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Bifidobacteria In Feces And Environmental Waters, Regina Lamendella, Jorge W. Santo Domingo, Catherine A. Kelty, Daniel B. Oerther

Civil, Architectural and Environmental Engineering Faculty Research & Creative Works

Bifidobacteria have been recommended as potential indicators of human fecal pollution in surface waters even though very little is known about their presence in nonhuman fecal sources. The objective of this research was to shed light on the occurrence and molecular diversity of this fecal indicator group in different animals and environmental waters. Genus- and species-specific 16S rRNA gene PCR assays were used to study the presence of bifidobacteria among 269 fecal DNA extracts from 32 different animals. Twelve samples from three wastewater treatment plants and 34 water samples from two fecally impacted watersheds were also tested. The species-specific assays …

Fibulin-2 Is Dispensable For Mouse Development And Elastic Fiber Formation, Francois-Xavier Sicot, Takeshi Tsuda, Dessislava Markova, John Klement, Machiko Arita, Rui-Zhu Zhang, Te-Cheng Pan, Robert P. Mecham, David E. Birk, Mon-Li Chu

Department of Dermatology and Cutaneous Biology Faculty Papers

Fibulin-2 is an extracellular matrix protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development. To examine its role in vivo, we generated mice that do not express the fibulin-2 gene (Fbln2) through homologous recombination of embryonic stem cells. Unexpectedly, the …

Arsenic As An Endocrine Disruptor: Arsenic Disrupts Retinoic Acid Receptor–And Thyroid Hormone Receptor–Mediated Gene Regulation And Thyroid Hormone–Mediated Amphibian Tail Metamorphosis, Jennifer C. Davey, Athena P. Nomikos, Manida Wungjiranirun, Jenna R. Sherman, Liam Ingram, Cavus Batki, Jean P. Lariviere, Joshua W. Hamilton

Dartmouth Scholarship

Background:

Chronic exposure to excess arsenic in drinking water has been strongly associated with increased risks of multiple cancers, diabetes, heart disease, and reproductive and developmental problems in humans. We previously demonstrated that As, a potent endocrine disruptor at low, environmentally relevant levels, alters steroid signaling at the level of receptor-mediated gene regulation for all five steroid receptors.

Objectives:

The goal of this study was to determine whether As can also disrupt gene regulation via the retinoic acid (RA) receptor (RAR) and/or the thyroid hormone (TH) receptor (TR) and whether these effects are similar to previously observed effects on steroid …

Abeta42 Mutants With Different Aggregation Profiles Induce Distinct Pathologies In Drosophila., Koichi Iijima, Hsueh-Cheng Chiang, Stephen A Hearn, Inessa Hakker, Anthony Gatt, Christopher Shenton, Linda Granger, Amy Leung, Kanae Iijima-Ando, Yi Zhong

Department of Biochemistry and Molecular Biology Faculty Papers

Aggregation of the amyloid-beta-42 (Abeta42) peptide in the brain parenchyma is a pathological hallmark of Alzheimer's disease (AD), and the prevention of Abeta aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Abeta can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Abeta42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Abeta42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Abeta42Arc) and an artificial mutation (Abeta42art) that …

Trk: A Neuromodulator Of Age-Specific Behavioral And Neurochemical Responses To Cocaine In Mice., Michelle Niculescu, Shane A Perrine, Jonathan S Miller, Michelle E Ehrlich, Ellen M Unterwald

Farber Institute for Neuroscience Faculty Papers

Responses to psychostimulants vary with age, but the molecular etiologies of these differences are largely unknown. The goal of the present research was to identify age-specific behavioral and molecular adaptations to cocaine and to elucidate the mechanisms involved therein. Postweanling, periadolescent, and adult male CD-1 mice were exposed to cocaine (20 mg/kg) for 7 d. The rewarding effects of cocaine were assessed, as were the response to a Trk antagonist and the regulation of dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32). Cocaine was rewarding in both periadolescent and adult mice using a conditioned place preference procedure. In contrast, postweanling mice …

