Postdoctoral esea - Denver, Colorado · Postdoctoral fellows are an integral part of the University community, contributing both to our fields of study and to the overall collaborative

Postdoctoral Research Day 2016

The PDRD Committee 2016

Carol Kiekhaefer, Ph.D.; PDRD Chair

Erica Alexeev, Ph.D.

Charmion Cruickshank-Quinn, Ph.D.

Brianna Klein Ph.D.

Sarala Pradhan Ph.D.

Kevin Quinn, Ph.D.

Teisha Rowland, Ph.D.


Table of Contents

Page Number Information

2 Keynote Speaker Information

3 Welcome Message from the Postdoc President

4 Event Contributors

5 Meet the Postdoc Director and the Graduate School Dean

6 Upcoming Postdoctoral Events

7 SciPhD Workshop Information

8 - 9 Oral Presentation Schedule

10 - 11 Poster Presentation Schedule

12 - 21 Oral Presentation Abstracts (alphabetical)

22 - 40 Poster Presentation Abstracts (alphabetical)

Back Cover of Booklet Schedule of the Day’s Events


KEYNOTE SPEAKER

Joseph A. Schwarcz, PhD

Joseph Schwarcz is Director of McGill University’s “Office for Science and Society.” He is well known for his informative and entertaining public lectures on topics ranging from the chemistry of love to the science of aging. Professor Schwarcz has received numerous awards for teaching chemistry and for interpreting science for the public and is the only non-American ever to win the American Chemical Society’s prestigious Grady-Stack Award for demystifying chemistry. He hosts "The Dr. Joe Show" on Montreal's CJAD and has appeared hundreds of times on The Discovery Channel, CTV, CBC, TV Ontario and Global Television. Dr. Schwarcz also writes a newspaper column entitled “The Right Chemistry” and has authored a number of books, “Radar, Hula Hoops and Playful Pigs,” “The Genie in the Bottle,” "That's The Way The Cookie Crumbles," “Dr. Joe And What You Didn’t Know,” “The Fly In The Ointment” “Let Them Eat Flax” “An Apple A Day,” “Brain Fuel,” “Science, Sense and Nonsense,” “Dr. Joe’s Brain Sparks,” “Dr. Joe’s Health Lab, ” “The Right Chemistry,” “Is That a Fact and his latest, released in May is “Monkeys, Myths and Molecules.” He is also an amateur conjurer and often spices up his presentations with a little magic. Dr. Schwarcz was awarded the 2010 “Montreal Medal” which is the Canadian Chemical Institute’s premier prize recognizing lifetime contributions to chemistry in Canada. In 2015 he was named winner of the Balles Prize for critical thinking by the US based Committee for Skeptical Inquiry.


Welcome


The University of Colorado Denver|Anschutz Medical Campus Postdoctoral Association would like to welcome you to our seventh annual Postdoctoral Research Day!

Today we focus on the contributions of the over 300 postdocs affiliated with the University of Colorado Denver|Anschutz Medical Campus. Postdoctoral fellows are an integral part of the University community, contributing both to our fields of study and to the overall collaborative environment. Today we are proud to showcase our current independent research activities in over 70 posters and short talks given by postdoctoral fellows from both campuses. Postdoctoral Research Day also includes a strong career development component. We are pleased to have Dr. Joe Schwarcz, Director of McGill University’s Office of Science and Society, as this year’s keynote speaker. We are also excited for this year’s career development workshop, presented by SciPhD co-founders Drs. Randy Ribaudo and Larry Petcovic, which will focus on “Preparing Scientists for Professional Careers”. Everyone is also invited to attend the awards ceremony and closing reception at the end of the day for a great chance to network and develop collaborations.

All postdoctoral fellows are automatically full members of the Postdoctoral Association (PDA).

