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Active Use Of Coyotes (Canis Latrans) To Detect Bovine Tuberculosis In Northeastern Michigan, Usa, Are R. Berentsen, Michael R. Dunbar, Shylo R. Johnson, S. Robbe-Austerman, L. Martinez, R. L. Jones

United States Department of Agriculture Wildlife Services: Staff Publications

Bovine tuberculosis (bTB) is endemic in white-tailed deer (Odocoileus virginianus) in northeastern Michigan, USA, and research suggests transmission to cattle. Prevalence of the disease in deer is estimated at 1.8%, but as prevalence decreases the difficulty of detection increases. Research suggests coyotes (Canis latrans) have a higher prevalence of bTB in Michigan than deer and sampling coyotes may be a more efficient surveillance tool to detect presence or spread of the disease. Coyotes possess suitable ecological characteristics to serve as a sentinel species, assuming transmission between coyotes is not significant. The question of whether free-ranging coyotes shed Mycobacterium bovis, the …

White-Tailed Deer Incidents With U.S. Civil Aircraft, Kirsten M. Biondi, Jerrold L. Belant, James A. Martin, Travis L. Devault, Guiming Wang

United States Department of Agriculture Wildlife Services: Staff Publications

Aircraft incidents with ungulates cause substantial economic losses and pose risks to human safety. We analyzed 879 white-tailed deer (Odocoileus virginianus) incidents with United States civil aircraft from 1990 to 2009 reported in the Federal Aviation Administration National Wildlife Strike Database. During that time, deer incidents followed a quadratic response curve, peaking in 1994 and declining thereafter. There appeared to be some seasonal patterning in incident frequency, with deer incidents increasing overall from January to November, and peaking in October and November (30.7%). Most incidents (64.8%) occurred at night, but incident rates were greatest (P 0.001) at dusk. Landing-roll represented …

The Carrot Or The Stick? Evaluation Of Education And Enforcement As Management Tools For Human-Wildlife Conflicts., Sharon Baruch-Mordo, Stewart W. Breck, Kenneth R. Wilson, John Broderick

United States Department of Agriculture Wildlife Services: Staff Publications

Evidence-based decision-making is critical for implementing conservation actions, especially for human-wildlife conflicts, which have been increasing worldwide. Conservation practitioners recognize that long-term solutions should include altering human behaviors, and public education and enforcement of wildlife-related laws are two management actions frequently implemented, but with little empirical evidence evaluating their success. We used a system where human-black bear conflicts were common, to experimentally test the efficacy of education and enforcement in altering human behavior to better secure attractants (garbage) from bears. We conducted 3 experiments in Aspen CO, USA to evaluate: 1) on-site education in communal dwellings and construction sites, 2) …

Acetaminophen And Zinc Phosphide For Lethal Management Of Invasive Lizards Ctenosaura Similis , Michael L. Avery, John D. Eisemann, Kandy L. Keacher, Peter J. Savarie

United States Department of Agriculture Wildlife Services: Staff Publications

Reducing populations of invasive lizards through trapping and shooting is feasible in many cases but effective integrated management relies on a variety of tools, including toxicants. In Florida, using wild-caught non-native black spiny-tailed iguanas Ctenosaura similis, we screened acetaminophen and zinc phosphide to determine their suitability for effective population management of this prolific invasive species. Of the animals that received acetaminophen, none died except at the highest test dose, 240 mg per lizard, which is not practical for field use. Zinc phosphide produced 100% mortality at dose levels as little as 25 mg per lizard, equivalent to about 0.5% in …

Hair Of The Dog: Obtaining Samples From Coyotes And Wolves Noninvasively, David E. Ausband, Julie K. Young, Barbara Fannin, Michael S. Mitchell, Jennifer L. Stenglen, Lizette P. Waits, John A. Shivik