Adenomatous Polyposis Coli Is Present Near The Minimal Level Required For Accurate Graded Responses To The Wingless Morphogen, Hassina Benchabane, Edward G. Hughes, Carter M. Takacs, Jason R. Baird, Yashi Ahmed

Dartmouth Scholarship

The mechanisms by which the Wingless (Wg) morphogen modulates the activity of the transcriptional activator Armadillo (Arm) to elicit precise, concentration-dependent cellular responses remain uncertain. Arm is targeted for proteolysis by the Axin/Adenomatous polyposis coli (Apc1 and Apc2)/Zeste-white 3 destruction complex, and Wg-dependent inactivation of destruction complex activity is crucial to trigger Arm signaling. In the prevailing model for Wg transduction, only Axin levels limit destruction complex activity, whereas Apc is present in vast excess. To test this model, we reduced Apc activity to different degrees, and analyzed the effects on three concentration-dependent responses to Arm signaling that specify distinct …

Diverse Antidepressants Increase Cdp-Diacylglycerol Production And Phosphatidylinositide Resynthesis In Depression-Relevant Regions Of The Rat Brain, Kimberly R. Tyeryar, Habiba O.U. Vongtau, Ashiwel S. Undieh

College of Pharmacy Faculty Papers

BACKGROUND: Major depression is a serious mood disorder affecting millions of adults and children worldwide. While the etiopathology of depression remains obscure, antidepressant medications increase synaptic levels of monoamine neurotransmitters in brain regions associated with the disease. Monoamine transmitters activate multiple signaling cascades some of which have been investigated as potential mediators of depression or antidepressant drug action. However, the diacylglycerol arm of phosphoinositide signaling cascades has not been systematically investigated, even though downstream targets of this cascade have been implicated in depression. With the ultimate goal of uncovering the primary postsynaptic actions that may initiate cellular antidepressive signaling, we …

Repeat Length And Rna Expression Level Are Not Primary Determinants In Cug Expansion Toxicity In Drosophila Models, Gwenn Le Mée, Nader Ezzeddine, Michèle Capri, Ounissa Aït-Ahmed

Faculty, Staff and Student Publications

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity …

Pioglitazone Inhibition Of Lipopolysaccharide-Induced Nitric Oxide Synthase Is Associated With Altered Activity Of P38 Map Kinase And Pi3k/Akt, Bin Xing, Tao Xin, Randy Lee Hunter, Guoying Bing

Neuroscience Faculty Publications

BACKGROUND: Previous studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-gamma)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-gamma agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone. …

Interspecific Hybridization Yields Strategy For South Pacific Filariasis Vector Elimination, Corey L. Brelsfoard, Yves Séchan, Stephen L. Dobson

Entomology Faculty Publications

BACKGROUND: Lymphatic filariasis (LF) is a leading cause of disability in South Pacific regions, where >96% of the 1.7 million population are at risk of LF infection. As part of current global campaign, mass drug administration (MDA) has effectively reduced lymphatic filiariasis prevalence, but mosquito vector biology can complicate the MDA strategy. In some regions, there is evidence that the goal of LF elimination cannot be attained via MDA alone. Obligate vector mosquitoes provide additional targets for breaking the LF transmission cycle, but existing methods are ineffective for controlling the primary vector throughout much of the South Pacific, Aedes polynesiensis. …

Amp-Activated Protein Kinase Regulates Co2-Induced Alveolar Epithelial Dysfunction In Rats And Human Cells By Promoting Na,K-Atpase Endocytosis, István Vadász, Laura A. Dada, Arturo Briva, Humberto E. Trejo, Lynn C. Welch, Jiwang Chen, Peter T. Toth, Emilia Lecuona, Lee A. Witters, Paul T. Schumacker, Navdeep S. Chandel, Werner Seeger, Jacob I. Sznajder

Dartmouth Scholarship

Hypercapnia (elevated CO₂ levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO₂-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO₂ levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO₂-triggered increase in intracellular Ca²⁺ concentration and Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β). Chelating intracellular Ca²⁺ or abrogating CaMKK-β function by gene silencing or …