As the primary University-recognized group for postdocs, the PDA gives postdocs a voice and the ability to change the University for the benefit of all. We hope that all CU Denver|Anschutz Medical Campus postdocs will have a chance to join us at PDA meetings and events in the coming years and share ideas for how to make the postdoctoral experience here the best possible! Best wishes for your continued success,

Jessica Finlay-Schultz, Ph.D. President, Postdoctoral Association University of Colorado Denver|Anschutz Medical Campus



Has been made possible by generous contributions from:

CU Denver / Anschutz Administration and Affiliates

The Graduate School Office of the Provost

The Vice Chancellor for Research Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology

National Jewish Health

Continuing Postdoctoral Advocates Contributed all 7 years

Colorado Clinical and Translational Sciences Institute (CCTSI)

Promega

External Corporate Donors Rocky Mountain Scientific Instrument Maintenance, LLC

Novus Biologicals Illumina Olympus Qiagen

IDT

External Companies Providing Product

Etai’s Bakery Cafe Starbucks

Dazbog Coffee Cedar Creek Pub

And also generous advisory contribution from

Bruce Mandt, Ph.D., Director of the Postdoctoral and Career Development Office;

Jessica Finlay-Schultz, Ph.D. President, Postdoctoral Association; Susan Nagel and Pat Goggans, Graduate School;

Emily Driver, Ph.D., Atif Shaikh, Ph.D., and Priyadarshini Pantham, Ph.D.


Meet Bruce The new Director of the Postdoctoral and Career Development Office

Some things are meant to be. At least that’s how Dr. Bruce Mandt feels about his recent appointment as Director of the Postdoctoral and Career Development Office (“The Director”) within the CU Denver|Anschutz Graduate School. Dr. Mandt has spent almost the entirety of his professional life at CU Denver|Anschutz, and has amassed a wealth of knowledge about what life is like as a graduate student, postdoctoral fellow, and faculty member at this institution. Faced with a challenging job market, Dr. Mandt explored a myriad of career options before ultimately realizing that his true passion lies in helping others – which is good, because that’s the single most important aspect of his new position! Dr. Mandt’s primary role will be to assist graduate students, postdoctoral fellows, and early stage researchers on both campuses in the growth of their careers and entry into the job market. As “The Director,” Dr. Mandt is very excited that he now gets to focus his efforts on helping graduate students and postdoctoral fellows reach their career goals, and he encourages you to let him know how he can assist you.

Meet Dean Engelke Dean of UC Denver Graduate School

Dr. David Engelke was recently appointed dean of the graduate school at the University of Colorado Denver in September 2015. He oversees educational programs for over 6,000 graduate students and has been working closely with both the Denver and the Anschutz Medical Campus as well as with the Postdoctoral Association. He earned a B.S. in Biochemistry at the University of Wisconsin, Madison and a Ph.D. in Biological Chemistry at Washington University (St. Louis) working with Robert Roeder. After postdoctoral studies with John Abelson at UC San Diego and Caltech, Dr. Engelke was faculty of Biological Chemistry at the University of Michigan. There he served in various roles as director of graduate programs and associate deans in the Medical School and Graduate School. Currently Dr. Engelke is Professor of Chemistry at UCD and Professor of Biochemistry and Molecular Genetics at AMC where his research interests center on the biosynthesis and functions of small RNAs in eukaryotic nuclei. Dr. Engelke’s strengths include his stature as a scientist, his enthusiasm for graduate education and his leadership abilities.


Upcoming Networking and Professional Development Opportunities

3/12/16 – Getting your First Job: Practical Elements of Successful Job Hunting.

This SciPhD workshop will follow-up on today’s workshop. 8:30am – 5pm; AO1, Rm 1601. Register at https://gs.ucdenver.edu/limesurvey/index.php/519121. Lunch provided for registrants.

3/14/16 – Postdoctoral Association (PDA) monthly meeting. 4 – 5pm; A01, Rm 1601. Participate in shaping your experience at CU Denver|Anschutz and gain marketable career

experience.

3/17/16 – PDA sponsored Postdoc Seminar Series. 12 – 1pm; RC-1 South, Rm 5105. Practice your scientific communication skills, learn about your fellow postdocs’ research, and

expand your professional network – Plus, free pizza!

