Microorganisms are essential in mitigating excess carbon and play a notable role in the breakdown of organic material. This process, decomposition, is essential in forested ecosystems where microorganisms can recycle nutrients and store carbon in soil organic matter or release it through respiration. Fungi participate in decomposition through the release of enzymes responsible for carrying out the chemical reactions that break down plant material. Species introductions have the potential to alter decomposition dynamics. In the Midwestern US, the invasive shrub …

Key Enzymes In Cancer: Mechanism Of Action And Inhibition With Anticancer Agents, Debasish Bandyopadhyay, Gabriel Lopez, Stephanie Cantu, Samantha Balboa, Annabel Garcia, Christina Silva, Diandra Valdes

Chemistry Faculty Publications and Presentations

Several enzymes play significant role in different stages of cancer including proliferation, invasion, metastasis and angiogenesis. Mechanism of actions of a few key enzymes along with their inhibitors have been discussed with particular attentation to riboneucleotide reductase, thymidylate synthease, topoisomerase II, interleukins, cell survival proteins and aminopeptidase N.

Effects Of Storing Three Phytase Sources Over 90 Days Under High Temperature And Humidity On Phytase Stability, Growth Performance, And Bone Mineralization Of Nursery Pigs, C. M. Vier, M. B. Menegat, K. M. Gourley, S. S. Dritz, M. D. Tokach, J. R. Bergstrom, R. D. Goodband, J. M. Derouchey, J. C. Woodworth

Kansas Agricultural Experiment Station Research Reports

A study was performed to evaluate the effects of storing three commercially available phytase products for 90 d, simulating summer conditions on phytase stability, growth performance, and bone mineralization of nursery pigs. The phytase products [HiPhos GT (20,000 FYT/g, DSM Nutritional Products, Parsippany, NJ); Axtra Phy TPT (20,000 FTU/g, Dupont, Wilmington, DE); and Quantum Blue G (40,000 FTU/g, AB Vista, Plantation, FL)] were left as pure forms or blended in a vitamin and trace mineral (VTM) premix for a 90 d period in an environmentally controlled chamber set at 85°F and 75% humidity. Sampling occurred on d 0, 30, 60, …

Regulation Of Katanin Activity On Microtubules, Madison A. Tyler

Masters Theses

The cytoskeleton is a dynamic network of microtubules constantly being reorganized to meet the spatiotemporal demands of the cell. Microtubules are organized into subcellular highways to control cell processes such as cell division, cargo transport, and neuronal development and maintenance. Reorganization of this intricate network is tightly regulated by various stabilizing and destabilizing microtubule-associated proteins that decorate the network. Katanin p60 is a microtubule destabilizing enzyme from the ATPases Associated with various Activities (AAA+) family. It can both sever and depolymerize microtubules. In order to sever microtubules, katanin recognizes the tubulin carboxy-terminal tails (CTTs) and hydrolyzes ATP. Using super-resolution microscopy …

Relationship Between Structure And Function In Nickel Proteins And Enzymes, Carolyn Carr

Doctoral Dissertations

Nickel is a rarely used but biologically important metal that is utilized in all three domains of life. In nickel utilizing organisms there is a corresponding trafficking system specifically designed to capture nickel, deliver, and export excess nickel to prevent toxic effects. It is critical to understand the mechanisms by which organisms achieve metal selectivity to duplicate or disrupt this process for the benefit of human health and to further understanding of regulation mechanisms in biology. RcnR is a Ni(II) and Co(II) responsive transcriptional regulator in E. coli. The research reported in this dissertation focuses on the relationship between …

A Study On The Interactions Of Trehalose With Model Folate Compounds, Carolyn R Ware

Chancellor’s Honors Program Projects

No abstract provided.

The Effects Of Xylanase Or An Algal Sourced Zinc Polysaccharide Complex On The Performance Of Laying Hens Fed A Corn And Soybean Diet, Ashley Bigge

Department of Animal Science: Dissertations, Theses, and Student Research

Two studies were conducted to determine the effects of two different feed additives on the performance of laying hens. Study 1 examined the effects of non-starch polysaccharide degrading enzyme inclusion in a low energy diet. Study 2 examined the effects of supplementing a paramylon zinc polysaccharide complex at increasing concentrations. Both studies utilized a randomized complete block design for treatment assignment, and data were analyzed using the Glimmix procedure in SAS 9.4 for windows.