United States Department of Agriculture Wildlife Services: Staff Publications

Canids can be difficult to detect and their populations difficult to monitor. We tested whether hair samples could be collected from coyotes (Canis latrans) in Texas, USA and gray wolves (C. lupus) in Montana, USA using lure to elicit rubbing behavior at both man-made and natural collection devices. We usedmitochondrial and nuclearDNA to determine whether collected hair samples were from coyote, wolf, or nontarget species. Both coyotes and wolves rubbed on man-made barbed surfaces but coyotes in Texas seldom rubbed on hanging barbed surfaces. Wolves in Montana showed a tendency to rub at stations where natural material collection devices (sticks …

Nilgai Antelope In Northern Mexico As A Possible Carrier For Cattle Fever Ticks And Babesia Bovis And Babesia Bigemina., E M. Cardenas-Canales, J. Alfonso Ortega-Santos, Tyler A. Campbell, Zeferino Garcia-Vaquez, Antonio Cantu-Covarrubias, Julio V. Figueroa-Millian, Randy W. Deyoung, David G. Hewitt, Fred C. Bryant

United States Department of Agriculture Wildlife Services: Staff Publications

Of 20 blood samples from nilgais from Me´ xico, five were polymerase chain reaction-positive for Babesia bigemina and one for Babesia bovis. Positive samples had the expected 170 (B. bigemina) and 291 (B. bovis) base pairs and were identical to Gen-Bank B. bigemina accession S45366 and B. bovis M38218.

Diseases And Parasites, Kurt C. Vercauteren, Tyler A. Campbell

United States Department of Agriculture Wildlife Services: Staff Publications

No abstract provided.

Efficacy Of The Boar-Operated-System To Deliver Baits To Feral Swine., Tyler A. Campbell, David B. Long, Giovanna Massei

United States Department of Agriculture Wildlife Services: Staff Publications

Feral swine (Sus scrofa) pose a significant disease threat to livestock and humans. Emerging technologies to reduce feral swine disease transmission risks include fertility control, vaccination, and toxicants. However, for these technologies to be appropriate for field application, a feral swine-specific oral delivery system is needed. We used two field trials to generate information related to appropriate field application of the Boar-Operated-System (BOSTM), an oral delivery system designed to provide bait access only to feral swine. Our objectives were to determine whether pre-baiting BOSTM units increased bait removal and to evaluate the proportion of feral swine and non-target animals that …

Absence Of Mycobacterium Bovis In Feral Swine (Sus Scrofa) From The Southern Texas Border Region., Tyler A. Campbell, David B. Long, Luiz R. Bazan, Bruce V. Thomsen, Suelee Robbe-Austerman, Ronald B. Davey, Liza A. Soliz, Seth Swafford, Kurt C. Vercauteren

United States Department of Agriculture Wildlife Services: Staff Publications

Free-ranging wildlife, such as feral swine (Sus scrofa), harbor a variety of diseases that are transmissible to livestock and could negatively impact agricultural production. Information is needed regarding the exposure and infection rates of Mycobacterium bovis and many other diseases and parasites in feral swine occurring in the Texas border region. Our main objective was to determine exposure rates and possible infection rates of M. bovis in feral swine by opportunistically sampling animals from the Texas border region. From June to September 2010, we obtained samples from 396 feral swine and tested 98 samples for M. bovis by histopathology and …

An Ideal Combination For Marine Turtle Conservation: Exceptional Nesting Season, With Low Nest Predation Resulting From Effective Low-Cost Predator Management, Richard M. Engeman, R. Erik Martin, John Woolard, Margo Stahl, Charles Pelizza, Anthony Duffiney, Bernice Constantin

United States Department of Agriculture Wildlife Services: Staff Publications

We examined impacts from effective predator management on nesting success of marine turtles in an exceptional nesting year at Hobe Sound National Wildlife Refuge, Florida, USA, a beach with a high density of nesting marine turtles that has a history of severe nest predation. Historically up to 95% of nests were predated, primarily by raccoons Procyon lotor and, more recently, armadillos Dasypus novemcinctus. Predator control was identified as the most important conservation tool for marine turtle reproduction. Predator management by refuge staff as ancillary duties typically only held predation levels to c. 50%. However, when experts in predator control …

Volume, Texture, And Molecular Mechanism Behind The Collapse Of Bread Made With Different Levels Of Hard Waxy Wheat Flours, Shivananda K. Garimella Purna, Rebecca A. Miller, Paul A. Seib, Robert A. Graybosch, Yong-Cheng Shi