3/18/16 – Association of International Researchers: Essence of South Asia. 3:30 – 5pm; RC-2, Krugman Conference Hall Experience life in Afghanistan, Bangladesh, India, Nepal, and Pakistan, expand your cultural

awareness, and sample some really great food!

3/24/16 – PDA Coffee Hour. 2 – 3pm, RC-1 North, Rm 6107. Get out of the lab and grab a cup of coffee with your fellow postdocs. This is a great

opportunity to connect with your peers and practice your small talk – an often underrated career and professional skill!

3/30/16 – Careers in Science Club Seminar: Dean Dave Engelke. 2 – 3pm; RC-1 North, Rm 6107. Learn about life in administration and make a valuable professional connection.

In addition, check out the newly redesigned Postdoctoral and Career Development Office website at http://www.ucdenver.edu/faculty-staff/postdoctoral/Pages/default.aspx.

This the home for all resources related to career development, networking, and finding your next career opportunity. Visit often, and find out how to make the most of your time at CU Denver|Anschutz!


WORKSHOP

SciPhD - Preparing Scientists for Professional Careers

Randy Ribaudo, Ph.D. and Larry Petcovic, Ph.D.

Being competitive and successful as a professional scientist, regardless of whether in an industry or academic setting requires mastery of scientific, business and social skills. Running an effective laboratory operation is like running a small business, and can benefit from applying best practices that have been developed by industry. In “Preparing for Professional Careers” we will look at how your scientific/technical skills combined with your business skills and social skills together make up the three identities that define your brand. The same competencies that industry requires in order to be successful are equally important in developing and co-existing in a high-performing team in academic settings.

In the first 1.5 hour session we will look at the 24 competencies that industry has identified as critical in being competitive and successful, and how they relate to scientists own past experiences during their graduate and post-graduate education. We will also look at different kinds of jobs that are available both in academia and industry.

In the second 1.5 hour session we will discuss how to identify business and social competencies in job ads, and how to use that information to develop targeted resumes that emphasize all three identities (scientific, business, social) in the context of what the hiring institution is seeking. We will also discuss how to build an effective network, and how to leverage that network to identify and research jobs, and get your resume on the hiring manager’s desk. Finally we will discuss how to prepare for interviews and effectively apply the business and social skills discussed in the first session to demonstrate why you are a good fit for the position.

Find out more at sciphd.com/.


ORAL PRESENTATIONS (Three Sessions) 1:00PM - 2:30PM

Basic Research

Academic Office 1, Room 2101

Time Presenter Abstract Title

1:00-1:10pm MA Crawford Redox-Active Sensing by Bacterial DksA Transcription Factor is Determined by Cysteine and Zinc Content

1:13-1:23pm ML Goodall The Autophagy Machinery Controls Cell Death Switching between Apoptosis and Necroptosis

1:26-1:36pm B Hiester L-type Voltage-Gated Calcium Channels Regulate Activity-Induced Fusion of Postsynaptic Recycling Endosomes

1:39-1:49pm S Jain Apoptosis During Extreme Cold Ischemia and Rewarming Involves a Caspase Independent Pathway

1:52-2:02pm CL Jones ETV6 Represses Pax5 in Early B Cell Development

2:06-2:15pm AL Stefanski Muc5b is an Endogenous Ligand of Siglec-F Expressed on Alveolar Macrophage and Siglec-F Activation Modulates Cellular Function

Clinical Research

Research Complex 2, Room 10105


1:00-1:10pm A Bergouignan

Effect of Frequent Interruptions Of Prolonged Sitting On Energy Expenditure And Substrate Oxidation In Overweight Adults

1:13-1:23pm H Li Molecular Analysis Identifies a Potential Epigenetic Role for AP-2a in Branchio-Oculo-Facial Syndrome

1:26-1:36pm N Mukherjee Strategic Use of BCL-2 Inhibitors to Target Melanoma Cells and Melanoma Initiating Cells

1:39-1:49pm YM Nkrumah-Elie

Unraveling Non-Genetic Mechanisms of Asthma: Untargeted Metabolomics of Identical Twins Discordant for Asthma

