HATRIC-LRC

The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and anti-tumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and co-localization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG treated gliomas displayed decreased numbers of Interleukin-35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control anti-tumor immunity.

Francis P Roche1, Ilkka Pietilä2, Hiroshi Kaito3, Elisabet O Sjöström1, Nadine Sobotzki4, Oriol Noguer1, Tor Persson Skare1, Magnus Essand1, Bernd Wollscheid5, Michael Welsh2, and Lena Claesson-Welsh1,*

1. 
   
   ¹Department of Immunology, Uppsala University
2. 
   
   ²Department of Medical Cell Biology, Uppsala University
3. 
   
   ³Department of Immunology, Uppsala university
4. 
   
   ⁴Department of Health Sciences, ETH Zurich
5. 
   
   ⁵Department of Health Sciences, ETH Zürich

1. ↵* Corresponding Author:

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.

Lisa Lasswitz¹Naresh Chandra^2,3Niklas Arnberg^2,3Gisa Gerold^1,2,4

¹ Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, 30625 Hannover, Germany

² Department of Clinical Microbiology, Virology, Umeå University, SE-90185 Umeå, Sweden

³ Molecular Infection Medicine Sweden (MIMS), Umeå University, SE-90185 Umea, Sweden

⁴ Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, SE-90185 Umea, Sweden

Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC-based ligand receptor capture (HATRIC-LRC), a chemoproteomic technology that successfully identifies target receptors for orphan ligands on living cells ranging from small molecules to intact viruses. HATRIC-LRC combines a click chemistry-based, protein-centric workflow with a water-soluble catalyst to capture ligand-receptor interactions at physiological pH from as few as 1 million cells. We show HATRIC-LRC utility for general antibody target validation within the native nanoscale organization of the surfaceome, as well as receptor identification for a small molecule ligand. HATRIC-LRC further enables the identification of complex extracellular interactomes, such as the host receptor panel for influenza A virus (IAV), the causative agent of the common flu.

Nadine Sobotzki, Michael A. Schafroth, Alina Rudnicka, Anika Koetemann, Florian Marty, Sandra Goetze, Yohei Yamauchi, Erick M. Carreira and Bernd Wollscheid

Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein–coupled receptor (GPCR) leading to PLCβ activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome.

Zhigang Li, Joseph J. Zeppa, Mark A. Hancock, John K. McCormick, Terence M. Doherty, Geoffrey N. Hendy and Joaquín Madrenas

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

• Effect of cytostatic proline rich polypeptide-1 on tumor suppressors of inflammation pathway signaling in chondrosarcoma
• Epigenetic regulation of embryonic stem cell marker miR302C in human chondrosarcoma as determinant of antiproliferative activity of proline-rich polypeptide 1
• Restoration of desmosomal junction protein expression and inhibition of H3K9‑specific histone demethylase activity by cytostatic proline‑rich polypeptide‑1 leads to suppression of tumorigenic potential in human chondrosarcoma cells
• Analysis of IL6‑protein complexes in chondrosarcoma
• Epigenetic control of cancer by neuropeptides (Review)

Karina Galoian, Silva Abrahamyan, Gor Chailyan, Amir Qureshi, Parthik Patel, Gil Metser, Alexandra Moran, Inesa Sahakyan, Narine Tumasyan, Albert Lee, Tigran Davtyan, Samvel Chailyan and Armen Galoyan

The majority of antibody therapeutics have been isolated from target-led drug discovery, where many years of target research preceded drug program initiation. However, as the search for validated targets becomes more challenging and target space becomes increasingly competitive, alternative strategies, such as phenotypic drug discovery, are gaining favour. This review highlights successful examples of antibody phenotypic screens that have led to clinical drug candidates. We also review the requirements for performing an effective antibody phenotypic screen, including antibody enrichment and target identification strategies. Finally, the future impact of phenotypic drug discovery on antibody drug pipelines will be discussed.

Section editors: Neil O Carragher – Institute of Genetics and Molecular Medicine, Cancer Research UK Edinburgh Centre, University of Edinburgh, Edinburgh, United Kingdom. Jonathan A. Lee – Quantitative Biology, Eli Lilly and Company, Indianapolis, Indiana. Ellen L. Berg – BioMAP Systems Division of DiscoverX, DiscoverX Corporation.

Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and INS-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the>INS receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells.

Conclusion

The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3.

Relevance

The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.

Li Y¹, Ozment T², Wright GL¹, Peterson JM^1,3.

MChemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.

Manish P. Ponda^a and Jan L. Breslow^a,¹

a Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University, New York, NY 10065

Contributed by Jan L. Breslow, September 23, 2016 (sent for review August 1, 2016; reviewed by Myron Cybulsky and Carl F. Nathan)

Target-blind activity-based screening of molecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41’s binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue–NP41-bound nerves, followed by hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits α4 and α2, nidogen 1, and decorin (FDR-adjusted P value < 10⁻⁷) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-α4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve “ghosts” months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.