Department of Agronomy and Horticulture: Faculty Publications

Physico-chemical properties of bread baked by partially replacing normal wheat (Triticum aestivum L.) flour (15, 30, and 45%) with two hard waxy wheat flours were investigated. Substitution with waxy wheat flour resulted in higher loaf volume and softer loaves. However, substitution at >30% resulted in excessive post-bake shrinkage and a ‘key-hole’ shape with an open crumb structure. Bread crumb microstructure indicated a loss of starch granule rigidity and fusing of starch granules. The cells in the interior of the bread did not become gas-continuous and as a result, shrunk as the loaf cooled. Soluble starch content was significantly higher in …

Registration Of ‘Nh03614 Cl’ Wheat, P. Stephen Baenziger, Robert A. Graybosch, Lenis Alton Nelson, Teshome H. Regassa, Robert N. Klein, D. D. Baltensperger, Dipak K. Santra, A. M. H. Ibrahim, W. Berzonsky, J. M. Krall, Lei Xu, Stephen N. Wegulo, M. L. Bernards, Y. Jin, J. Kolmer, J. H. Hatchett, M.-S. Chen, G. Bai

Department of Agronomy and Horticulture: Faculty Publications

‘NH03614 CL’ (Reg. No. CV-1051, PI 653833) hard red winter wheat (Triticum aestivum L.) was developed cooperatively by the Nebraska Agricultural Experiment Station and the USDA-ARS and released in 2008 by the developing institutions and the South Dakota Agricultural Experiment Station and the Wyoming Agricultural Experiment Station. In addition to researchers at the releasing institutions, USDA-ARS researchers at Manhattan, KS and St. Paul, MN participated in the development of NH03614 CL. NH03614 CL was selected from the cross ‘Wesley’ sib//‘Millennium’ sib/‘Above’ sib that was made in the spring of 1997 to develop new herbicide-tolerant cultivars. NH03614 CL was selected …

Hyaluronic Acid And Fibrin Hydrogels With Concentrated Dna/ Pei Polyplexes For Local Gene Delivery, Yuguo Lei, Maha Rahim, Quinn Ng, Tatiana Segura

Department of Chemical and Biomolecular Engineering: Faculty Publications

Local delivery of DNA through a hydrogel scaffold would increase the applicability of gene therapy in tissue regeneration and cancer therapy. However, the delivery of DNA/cationic polymer nanoparticles (polyplexes) using hydrogels is challenging due to the aggregation and inactivation of polyplexes during their incorporation into hydrogel scaffolds. We developed a novel process (termed Caged Nanoparticle Encapsulation or CnE) to load concentrated and unaggregated nonviral gene delivery nanoparticles into various hydrogels. Previously, we showed that PEG hydrogels loaded with DNA/PEI polyplexes through this process were able to deliver genes both in vitro and in vivo. In this study, we found that …

Experimental Validation Of A Fundamental Model For Pcr Efficiency, Tobias M. Louw, Christine S. Booth, Elsje Pienaar, Joel R. Termaat, Scott E. Whitney, Hendrik J. Viljoen

Department of Chemical and Biomolecular Engineering: Faculty Publications

Recently a theoretical analysis of PCR efficiency has been published by Booth et al., (2010). The PCR yield is the product of three efficiencies: (i) the annealing efficiency is the fraction of templates that form binary complexes with primers during annealing, (ii)the polymerase binding efficiency is the fraction of binary complexes that bind to polymerase to form ternary complexes and (iii)the elongation efficiency is the fraction of ternary complexes that extend fully. Yield is controlled by the smallest of the three efficiencies and control could shift from one type of efficiency to another over the course of a PCR experiment. …

Could Metabolic Syndrome, Lipodystrophy, And Aging Be Mesenchymal Stem Cell Exhaustion Syndromes?, Eduardo Mansilla, Vanina Díaz Aquino, Daniel Zambón, Gustavo Horacio Marin, Karina Mártire, Gustavo Roque, Thomas Ichim, Neil H. Riordan, Amit Patel, Flavio Sturla, Gustavo F. Larsen, Rubén Spretz, Luis Núñez, Carlos Soratti, Ricardo Ibar, Michiel Van Leeuwen, José María Tau, Hugo Drago, Alberto Maceira

