Digital Commons Network^™

A Versatile Assay For The Identification Of Rna Silencing Suppressors Based On Complementation Of Viral Movement, Jason G. Powers, Tim L. Sit, Feng Qu, T. Jack Morris, Kook-Hyung Kim, Steven A. Lommel

Nebraska Center for Virology: Faculty Publications

The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVΔ92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of …

Hiv-1 Tat And Aids-Associated Cancer: Targeting The Cellular Anti-Cancer Barrier?, Giuseppe Nunnari, Johanna A Smith, René Daniel

Department of Medicine Faculty Papers

The acquired immunodeficiency syndrome (AIDS) is accompanied by a significant increase in the incidence of neoplasms. Several causative agents have been proposed for this phenomenon. These include immunodeficiency and oncogenic DNA viruses and the HIV-1 protein Tat. Cancer in general is closely linked to genomic instability and DNA repair mechanisms. The latter maintains genomic stability and serves as a cellular anti-cancer barrier. Defects in DNA repair pathway are associated with carcinogenesis. This review focuses on newly discovered connections of the HIV-1 protein Tat, as well as cellular co-factors of Tat, to double-strand break DNA repair. We propose that the Tat-induced …

Upa Is Upregulated By High Dose Celecoxib In Women At Increased Risk Of Developing Breast Cancer., Wenyi Qin, Weizhu Zhu, John E Hewett, George Rottinghaus, Yin-Chieh Chen, John T Flynn, Beth Kliethermes, Ferdinando Mannello, Edward R Sauter

Department of Physiology Faculty Papers

BACKGROUND: While increased urokinase-type plasminogen activator (uPA) expression in breast cancer tissue is directly associated with poor prognosis, recent evidence suggests that uPA overexpression may suppress tumor growth and prolong survival. Celecoxib has been shown to have antiangiogenic and antiproliferative properties. We sought to determine if uPA, PA inhibitor (PAI)-1 and prostaglandin (PG)E2 expression in nipple aspirate fluid (NAF) and uPA and PGE2 expression in plasma were altered by celecoxib dose and concentration in women at increased breast cancer risk.

METHODS: NAF and plasma samples were collected in women at increased breast cancer risk before and 2 weeks after taking …

Inhibition Of Oncogene-Induced Inflammatory Chemokines Using A Farnesyltransferase Inhibitor., Katharine C Degeorge, Brent R Degeorge, James S Testa, Jay L Rothstein

Department of Microbiology and Immunology Faculty Papers

ABSTRACT: BACKGROUND: Farnesyltransferase inhibitors (FTI) are small molecule agents originally formulated to inhibit the oncogenic functions of Ras. Although subsequent analysis of FTI activity revealed wider effects on other pathways, the drug has been demonstrated to reduce Ras signaling by direct measurements. The purpose of the current study was to determine if FTI could be used to inhibit the inflammatory activities of a known Ras-activating human oncoprotein, RET/PTC3. RET/PTC3 is a fusion oncoprotein expressed in the thyroid epithelium of patients afflicted with thyroid autoimmune disease and/or differentiated thyroid carcinoma. Previous studies have demonstrated that RET/PTC3 signals through Ras and can …

Evaluation Of Serological Tests For The Diagnosis Of Visceral Leishmaniasis, Selçuk Kiliç, Ayşegül Taylan Özkan, Cahi̇t Babür, Gönül Tanir, Henk Dfh Schallig

Turkish Journal of Medical Sciences

Aim: Visceral leishmaniasis (VL) is a zoonosis that affects both animals and man. VL is seen sporadically in the Aegean, Mediterranean, Black Sea and central Anatolian regions of Turkey. The aim of this study was to evaluate serological tests for the diagnosis of VL. Materials and Methods: The performance of two agglutination tests based on freeze-dried whole promastigote Leishmania antigen, i.e. Fast Agglutination Screening Test (FAST) and Direct Agglutination Test (DAT), an Indirect Immunofluorescent Antibody Test (IFAT), and an Enzyme-Linked Immunosorbent Assay (ELISA) using soluble antigen were compared to parasitological diagnosis in the serum specimens of 59 patients with the …