The first study took place July 2014 to March 2015 and consisted of two phases. Each phase tested a positive control, negative control with lower ME …

Enzymatically Active Microspheres For Self-Propelled Colloidal Engines, Jungeun Park

Dissertations and Theses

Micro- and nano-motors have attracted numerous attentions from various scientific areas due to their potential applications. Most studies on self-propelled colloidal engines have exploited catalytic decomposition of hydrogen peroxide to drive the motor. Since the hydrogen peroxide is caustic, it is not suitable to use in biological applications, encouraging people to develop “greener” fuels. The aim of this research is to study a new transduction mechanism for self-propulsion not tied to hydrogen peroxide, and which can in particular be used with biological molecules as fuels. In this study, we focus on making particles with enzymatic activity which can effectively decompose …

The Urea Carboxylase And Allophanate Hydrolase Activities Of Urea Amidolyase Are Functionally Independent, Yi Lin, Cody J. Boese, Martin St. Maurice

Biological Sciences Faculty Research and Publications

Urea amidolyase (UAL) is a multifunctional biotin-dependent enzyme that contributes to both bacterial and fungal pathogenicity by catalyzing the ATP-dependent cleavage of urea into ammonia and CO₂. UAL is comprised of two enzymatic components: urea carboxylase (UC) and allophanate hydrolase (AH). These enzyme activities are encoded on separate but proximally related genes in prokaryotes while, in most fungi, they are encoded by a single gene that produces a fusion enzyme on a single polypeptide chain. It is unclear whether the UC and AH activities are connected through substrate channeling or other forms of direct communication. Here, we use …

Insights From Molecular Dynamics On Substrate Binding And Effects Of Active Site Mutations In Delta1-Pyrroline-5-Carboxylate Dehydrogenase, Bogdan F. Ion, Mohamed M. Aboelnga, James W. Gauld

Chemistry and Biochemistry Publications

The NAD+-dependent enzyme, 1-pyrroline-5-carboxylate dehydrogenase (P5CDH), has an important role in proline and hydroxyproline catabolism for humans. Specifically, this aldehyde dehydrogenase is responsible for the oxidation of both L-glutamate- -semialdehyde (GSA) and 4-erythro-hydroxy-L-glutamate- -semialdehyde (4-OH-GSA) to their respective L-glutamate product forms. We have performed a detailed molecular dynamics (MD) study of both the reactant and product complex structures of P5CDH to gain insights into ligand binding (i.e., GSA, 4-OH-GSA, NAD+, GLU) in the active site. Moreover, our investigations were further extended to examine the structural impact of S352L, S352A, and E314A mutations on the deficiency in the P5CDH enzymatic activity. …

Microbial Extracellular Enzymes In Marine Sediments: Methods Development And Potential Activities In The Baltic Sea Deep Biosphere, Jenna Marie Schmidt

Masters Theses

The deep biosphere is defined as the subsurface ecosystem in which little energy is available to microorganisms and microorganisms can live for thousands of years. Heterotrophic microbes survive in the deep biosphere even though organic matter is limited and highly recalcitrant in nature. Measuring microbial extracellular enzyme activity provides a potential means to evaluate the rate at which microorganisms are performing carbon remineralization in the energy limited sediment beneath the seafloor. Extracellular enzymes breakdown organic compounds so that the nutrients can move inside the cell and be used for energy. This study explored the role extracellular enzymes play in the …

Mutant Study Of Sinorhizobium Meliloti Proline Utilization A (Puta), Jacob E. Wilkinson, John J. Tanner, Donald F. Becker