Department of Chemical and Biomolecular Engineering: Faculty Publications

One of the most important and complex diseases ofmodern society is metabolic syndrome. This syndrome has not been completely understood, and therefore an effective treatment is not available yet. We propose a possible stem cell mechanism involved in the development ofmetabolic syndrome. This way of thinking lets us consider also other significant pathologies that could have similar etiopathogenic pathways, like lipodystrophic syndromes, progeria, and aging. All these clinical situations could be the consequence of a progressive and persistent stemcell exhaustion syndrome (SCES). Themain outcome of this SCES would be an irreversible loss of the effective regenerative mesenchymal stem cells (MSCs) …

Improved Cellular Infiltration Into Nanofibrous Electrospun Crosslinked Gelatin Scaffolds Templated With Micrometer Sized Polyethylene Glycol Fibers, Maciej Skotak, Jorge Ragusa, Daniela Gonzalez, Anuradha Subramanian

Department of Chemical and Biomolecular Engineering: Faculty Publications

Gelatin-based nanofibrous scaffolds with a mean fiber diameter of 300 nm were prepared with and without micrometer-sized polyethylene glycol (PEG) fibers that served as sacrificial templates. Upon fabrication of the scaffolds via electrospinning, the gelatin fibers were crosslinked with glutaraldehyde, and the PEG templates were removed using tert-butanol to yield nanofibrous scaffolds with pore diameters ranging from 10 to 100 μm, as estimated with mercury intrusion porosimetry. Non-templated gelatin-based nanofibrous matrices had an average pore size of 1 μm. Fibroblasts were seeded onto both types of the gelatin-based nanfibrous surfaces and cultured for 14 days. For comparative purposes, chitosan-based and …

Functional Factor Viii Made With Von Willebrand Factor At High Levels In Transgenic Milk, Steven W. Pipe, Hongzhi Miao, Stephen P. Butler, Jennifer Calcaterra, William H. Velander

Department of Chemical and Biomolecular Engineering: Faculty Publications

Background—Current manufacturing methods for recombinant human Factor VIII (rFVIII) within mammalian cell cultures are inefficient which limit the abundance needed for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary gland of mice, rabbits, sheep and pigs. Unfortunately, it is secreted into milk with low specific activity due in part to the labile, heterodimeric structure that results from furin processing of its B domain.

Objectives—To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering.

Methods—Transgenic mice were made with a mammary specific FVIII gene (226/N6) bioengineered …

Induction Of Tolerance By Oral Administration Of Factor Viii And Treatment Of Hemophila, Oral Alpan, Tirumalai Kamala, Polly Matzinger, William Hugold Velander

Department of Chemical and Biomolecular Engineering: Faculty Publications

Disclosed herein is a simple method for the treatment of antigen-deficiency diseases, by orally administering to a Subject a therapeutically effective amount of the deficient anti gen, wherein the antigen is not present in a liposome. In one embodiment, the method increases hemostasis in a subject having hemophilia A or B, by orally administering to the hemophiliac atherapeutically effective amount of the appropriate clotting factor other than in a liposome, Sufficient to induce oral tolerance and Supply exogenous clotting factor to the subject.

Process Development And Cgmp Manufacturing Of A Recombinant Ricin Vaccine: An Effective And Stable Recombinant Ricin A-Chain Vaccine—RveCTm, Michael M. Meagher, Javier G. Seravalli, S. Todd Swanson, Roger G. Ladd, Yogender P. Khasa, Mehmet Inan, Jay C. Harner, Scott K. Johnson, Kevin E. Van Cott, Changhong Lindsey, Robert Wannemacher, Leonard A. Smith

Papers in Biomolecular Engineering

Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A-chain (RTA), which is an N-glycosidase enzyme, and the B-chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.), a recombinant truncated form of RTA (rRTA1-33/ 44-198 protein, herein denoted RVEa^TM) expressed in Escherichia coli using a codon-optimized gene was shown to be non-toxic, stable, and protective against a ricin challenge in mice. Here, we …

The Role Of Transition Metal Homeostasis In Plant Seed Development, Elsbeth L. Walker, Brian M. Waters

Department of Agronomy and Horticulture: Faculty Publications

Copyright © 2011 Elsevier Ltd. Used by permission.