Cisplatin Causes Oxidation In Rat Liver Tissues: Possible Protective Effects Of Antioxidant Food Supplementation, Aslihan Avci, Recep Çeti̇n, İmge B. Ergüder, Erdi̇nç Devri̇m, Bülent Kiliçoğlu, Özden Çandir, H. Serdar Öztürk, İlker Durak

Turkish Journal of Medical Sciences

Aims: In this study, we aimed to investigate the possible molecular mechanism of cisplatin hepatotoxicity and to establish whether some natural antioxidant foods, namely dried black grape and tomato, may provide protection against cisplatin hepatotoxicity. Materials and Methods: Twenty-eight rats were used throughout the study. Cisplatin was administered intraperitoneally (i.p.) in a single dose (10 mg/kg). Antioxidant food supplementation was started three days before cisplatin treatment. There were 7 animals in each group (control, cisplatin, cisplatin plus dried black grape and cisplatin plus tomato juice). Rats were sacrificed 72 h after the treatment. The livers were removed and prepared for …

Stressor- And Corticotropin Releasing Factor-Induced Reinstatement And Active Stress-Related Behavioral Responses Are Augmented Following Long-Access Cocaine Self-Administration By Rats, John R. Mantsch, David A. Baker, David M. Francis, Eric S. Katz, Michael A. Hoks, Joseph P. Serge

Biomedical Sciences Faculty Research and Publications

Rationale Stressful events during periods of drug abstinence likely contribute to relapse in cocaine-dependent individuals. Excessive cocaine use may increase susceptibility to stressor-induced relapse through alterations in brain corticotropin-releasing factor (CRF) responsiveness.

Objectives This study examined stressor- and CRF-induced cocaine seeking and other stress-related behaviors in rats with different histories of cocaine self-administration (SA).

Materials and methods Rats self-administered cocaine under short-access (ShA; 2 h daily) or long-access (LgA; 6 h daily) conditions for 14 days or were provided access to saline and were tested for reinstatement by a stressor (electric footshock), cocaine or an icv injection of CRF and …

A Stable Explant Culture Of Her2/Neu Invasive Carcinoma Supported By Alpha-Smooth Muscle Actin Expressing Stromal Cells To Evaluate Therapeutic Agents, Marie P. Piechocki

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

To gain a better understanding of the effects of therapeutic agents on the tumor microenvironment in invasive cancers, we developed a co-culture model from an invasive lobular carcinoma. Tumor cells expressing HER2/neu organize in nests surrounded by alpha-Smooth Muscle Actin (α-SMA) expressing tumor stroma to resemble the morphology of an invading tumor. This co-culture, Mammary Adenocarcinoma Model (MAM-1) maintains a 1:1 ratio of HER2/neu positive tumor cells to α-SMA-reactive stromal cells and renews this configuration for over 20 passages in vitro.

Methods

We characterized the cellular elements of the MAM-1 model by microarray analysis, and immunocytochemistry. We developed …

Autoimmune-Induced Preferential Depletion Of Myelin-Associated Glycoprotein (Mag) Is Genetically Regulated In Relapsing Eae (B6 × Sjl) F1 Mice, Dusanka S. Skundric, Rujuan Dai, Vaagn L. Zakarian, Weili Zhou

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

Experimental autoimmune encephalomyelitis (EAE) is commonly used to investigate mechanisms of autoimmune-mediated damage to oligodendrocytes, myelin, and axons in multiple sclerosis (MS). Four distinct autoimmune mechanisms with subsequently distinct patterns of demyelination have been recognized in acute MS lesions. EAE correlates for those distinct patterns of MS lesions are unknown. An excessive loss of myelin-associated glycoprotein (MAG), as a result of distal oligodendrogliopathy, is found exclusively in the subtype III lesion. We sought to answer if types of demyelination in acute lesions during onset and relapse of EAE can replicate the specific patterns observed in MS acute lesions. …

Reduced Acute Inflammatory Responses To Microgel Conformal Coatings, Amanda W. Bridges, Neetu Singh, Kellie L. Burns, Julia E. Babensee, L. Andrew Lyon, Andrés J. García