UCARE Research Products

The purpose of this project is to purify and characterize the reaction kinetics of mutant versions the enzyme Proline Utilization A (PutA) in Sinorhizobium meliloti. The enzyme catalyzes the first step in proline metabolism. It has two active sites. The first is proline dehydrogenase (PRODH) which converts proline to pyrroline-5-carboxylate (P5C). The second is P5C dehydrogenase (P5CDH) which converts P5C to glutamate. Although many bacterial organisms have PutA, there are still significant interspecies variations, resulting in an entire family of PutA enzymes. The main difference is the length of the amino acid sequence. This affects the protein’s structure or …

Analysis Of Morphology And Secretion Mutants In Aspergillus Nidulans, Shelby N. Holaday, Steven D. Harris

UCARE Research Products

Filamentous fungi are important economically and medically due to their capacity to produce secondary metabolites or as human and animal pathogens. The genes and molecular mechanism responsible for secretion is poorly understood. Using classical genetics, we derived temperature sensitive (Ts) fungal mutants in Aspergillis nidulans. These mutants were then analyzed for their secondary metabolite secretion capacity.

In this study, we used the Ts strains of A. nidulans to determine how knockouts affect morphological and protein secretion by examining the phenotypes under the microscope and also by staining the mutants with Congo red staining. Mutants were stained with Congo red to …

Identifying Non-Classical Active Sites As A Tool For Enzyme Inhibition, Marisol Serrano

Open Access Theses & Dissertations

Chagas disease, caused by the parasite Trypanosoma cruzi, is an endemic life-threatening disease that affects mainly the heart. It remains the leading cause of heart failure in Latin American countries. Since current treatments against this parasite are highly toxic and somewhat ineffective, novel and more efficacious types of interventions are desired. Cruzain, identified as the major cathepsin for T. cruzi, plays a major role in the parasite's life cycle; making this enzyme very attractive for potential trypanocidal drugs discovery. The recombinant cruzain is synthesized as a zymogenic pro-protein (PCZN) which possesses a pro domain and a catalytic domain. In this …

Gross Responses And Apparent Ileal Digestibility Of Amino Acids And Minerals In Broiler Chicken Fed Vegetable-Based Starter Diets Supplemented With Microbial Enzymes, Mohammad Abul Hossain, Paul Ade Iji, Al-Farooq Mohammad Fakhrul Islam

Turkish Journal of Veterinary & Animal Sciences

This study was conducted to investigate the effects of diet and exogenous enzymes on growth responses and micronutrient digestibility of broilers. Day-old broilers (n = 256) were distributed into a 2 x 2 factorial design and were fed on two basal diets: soybean (T1) or canola (T2) meals as such, or supplemented with enzymes up to 21 days. Feed intake (FI) and live weight (LW) to 21 days on the T2 diet was higher (P < 0.001) than that on T1. Feed conversion ratio (FCR) was better (P < 0.01) on T1 than on T2 at 21 days. Enzymes generally improved (P < 0.001) FI, FCR, and LW. The digestibility of histidine was higher (P < 0.05) on T2, but lysine digestibility was higher (P < 0.01) on T1 at 21 days. Histidine, threonine, lysine, valine, isoleucine, and phenylalanine digestibility was improved (P < 0.05) in the supplemented diets. Mineral digestibility was unaffected (P > 0.05) by diet, but enzymes increased (P < 0.05) digestibility of P, K, Mn, and Cu at 21 days. The digestibility of Cu, Zn, and Mg was higher (P < 0.05) on T2, whereas Ca digestibility was greater (P < 0.05) on the T1 diet. Broilers' growth responded positively to enzyme diets, probably due to improvement in nutrient digestibility.

Comparison Of The Efficiency Of Concentrated Soluble Recombinant Phospholipase D And Natural Phospholipase D Enzymes, Asli Sakmanoğlu, Osman Ergani̇ş

Turkish Journal of Veterinary & Animal Sciences

Phospholipase D (PLD) is a major virulence determinant of Corynebacterium pseudotuberculosis. Various studies have focused on the expression of recombinant PLD (rPLD) enzyme in different Escherichia coli strains, but generally a high yield of insoluble protein was reported. The aims of this study were to express soluble rPLD by different methods in E. coli. The rPLD and natural PLD (dPLD) enzymes were concentrated using an ultramembrane cassette system after the efficiencies of these concentrated enzymes were compared. The rPLD enzyme was expressed in One Shot®BL21(DE3) E. coli when induced by IPTG in TY medium. Soluble dPLD and rPLD enzyme hemolytic …