Moving Magnesium In Plant Cells, Brian M. Waters

Department of Agronomy and Horticulture: Faculty Publications

Magnesium (Mg) is among the most abundant mineral elements in plants, yet the knowledge of which genes control its accumulation in specific tissues and organelles lags behind that of many other mineral elements. Only in recent years has identification of important molecular players begun to take shape. In this issue of New Phytologist, Conn et al. (pp. 583–594) shed additional light on two Mg transporters that play important roles in accumulation of Mg in leaf cell vacuoles. Using subcellular-level ion measurements on leaves, gene expression measurements after single-cell sampling, a genetic approach, and clever use of calcium (Ca) and …

Effect Of Flow Gradient, Roi Size And Random Scatterer Movement During Speckle Size Estimation Based Blood Flow Measurement, Tiantian Xu, Greg Bashford

Department of Agricultural and Biological Systems Engineering: Faculty Publications

Conventional blood flow velocity measurement using ultrasound is capable of resolving the axial component (i.e., that aligned with the ultrasound propagation direction) of the blood flow velocity vector. However, these Doppler-based methods are incapable of detecting blood flow in the direction normal to the ultrasound beam. An algorithm which measures the lateral blood flow velocity using speckle size change with scan velocity was developed in our previous studies. This method uses the apparent speckle size change that occurs when scatterers are moving relative to the spatial rate of A-line acquisition. Our previous results showed that the estimation error of this …

Evaluation Of Select Sensors For Real-Time Monitoring Of Escherichia Coli In Water Distribution Systems, Syreeta L. Miles, Ryan G. Sinclair, Mark R. Riley, Ian L. Pepper

Department of Agricultural and Biological Systems Engineering: Faculty Publications

This study evaluated real-time sensing of Escherichia coli as a microbial contaminant in water distribution systems. Most sensors responded to increased E. coli concentrations, showing that select sensors can detect microbial water quality changes and be utilized as part of a contaminant warning system.

Biological Approaches For Addressing The Grand Challenge Of Providing Access To Clean Drinking Water, Mark R. Riley, Charles P. Gerba, Menachem Elimelech

Department of Agricultural and Biological Systems Engineering: Faculty Publications

The U.S. National Academy of Engineering (NAE) recently published a document presenting “Grand Challenges for Engineering”. This list was proposed by leading engineers and scientists from around the world at the request of the U.S. National Science Foundation (NSF). Fourteen topics were selected for these grand challenges, and at least seven can be addressed using the tools and methods of biological engineering. Here we describe how biological engineers can address the challenge of providing access to clean drinking water. This issue must be addressed in part by removing or inactivating microbial and chemical contaminants in order to properly deliver water …

Identification Of Holocarboxylase Synthetase Chromatin Binding Sites In Human Mammary Cell Lines Using The Damid Technology, Dipika Singh, Angela K. Pannier, Janos Zempleni

Department of Agricultural and Biological Systems Engineering: Faculty Publications

Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to Dam for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously …

Flocculation Of Wall-Deficient Cells Of Chlamydomonas Reinhardtii Mutant Cw15 By Calcium And Methanol, Matt Scholz, Takanori Hoshino, Mark R. Riley, Joel Cuello

Department of Agricultural and Biological Systems Engineering: Faculty Publications

Flocculation is a common and inexpensive method for harvesting algae from solution. After nitrogen starvation, it was shown that 83 + 3% of the wall-deficient cells of the cw 15 mutant of Chlamydomonas reinhardtii flocculated from 12 mL samples within 15 min after the addition of 15 mM calcium chloride at pH 8.4. Only 24 2% of the wildtype strain flocculated under these conditions, thus demonstrating how a simple mutation might facilitate process design. The data suggested that algae grown in waters with similar calcium concentrations (e.g. certain wastewaters) might be harvested through simple pH adjustment. It was also discovered …