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Implantation of synthetic materials into the body elicits inflammatory host responses that limit medical device integration and biological performance. This inflammatory cascade involves protein adsorption, leukocyte recruitment and activation, cytokine release, and fibrous encapsulation of the implant. We present a coating strategy based on thin films of poly(N-isopropylacrylamide) hydrogel microparticles (i.e. microgels) cross-linked with poly(ethylene glycol) diacrylate. These particles were grafted onto a clinically relevant polymeric material to generate conformal coatings that significantly reduced in vitro fibrinogen adsorption and primary human monocyte/macrophage adhesion and spreading. These microgel coatings also reduced leukocyte adhesion and expression of pro-inflammatory cytokines (TNF-alpha, IL-1 beta, …

Hiv-1 Tat Protein Alter The Tight Junction Integrity And Function Of Retinal Pigment Epithelium: An In Vitro Study., Ling Bai, Zhenping Zhang, Hui Zhang, Xiumei Li, Qiurong Yu, Haotian Lin, Wenhui Yang

Department of Medicine Faculty Papers

BACKGROUND: How HIV-1 enter into the eyes remains obscure. We postulated that HIV-1 Tat protein can alter the expression of specific tight-junction proteins and disturb the blood retinal barrier, and contributes to HIV trafficking into the eyes. This study is to determine the effects of HIV-1 Tat proteins on the barrier function and tight-junction protein expression of retinal pigment epithelial cell (RPE). METHODS: A human RPE cell line (D407) cultured on microporous filter-supports was used. After treating with HIV-1 Tat protein, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of the RPE cells …

The Notch Signaling Pathway Drives Astrogliogenesis By Regulating The Expression Of The Transcription Factors Nfia And Sox9, Derek Janssens, Merritt Taylor

Student Summer Scholars Manuscripts

In the developing nervous system the Notch signaling pathway has recently been shown to be necessary and sufficient for neural stem cells to differentiate into astroctyes, a process called astrogliogenesis. However, the mechanism of how Notch signaling regulates this process has not yet been characterized. Our lab is examining whether Notch signaling controls the expression of the genes NFIA and Sox9 which are known to regulate astogliogenesis. These candidate genes were selected because their DNA sequences have characteristics that indicate their expression could be controlled by Notch through a transcriptional complex of Notch and other proteins, including the DNA binding …

Micrornas (Mirnas) In Neurodegenerative Diseases, Peter T. Nelson, Wang-Xia Wang, Bernard W. Rajeev

Sanders-Brown Center on Aging Faculty Publications

Aging-related neurodegenerative diseases (NDs) are the culmination of many different genetic and environmental influences. Prior studies have shown that RNAs are pathologically altered during the inexorable course of some NDs. Recent evidence suggests that microRNAs (miRNAs) may be a contributing factor in neurodegeneration. miRNAs are brain-enriched, small (~22 nucleotides) non-coding RNAs that participate in mRNA translational regulation. Although discovered in the framework of worm development, miRNAs are now appreciated to play a dynamic role in many mammalian brain-related biochemical pathways, including neuroplasticity and stress responses. Research about miRNAs in the context of neurodegeneration is accumulating rapidly, and the goal of …

Sc Inbre: A Catalyst For Biomedical Research Development In South Carolina, Lucia A. Pirisi-Creek M.D, Scott Little, Ph.D.

Journal of the South Carolina Academy of Science

No abstract provided.

The Relationship Between Carnitine Levels And Lipid Peroxidation In Glial Brain Tumors, Ni̇lüfer Bayraktar, Hati̇ce Paşaoğlu, Aydin Paşaoğlu, Memduh Kaymaz, Gürsel Bi̇beroğlu, Sevsen Kulaksizoğlu

Turkish Journal of Medical Sciences

Aim: It is assumed that reactive oxygen species and lipid peroxidation cause cell membrane damage and play a role in oncogenesis. Carnitine and acyl esters, which play a role in intracellular short-, medium-, and long-chain fatty acid metabolism, are one of the defense mechanisms against free radical toxicity. It is thought that the protective effects of carnitine are related with its role in lipid metabolism. In our study, we analyzed the relation between malondialdehyde (MDA) and free carnitine, and C2, C3, C4, C5, C6, C8, C10, C12, C14:1, C14, C16:1, C16, C18:1, C18, C20:4 carnitine levels in glial tumors [glioblastoma …