Repression Of Hla By Rot Is Dependent On Sae In Staphylococcus Aureus, Dongmei Li, Ambrose Cheung

Dartmouth Scholarship

The regulatory locus sae is a two-component system in Staphylococcus aureus that regulates many important virulence factors, including alpha-toxin (encoded by hla) at the transcriptional level. The SarA homologs Rot and SarT were previously shown to be repressors of hla in selected S. aureus backgrounds. To delineate the interaction of rot and sae and the contribution of sarT to hla expression, an assortment of rot and sae isogenic single mutants, a rot sae double mutant, and a rot sae sarT markerless triple mutant were constructed from wild-type strain COL. Using Northern blot analysis and transcriptional reporter gene green fluorescent protein, …

Genome-Wide Analysis Of The Interaction Between The Endosymbiotic Bacterium Wolbachia And Its Drosophila Host, Zhiyong Xi, Laurent Gavotte, Yan Xie, Stephen L. Dobson

Entomology Faculty Publications

BACKGROUND: Intracellular Wolbachia bacteria are obligate, maternally-inherited, endosymbionts found frequently in insects and other invertebrates. The success of Wolbachia can be attributed in part to an ability to alter host reproduction via mechanisms including cytoplasmic incompatibility (CI), parthenogenesis, feminization and male killing. Despite substantial scientific effort, the molecular mechanisms underlying the Wolbachia/host interaction are unknown.

RESULTS: Here, an in vitro Wolbachia infection was generated in the Drosophila S2 cell line, and transcription profiles of infected and uninfected cells were compared by microarray. Differentially-expressed patterns related to reproduction, immune response and heat stress response are observed, including multiple genes that have …

Fak, Pdz-Rhogef And Rockii Cooperate To Regulate Adhesion Movement And Trailing-Edge Retraction In Fibroblasts, M. P. Iwanicki, T. Vomastek, R. W. Tilghman, K. H. Martin, J. Banerjee, P. B. Wedegaertner, J. T. Parsons

Faculty & Staff Scholarship

No abstract provided.

Il-21 Limits Peripheral Lymphocyte Numbers Through T Cell Homeostatic Mechanisms., Shrimati Datta, Nora E. Sarvetnick

Journal Articles: Regenerative Medicine

BACKGROUND: IL-21, a member of the common gamma-chain utilizing family of cytokines, participates in immune and inflammatory processes. In addition, the cytokine has been linked to autoimmunity in humans and rodents.

METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanism whereby IL-21 affects the immune system, we investigated its role in T cell homeostasis and autoimmunity in both non-autoimmune C57BL/6 and autoimmune NOD mice. Our data indicate that IL-21R knockout C57BL/6 and NOD mice show increased size of their lymphocyte population and decreased homeostatic proliferation. In addition, our experimental results demonstrate that IL-21 inhibits T cell survival. These data suggest that IL-21 acts …

Gene Targeting In Adult Rhesus Macaque Fibroblasts, Daniel T. Meehan, Mary Ann Zink, Melissa Mahlen, Marilu Nelson, Warren G. Sanger, Shoukhrat M. Mitalipov, Don P. Wolf, Michel M. Ouellette, Robert B. Norgren

Journal Articles: Genetics, Cell Biology & Anatomy

BACKGROUND: Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT) has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology.

RESULTS: A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow …

Targeting Nf-Kappab: A Promising Molecular Therapy In Inflammatory Arthritis., Jorge A. Roman-Blas, Sergio A. Jimenez

Scleroderma Center Faculty Papers

The nuclear factor-kappa B family of transcription factors is intimately involved in the regulation of the inflammatory responses that play a fundamental role in the damage of articular tissues. Thus, many studies have examined the important contributions of components of the NF-kappaB signaling pathways to the pathogenesis of various rheumatic diseases and their pharmacologic modulation. Currently available therapeutic agents including nonsteroidal anti-inflammatory drugs, corticosteroids, nutraceuticals, and disease-modifying antirheumatic drugs, as well as novel specific small-molecule inhibitors have been employed. In addition, promising nucleic acid-based strategies have shown encouraging results. However, further research will be needed before NF-kappaB-aimed strategies become an …