Genome-Wide Ribosome Profiling Reveals Complex Translational Regulation In Response To Oxidative Stress, Maxim V. Gerashchenko, Alexei V. Lobanov, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Information on unique and coordinated regulation of transcription and translation in response to stress is central to the understanding of cellular homeostasis. Here we used ribosome profiling coupled with next-generation sequencing to examine the interplay between transcription and translation under conditions of hydrogen peroxide treatment in Saccharomyces cerevisiae. Hydrogen peroxide treatment led to a massive and rapid increase in ribosome occupancy of short upstream ORFs, including those with non-AUG translational starts, and of the N-terminal regions of ORFs that preceded the transcriptional response. In addition, this treatment induced the synthesis of N-terminally extended proteins and elevated stop codon read-through …

Systems-Scale Analysis Reveals Pathways Involved In Cellular Response To Methamphetamine, Lijie Sun, Hong-Mei Li, Manfredo J. Seufferheld, Kent R. Walters Jr., Venu M. Margam, Amber Jannasch, Naomi Diaz, Catherine P. Riley, Weilin Sun, Yueh-Feng Li, William M. Muir, Jun Xie, Jing Wu, Fan Zhang, Jake Y. Chen, Eric L. Barker, Jiri Adamec, Barry R. Pittendrigh

Department of Biochemistry: Faculty Publications

Background: Methamphetamine (METH), an abused illicit drug, disrupts many cellular processes, including energy metabolism, spermatogenesis, and maintenance of oxidative status. However, many components of the molecular underpinnings of METH toxicity have yet to be established. Network analyses of integrated proteomic, transcriptomic and metabolomic data are particularly well suited for identifying cellular responses to toxins, such as METH, which might otherwise be obscured by the numerous and dynamic changes that are induced.

Methodology/Results: We used network analyses of proteomic and transcriptomic data to evaluate pathways in Drosophila melanogaster that are affected by acute METH toxicity. METH exposure caused changes in the …

Comparison Of Genomic And Proteomic Data In Recurrent Airway Obstruction Affected Horses Using Ingenuity Pathway Analysis®, Julien Racine, Vinzenz Gerber, Marybeth Miskovic Feutz, C. Paige Riley, Jiri Adamec, June E. Swinburne, Laurent L. Couetil

Department of Biochemistry: Faculty Publications

Background: Recurrent airway obstruction (RAO) is a severe chronic respiratory disease affecting horses worldwide, though mostly in the Northern hemisphere. Environmental as well as genetic factors strongly influence the course and prognosis of the disease. Research has been focused on characterization of immunologic factors contributing to inflammatory responses, on genetic linkage analysis, and, more recently, on proteomic analysis of airway secretions from affected horses. The goal of this study was to investigate the interactions between eight candidate genes previously identified in a genetic linkage study and proteins expressed in bronchoalveolar lavage fluid (BALF) collected from healthy and RAO-affected horses. The …

Development And Optimization Of An Lc-Ms/Ms-Based Method For Simultaneous Quantification Of Vitamin D2, Vitamin D3, 25- Hydroxyvitamin D2 And 25-Hydroxyvitamin D3, Jiri Adamec, Amber Jannasch, Jianjie Huang, Emily Hohman, James C. Fleet, Munro Peacock, Mario G. Ferruzzi, Berdine Martin, Connie M. Weaver

Department of Biochemistry: Faculty Publications

Simultaneous and accurate measurement of vitamin D and 25-hydroxyvitamin D in biological samples is a barrier limiting our ability to define “optimal” vitamin D status. Thus, our goal was to optimize conditions and evaluate an LC-MS method for simultaneous detection and quantification of vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃ in serum. Extraction and separation of vitamin D forms were achieved using acetone liquid–liquid extraction and by a reversed phase C8 column, respectively. Detection was performed on a triple quadrupole tandem mass spectrometer (QQQ-MS/MS) equipped with atmospheric pressure photo ionization source. The …