Acute Intoxication By Endosulfan: Any Role Of N-Acetylcysteine Therapy?, Ahmet Demi̇rcan, Eli̇f Çalidağ, Murat Özsaraç, Ayfer Keleş, Fi̇kret Bi̇ldi̇k

Turkish Journal of Medical Sciences

Reactive oxygen species may play a role in the mediation of acetaminophen (APAP) hepatotoxicity. N-acetylcysteine (NAC) is the standard therapy for treatment of the APAP overdose patient. The suspected mechanism of hepatic injury appears to be similar to that of APAP and the empiric use of NAC may be justified, especially in significant endosulfan poisoning. An 18-year-old male presented to the emergency department because of unconsciousness and seizure that developed after acute intentional ingestion of endosulfan. Severe diaphoresis was noted and he had a Glasgow Coma Score of 7 (E1M4V2). Within 8 hours of his arrival in the emergency department, …

A Novel Method For The Production Of Potentially Therapeutic Human Monoclonal Antibodies In Vitro, Adria L. Bykowski

MUSC Theses and Dissertations

We have developed a novel approach for the production of potentially therapeutic human monoclonal antibodies in vitro. This method holds great promise for generating therapeutic antibodies against infectious disease, cancer, and inflammation. The complex nature of current methods for producing human monoclonal antibodies is a major impediment to large scale passive transfer immunotherapy. Our novel technique utilizes Epstein-Barr virus immortalized tonsil or peripheral blood B cells from healthy human volunteers. These B cells are forced to undergo non-specific differentiation to become mature IgG antibody producing plasma "like" cells that continuously proliferate in vitro. Using this method, millions of transformed B …

Sr-Bi Protects Against Endotoxemia In Mice Through Its Roles In Glucocorticoid Production And Hepatic Clearance, Lei Cai, Ailing Ji, Frederick C. De Beer, Lisa R. Tannock, Deneys R. Van Der Westhuyzen

Internal Medicine Faculty Publications

Septic shock results from an uncontrolled inflammatory response, mediated primarily by LPS. Cholesterol transport plays an important role in the host response to LPS, as LPS is neutralized by lipoproteins and adrenal cholesterol uptake is required for antiinflammatory glucocorticoid synthesis. In this study, we show that scavenger receptor B-I (SR-BI), an HDL receptor that mediates HDL cholesterol ester uptake into cells, is required for the normal antiinflammatory response to LPS-induced endotoxic shock. Despite elevated plasma HDL levels, SR-BI–null mice displayed an uncontrollable inflammatory cytokine response and a markedly higher lethality rate than control mice in response to LPS. In addition, …

Analysis Of Hiv-1 Variable Loop 3-Specific Neutralizing Antibody Responses By Hiv-2/Hiv-1 Envelope Chimeras, Katie L. Davis

All ETDs from UAB

Defining the epitope specificities of the neutralizing antibody response to the HIV-1 envelope protein (Env) during natural human infection and in response to vaccination by HIV-1 immunogens could provide insight into the mechanisms of virus immune containment and facilitate vaccine design. Transplantation of discrete HIV-1 neutralizing epitopes into HIV-2 Env scaffolds may provide a sensitive, biologically functional context in which to quantify specific antibody reactivities in complex sera. Here we describe the development of a novel HIV-2 proviral scaffold (pHIV-2KR.X7) into which we have substituted the env V3 region of several laboratory and primary HIV-1 strains to yield a panel …

The Developmental Functions Of Bdnf And Mecp2 On Dendritic And Synaptic Structure, Christopher A. Chapleau

All ETDs from UAB

Mutations in the transcriptional repressor MeCP2 cause Rett Syndrome (RTT), a mental retardation disease associated with reduced dendritic architecture of cortical pyramidal neurons, in addition to a decrease in the number of dendritic spines. I present the first quantification analysis of spine density in postmortem human brain tissue, demonstrating that hippocampal CA1 pyramidal neurons have reduced spine densities in RTT patients throughout their lives. One gene that MeCP2 regulates is BDNF, an important developmental factor that modulates synaptic transmission and plasticity, as well as spine formation and maturation. In immature cultured hippocampal neurons, cell-autonomous expression of BDNF accelerates the differentiation …