An Argument For The Basic Legal Rights Of Farmed Animals, Steven M. Wise

Michigan Law Review First Impressions

The most abused beings in the United States are those whom we raise and kill for food. The numbers of dead are staggering. Most are victims of the severe and almost entirely unregulated practices that Americans permit on their factory farms. According to the United States Department of Agriculture’s National Agricultural Statistics Service, in 2007, a total of 10.4 billion land-based animals were killed by the American food industry. These included 9.4 billion broiler chickens, 450 million laying hens, 317 million turkeys, 121 million pigs, 39 million bovines, 28 million ducks, 10 million rabbits, and 4 million sheep and goats—fifty …

Of Persons And The Criminal Law: (Second Tier) Personhood As A Prerequisite For Victimhood, Luis E. Chiesa

Elisabeth Haub School of Law Faculty Publications

The trial of Michael Vick illustrates how our current criminal laws increasingly treat nonhuman creatures as "victims," with all of the consequences that this entails, including the possibility to order that restitution be paid to the animals. In light of these considerations, it is fair to say that from a purely descriptive point of view, nonhuman creatures can qualify for victimhood. This does not mean, however, that this conclusion is normatively appealing. Some have argued that this approach is profoundly misguided, given that the criminal law should only aim to safeguard the rights of humans.

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 …

Collective Behavior Of Stabilized Reaction-Diffusion Waves, Aaron J. Steele, Mark Tinsley, Kenneth Showalter

Faculty & Staff Scholarship

No abstract provided.

Phosphorylation Of Afap-110 Affects Podosome Lifespan In A7r5 Cells, A. Dorfleutner, Y. Cho, D. Vincent, J. Cunnick, H. Lin, S. A. Weed, C. Stehlik, D. C. Flynn

Faculty & Staff Scholarship

No abstract provided.

Eomesodermin, A Target Gene Of Pou4f2, Is Required For Retinal Ganglion Cell And Optic Nerve Development In The Mouse, Chai-An Mao, Takae Kiyama, Ping Pan, Yasuhide Furuta, Anna-Katerina Hadjantonakis, William H Klein

Faculty, Staff and Student Publications

The mechanisms regulating retinal ganglion cell (RGC) development are crucial for retinogenesis and for the establishment of normal vision. However, these mechanisms are only vaguely understood. RGCs are the first neuronal lineage to segregate from pluripotent progenitors in the developing retina. As output neurons, RGCs display developmental features very distinct from those of the other retinal cell types. To better understand RGC development, we have previously constructed a gene regulatory network featuring a hierarchical cascade of transcription factors that ultimately controls the expression of downstream effector genes. This has revealed the existence of a Pou domain transcription factor, Pou4f2, that …

Aging Predisposes Oocytes To Meiotic Nondisjunction When The Cohesin Subunit Smc1 Is Reduced, Vijayalakshmi V. Subramanian, Sharon E. Bickel

Dartmouth Scholarship

In humans, meiotic chromosome segregation errors increase dramatically as women age, but the molecular defects responsible are largely unknown. Cohesion along the arms of meiotic sister chromatids provides an evolutionarily conserved mechanism to keep recombinant chromosomes associated until anaphase I. One attractive hypothesis to explain age- dependent nondisjunction (NDJ) is that loss of cohesion over time causes recombinant homologues to dissociate prematurely and segregate randomly during the first meiotic division. Using Drosophila as a model system, we have tested this hypothesis and observe a significant increase in meiosis I NDJ in experimentally aged Drosophila oocytes when the cohesin protein SMC1 …

Krepa6 Is An Rna-Binding Protein Essential For Editosome Integrity And Survival Of Trypanosoma Brucei, Salvador Zipagun Tarun Jr., Achim Schnauffer, Nancy Lewis Ernst, Rosemary Proff, Junpeng Deng, William Hol, Kenneth Stuart, K.