Heather L. Glasgow

^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Michael A. Whitney

^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Larry A. Gross

^bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;

Beth Friedman

^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Stephen R. Adams

^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

Jessica L. Crisp

^bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;

Timon Hussain

^cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;

Andreas P. Frei

^dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;

Karel Novy

^dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;

Bernd Wollscheid

^dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland;

Quyen T. Nguyen

^cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093;

Roger Y. Tsien

^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093;

^bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093;

^eDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093

C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects throughout the body. Our initial work shows promise in the development of CTRP3-induced cellular processes as a means to combat nonalcoholic fatty liver disease (NAFLD). Clinically, NAFLD is defined as the excessive accumulation of fat in the liver, usually due to obesity, and NAFLD effects 1 in 10 Americans. Our previous data show that when high fat fed mice are treated with CTRP3 they are protected from developing NAFLD. However, the mechanism for this effect remains unclear. The purpose of this project was to identify the unknown receptor that mediates the hepatic actions of CTRP3.

Jonathan M Peterson, Health Sciences, East Tennessee State University, Johnson City, TN

This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in INS-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.

Andres Garelli, Fabiana Heredia, Andreia P. Casimiro, Andre Macedo, Catarina Nunes, Marcia Garcez, Angela R. Mantas Dias, Yanel A. Volonte, Thomas Uhlmann, Esther Caparros, Takashi Koyama & Alisson M. Gontijo

The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

Damaris Bausch-Fluck

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland

Andreas Hofmann

Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Thomas Bock

Current Address: European Molecular Biology Laboratory, Heidelberg, Germany

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Andreas P. Frei

Current Address: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, United States of America

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Ferdinando Cerciello

Current Address: James Thoracic Center, James Cancer Center, The Ohio State University Medical Center, Columbus, Ohio, United States of America

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Laboratory of Molecular Oncology, University Hospital Zurich, Zurich, Switzerland

Andrea Jacobs

Current Address: Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Hansjoerg Moest

Current Address: Novartis Institute of Biomedical Research, Novartis, Basel, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Ulrich Omasits

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland

Rebekah L. Gundry

Affiliation Department of Biochemistry, Medical College of Wisconsin, Wisconsin, Milwaukee, United States of America

Charles Yoon

Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada

Ralph Schiess

Current Address: ProteoMediX AG, Schlieren, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Alexander Schmidt

Current Address: Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland

Affiliation Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

Paulina Mirkowska

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland

Anetta Härtlová

Current Address: College of Life Sciences, University of Dundee, Dundee, United Kingdom

Affiliation Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic

Jennifer E. Van Eyk

Current Address: Cedars-Sinai, Clinical Biosystem Research Institute, Los Angeles, California, United States of America

Affiliation Department of Medicine, Biological Chemistry and Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America

Jean-Pierre Bourquin

Affiliation Oncology Research Laboratory, University Children Hospital Zurich, Zurich, Switzerland

Ruedi Aebersold

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland, Faculty of Science, University of Zurich, Zurich, Switzerland

Kenneth R. Boheler

Affiliations SCRMC, LKS Faculty of Medicine, Hong Kong University, Hong Kong, Hong Kong SAR, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America

Peter Zandstra

Affiliation Institute for Biomaterials & Biomedical Engineering, University of Toronto, Toronto, Canada

Bernd Wollscheid

* E-mail: wbernd@ethz.ch

Affiliations Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland, Department of Health Sciences and Technology, BMPP, ETH Zurich, Zurich, Switzerland

Competing Interests

The authors have declared that no competing interests exist.

Author Contributions

Conceived and designed the experiments: DBF BW KRB PZ RA. Performed the experiments: DBF AH TB APF FC AJ HM RLG CY RS AS PM AH. Analyzed the data: DBF UO. Contributed reagents/materials/analysis tools: AS APF FC TB BW. Wrote the paper: DBF BW. Supervised experiments: JVE JPB.

Omasits U, Ahrens CH, Müller S, Wollscheid B. Bioinformatics. 2013 Nov 21.

Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS—one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., INS) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.

Andreas P Frei, Hansjoerg Moest, Karel Novy & Bernd Wollscheid

Identifying ligands for receptors in live cells can provide valuable information, providing insight into signaling pathways as well as drug targets, but these interactions can be difficult to detect and quantify. Frei et al. now report a trifunctional chemoproteomic reagent called TRICEPS that binds glycosylated receptors on live cells and allows for the purification of the ligand-receptor complex and identification of receptor-derived peptides by MS. TRICEPS contains three functional groups: an N-hydroxysuccinimide ester for ligand conjugation, a protein..

Amy Donner