Calmodulin Binding To Cellular Flice Like Inhibitory Protein Modulates Fas-Induced Signaling And Tumorigenesis In Cholangiocarcinoma, Pritish Subhash Pawar

All ETDs from UAB

Cholangiocarcinoma, a fatal tumor arising from biliary epithelium, has very poor 5-year survival rate due to lack of early diagnosis and effective therapies. Induction of the Fas-mediated apoptosis is a promising therapeutic target in this tumor. Studies from our group and others have indicated that Fas-expression correlates inversely with disease progression and that CaM-antagonists induce apoptosis in cholangiocarcinoma cells in a Fas related manner. Further, we reported a direct and dynamic interaction of CaM and Fas and that CaM is recruited into the Fas-stimulated death inducing signaling complex (DISC), suggesting a cross talk between Fas and CaM pathways. The studies …

Reciprocal Regulation Of Par-4 And Caspase-8 In The Trail Signaling Pathway, Padhma Ranganathan

University of Kentucky Doctoral Dissertations

Par‐4 is a pro‐apoptotic tumor suppressor that is mutated, suppressed or inactivated in cancer. Par‐4 exploits components of the extrinsic pathway to cause apoptosis selectively of cancer cells. This study identified Par‐4 as an essential component of the apoptotic pathway induced by TRAIL, which selectively targets cancer cells. RNA interference‐mediated knockdown of Par‐4 rendered cancer cells unresponsive to TRAIL‐induced apoptosis. Cells with knocked‐down levels of Par‐4 were deficient in the activation of the apoptosis‐initiator caspase‐8 and the apoptosis‐effector caspase‐3 in response to TRAIL. Par‐4 was identified as a critical mediator of membrane translocation of caspase‐8 and the adapter protein FADD. …

Role Of Variant Sialylation In Regulating Tumor Cell Behavior, Faheem M. Shaikh

All ETDs from UAB

Many different tumors have been documented to have elevated levels of the enzyme ST6Gal I, a Golgi glycosyltransferase that adds α2-6 sialic acids to glycoproteins. Concurrently, upregulated ST6Gal I is associated with metastasis and poor patient prognosis. We initially showed that HD3 colonocytes made to express oncogenic-ras, a common mutation in colon carcinoma, have increased expression of ST6Gal I and elevated α2-6 sialylated integrins. Having established that ras regulates ST6Gal I expression in colon epithelial cells, we found that colonocytes carrying hyper α2-6 sialylated β1 integrins exhibit increased adhesion to and migration toward collagen I. Further, we found that β1 …

Antioxidant Effect Of Melatonin In Systemic Circulation Of Rats After Unilateral Testicular Torsion, Fi̇kret Erdemi̇r, Beki̇r Suha Parlaktaş, Hüseyi̇n Özyurt, Özgür Boztepe, Ömer Atiş, Semsetti̇n Şahi̇n

Turkish Journal of Medical Sciences

Aim: To investigate the antioxidant effects of melatonin in the systemic circulation of rats after unilateral testicular torsion. Materials and Methods: After the approval of the study by the Local Ethical Committee, 30 male Wistar albino rats, 5.5-6 months old and weighing 250-300 g, were used in the study. The rats were randomly divided into three groups, each consisting of 10 rats. In group 1, the control group, the left testis was excised after sham operation. In group 2, the torsion group, torsion was created by rotating the left testis 720° in clockwise direction and maintained for 2 h. In …

Il-10 And Il-13 Production By Peripheral Blood Mononuclear Cells In Patients With Diabetes Mellitus, Ali̇ İnal, Uğur Muşabak, Göksal Keski̇n, Levent Özsari, Ali̇ Şengül, Mustafa Kutlu, Çağatay Öktenli̇, Zeki̇ Yeşi̇lova