Characterization Of Cytidylyltransferase Enzyme Activity Through High Performance Liquid Chromatography, James Brault

Theses and Dissertations

The cytidylyltransferases are a family of enzymes that utilize cytidine 5â?? triphosphate (CTP) to synthesize molecules that are precursors to membrane phospholipids. There are four well known enzymes: CTP: phosphoethanolamine cytidylyltransferase (ECT), CTP: glycerol-3-phosphate cytidylyltransferase (GCT), 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), and CTP: phosphocholine cytidylyltransferase (CCT). Previously, a radioisotope tagging method was employed to study cytidylyltransferase catalysis. Using CCT as a model, a method utilizing high performance liquid chromatography (HPLC) was developed to replace the radioisotope scintillation technique. The development of this new HPLC method is cheaper, more efficient, and more accurate than the previously established method.

The targets of separation …

Storage Of Round And Square Switchgrass Bales: Effect Of Storage Time And Fungal Inoculation On Saccharification Efficiency, Noaa Frederick

Graduate Theses and Dissertations

To produce fermentable sugars from lignocellulosic biomass feedstock, severe pretreatment conditions are needed (either high acid concentration, temperature, or retention times). High severities can produce toxic byproducts which inhibit enzymatic hydrolysis or fermentation. In order to reduce pretreatment severities (and thus increase enzyme and fermentation efficiency), the white-rot fungus Pleurotus ostreastus was seeded into square and round bales of Kanlow switchgrass (Panicum virgastum L.) and left in the field over a period of 9 month. The laccase producing fungus is believed to selectively degrade lignin, a common plant structural polymer, which can function as an enzymatic inhibitor. Samples were taken …

Development Of A Novel Enzymatic Pre-Treatment For Lignocellulosic Biomass, Melissa Robins, Jenna Rickus

The Summer Undergraduate Research Fellowship (SURF) Symposium

Biofuels, fuels derived directly from living matter, present a renewable and environmentally friendly alternative to petroleum based fuels. Bioethanol produced from low input energy crops or agricultural waste is a promising fuel source because it does not interfere with the human food supply chain and the ethanol produced can be blended with gasoline. These potential sources of bioethanol are not yet commercially viable due to a polymer called lignin present in the plant’s cell wall which impedes the conversion of cellulose to glucose and the eventual fermentation of glucose to ethanol. Developing new methods for the pretreatment of lignocellulosic biomass …

Enzyme Catalyzed Alginate Nanogels For Drug Delivery, Danna Nichole Sharp

Masters Theses

Developing nanoscale carriers for the delivery of therapeutics is an important topic of investigation in current biomedical research. As opposed to traditional drug delivery systems, nanoscale systems offer enhanced tissue and cell permeation in addition to reducing drug elimination from the body. Biological based therapeutics such as DNA and proteins are now widely employed in medical applications and research has focused on using nanoscale drug delivery systems to administer these more effectively. Current synthesis methods of nanoscale biotherapeutic carriers face significant challenges. Among these are creating carriers with: sizes between 10-200 nm, low polydispersity, and non-cytotoxic materials. In this thesis, …

Expression, Localization, And Kinetic Characterization Of The Phospholipid Biosynthesis Enzyme Ctp: Phosphocholine Cytidylyltransferase From The Protozoan Parasite Leishmania Major, Justin Daniel Theodore Lange

Theses and Dissertations

The eukaryotic parasite Leishmania is the causative agent of the disease leishmaniasis. L. major is the most common of 21 species that causes visceral leishmaniasis in humans, and 30 that cause the same disease in other mammals. Visceral leishmaniasis causes fever, weight loss, and over a short amount of time, multiple organ failure, and has a 100% mortality rate within 2 years. This makes it the second largest parasitic killer in the world behind malaria. Over 90% of the worldâ??s cases of visceral leishmaniasis have been reported in underdeveloped countries of India, Bangladesh, Nepal, Sudan, Ethiopia and Brazil, with 500,000 …