Biology

Most mitochondrial mRNAs in kinetoplastid protozoa require post-transcriptional RNA editing that inserts and deletes uridylates, a process that is catalyzed by multiprotein editosomes. KREPA6 is the smallest of six editosome proteins that have predicted oligonucleotide-binding (OB) folds. Inactivation of KREPA6 expression results in disruption and ultimate loss of ∼20S editosomes and inhibition of procyclic form cell growth. Gel shift studies show that recombinant KREPA6 binds RNA, but not DNA, with a preference for oligo-(U) whether on the 3′ end of gRNA or as a (UU)12 homopolymer. Thus, KREPA6 is essential for the structural integrity and presence of ∼20S editosomes and …

Transcriptional Regulatory Network Analysis During Epithelial-Mesenchymal Transformation Of Retinal Pigment Epithelium., Craig H Pratt, Rajanikanth Vadigepalli, Praveen Chakravarthula, Gregory E Gonye, Nancy J Philp, Gerald B Grunwald

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

PURPOSE: Phenotypic transformation of retinal pigment epithelial (RPE) cells contributes to the onset and progression of ocular proliferative disorders such as proliferative vitreoretinopathy (PVR). The formation of epiretinal membranes in PVR may involve an epithelial-mesenchymal transformation (EMT) of RPE cells as part of an aberrant wound healing response. While the underlying mechanism remains unclear, this likely involves changes in RPE cell gene expression under the control of specific transcription factors (TFs). Thus, the purpose of the present study was to identify TFs that may play a role in this process.

METHODS: Regulatory regions of genes that are differentially regulated during …

Human Identity: The Question Presented By Human-Animal Hybridization, Jospeh Vining

Other Publications

What makes each of us, as individuals, human to one another, or, more generally, what makes an individual creature human? We have not often had to ask the question because of the species line based on reproductive capacity and incapacity, although "degrees of humanness" were explored in the various eugenic programs of the last century. Now the biotechnological possibility of fusing human and other forms of life is presenting the question in a new and serious way. If the traditional biological means of defining species are no longer reliable, what other criteria might determine what is "human" and what is …

Messenger Rna Half-Life Measurements In Mammalian Cells, Chyi-Ying A Chen, Nader Ezzeddine, Ann-Bin Shyu

Faculty, Staff and Student Publications

The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and "user-friendly" methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic …

Application Of In Vivo Induced Antigen Technology (Iviat) To Bacillus Anthracis, Sean M Rollins, Amanda Peppercorn, John S Young, Melissa Drysdale, Andrea Baresch, Margaret V Bikowski, David A Ashford, Conrad P Quinn, Martin Handfield, Jeffrey D Hillman, C Rick Lyons, Theresa M Koehler, Stephen B Calderwood, Edward T Ryan

Faculty, Staff and Student Publications

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced …

Large Scale Variation In Enterococcus Faecalis Illustrated By The Genome Analysis Of Strain Og1rf, Agathe Bourgogne, Danielle A Garsin, Xiang Qin, Kavindra V Singh, Jouko Sillanpaa, Shailaja Yerrapragada, Yan Ding, Shannon Dugan-Rocha, Christian Buhay, Hua Shen, Guan Chen, Gabrielle Williams, Donna Muzny, Arash Maadani, Kristina A Fox, Jason Gioia, Lei Chen, Yue Shang, Cesar A Arias, Sreedhar R Nallapareddy, Meng Zhao, Vittal P Prakash, Shahreen Chowdhury, Huaiyang Jiang, Richard A Gibbs, Barbara E Murray, Sarah K Highlander, George M Weinstock

Faculty, Staff and Student Publications

BACKGROUND: Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies.

RESULTS: The 2,739,625 base pair chromosome of OG1RF was found to contain approximately 232 kilobases unique to this strain compared to V583, the only publicly available sequenced strain. Almost no mobile genetic elements were found in OG1RF. The 64 areas of divergence were classified into three categories. First, OG1RF carries 39 unique regions, including 2 CRISPR loci and a new WxL locus. Second, we found nine replacements where a sequence …