Turkish Journal of Medical Sciences

Aim: It is generally accepted that proinflammatory cytokines secreted by mononuclear cells are responsible for pancreatic beta (B) cell destruction in animal models of autoimmune type-1 diabetes mellitus (DM). Several studies have shown that markers of inflammation and pro-inflammatory cytokines such as interleukin (IL)-10 and IL-13 associate with the metabolic syndrome, dyslipidemia, type-2 DM and type-1 DM. The aim of this study was to examine the in vitro production of IL-10 and IL-13 in cultures of peripheral lymphocytes obtained from patients with type-2 DM, type-1 DM and healthy controls. Materials and Methods: Twenty patients (10 females, 10 males) with type-2 …

Neural Tube Defects And 19 Bp Deletion Within Intron-1 Of Dihydrofolate Reductase Gene, Nejat Akar, Ece Akar, Yonca Eği̇n, Gülhi̇s Deda, Saadet Arsan, Mesi̇ha Eki̇m

Turkish Journal of Medical Sciences

Aims: Dihydrofolate reductase (DHFR) is necessary for the reduction of the ingested folates before they are used in the body metabolism. Thus, the DHFR enzyme has an important role in folate supplementation and the DHFR gene is a strong candidate for a teratogenic locus for neural tube defects (NTDs). There is a 19 bp deletion within the first intron of the DHFR gene, which may have an effect on folate reduction. We thus studied this mutation in Turkish spina bifida patients and their mothers to determine whether there is an association with the occurrence of NTD. Materials and Methods: The …

Gene Therapy Using Tet-Repressor System To Modulate Prostate Tumor Microenvironment, Nazita Yousefieh

Theses and Dissertations in Biomedical Sciences

Prostate cancer is the most commonly diagnosed malignancy in men in the United States and is projected to be the third most frequent cause of male cancer-related deaths in 2007 after lung and skin cancers. The initial treatment for prostate cancer at early stages is prostatectomy or radiation, which usually is curative. However, approximately 20% of patients are not cured by such treatments and their cancer recurs, sometimes with long latencies. In other patients prostate cancer is diagnosed only after the cancer has metastasized and there are no effective therapies at this stage. Therefore immunotherapy seems to be a promising …

Role Of The Reactive Oxygen Species Peroxynitrite In Traumatic Brain Injury, Ying Deng

University of Kentucky Doctoral Dissertations

Reactive oxygen species (ROS) is cytotoxic to the cell and is known to contribute to secondary cell death following primary traumatic brain injury (TBI). We described in our study that PN is the main mediator for both lipid peroxidation and protein nitration, and occurred almost immediately after injury. As a downstream factor to oxidative damage, the peak of Ca2+-dependent, calpainmediated cytoskeletal proteolysis preceded that of neurodegeneration, suggesting that calpain-mediated proteolysis is the common pathway leading to neuronal cell death. The time course study clearly elucidated the interrelationship of these cellular changes following TBI, provided window of opportunity for pharmacological intervention. …

United States Regulation Of Stem Cell Research: Recasting Government's Role And Questions To Be Resolved, Owen C. B. Hughes, Alan L. Jakimo, Michael J. Malinowski

Journal Articles

This article directly addresses the stem cell controversy, but also the broader history and norms regarding the roles of federal and state government in U.S. science research funding.

The Human Immunodeficiency Virus Type 1 Envelope Confers Higher Rates Of Replicative Fitness To Perinatally Transmitted Viruses Than To Nontransmitted Viruses, Xiaohong Kong, John T. West, Hong Zhang, Danielle M. Shea, Tendai J. M’Soka, Charles Wood

Nebraska Center for Virology: Faculty Publications

Selection of a minor viral genotype during perinatal transmission of human Immunodeficiency virus type 1

(HIV-1) has been observed, but there is a lack of information on the correlation of the restrictive transmission

with biological properties of the virus, such as replicative fitness. Recombinant viruses expressing the enhanced

green fluorescent protein or the Discosoma sp. red fluorescent (DsRed2) protein carrying the V1 to V5

regions of env from seven mother-infant pairs (MIPs) infected by subtype C HIV-1 were constructed, and

competition assays were carried out to compare the fitness between the transmitted and nontransmitted

viruses. Flow cytometry was used to …