1:52-2:02pm G Wang Rapid Harvesting of Highly Concentrated Tumor Cells from Bulk Human Blood with Buoyant Immuno-microbubbles


Translational Research

Research Complex 2, Room 3109


1:00-1:10pm EK Kleczko The Role of T Cells in the Progression of Autosomal Dominant Polycystic Kidney Disease

1:13-1:23pm R Kumar Thrombospondin-1 Deficient Bone Marrow and Pharmacological Inhibition of Transforming Growth Factor-Beta1 Activation Protects against Schistosomiasis Pulmonary Hypertension

1:26-1:36pm B Nedumaran Generation of TRPV1- and Chat-knock-in Reporter Mice to Study Urinary Bladder Pain Syndrome

1:39-1:49pm S Rakete Identification of Novel Amino Acid Modifications in Human Lens Protein

1:52-2:02pm TJ Rowland Characterization of a Cardiomyopathy Using Cardiomyocytes Differentiated from Patient-Derived Induced Pluripotent Stem Cells

2:05-215pm H Ye Metabolic Adaptations of Leukemia Stem Cells to an Adipose Tissue Microenvironment Niche

2:18-2:28pm T Yu Oral Ghrelin Receptor Facilitate Fat Taste Sensitivity in Mice


POSTER PRESENTATIONS

2:30PM - 4:30PM - Krugman Hall Session A: 2:30 – 3:15pm Session B: 3:15 – 4:00pm

Name Rotation Poster # Title BM Akiyama

A 01 Structure and dynamics of a nuclease-resistant RNA from arthropod-borne Flaviviruses

EE Alexeev B 02 Microbiota-Derived Tryptophan Metabolites Activate Aryl Hydrocarbon Receptor and Induce IL-10 Receptor Expression in Intestinal Epithelia

D Beqollari A 03 Rad is an agent of skeletal muscle atrophy LK Bogren B 04 Metabolic Processes and Physiological Parameters Involved

with the Ischemia/Reperfusion Protective Phenotype in Hibernators

DR Bruns A 05 Interleukin-19: A Novel Sexually Dimorphic Cardiac Cytokine JL Christenson

B 07 MMTV-PyMT and Derived Met-1 Cells as Immunocompetent Models of Androgen Receptor-Positive Triple-Negative Breast Cancer

SE Clark B 08 Unique Requirements for Listeria Induction of Natural Killer Cell IL-10 Production

VF Curtis A 09 The Influence of Neddylation and the NF-Mucosal Inflammatory Response

BJ Davenport

B 10 The L-arginine Metabolic Axis in Macrophages Regulates Pathogenicity of Cutaneous Leishmaniasis

DR Eiler A 11 Biochemical, Biophysical, and Infection-Based Studies of the ciRNA-RNase L Complex

NL Farnsworth

B 12 Connexin36 Gap Junction Coupling Protects Against Cytokine--Cell Death and Dysfunction with Ca2+ Dependence

S Ghosh A 13 Targeting Epigenetic regulator WD-Repeat Containing protein 5 (WDR5) in Myc-driven Medulloblastoma

MA Gordon B 14 Targeting the long noncoding RNA MALAT1 in ovarian cancer JD Hintzsche

A 15 Characterizing the Mutational Landscape of 418 Melanoma Tumors using Whole Exome Sequencing

R Hussain B 16 Deletion of Integrin Linked Kinase (ILK) in neural progenitor cells disturbs neuron glia balance during early development

X Jiang A 17 Serum Circulating Factors in Children with Dilated Cardiomyopathy Are Important In Modulating Pathological Responses In Cadiomyocytes

WK Jubair B 18 Dysbiosis alters mucosal immunity towards development of rheumatoid arthritis

AC Keller A 19 Manipulation of NOS activity in the context of glucose stress alters AMPK and mitochondrial oxphos protein content

JM Lanis B 20 Tryptophan Metabolite Activation of the Aryl Hydrocarbon Receptor Regulates IL-10 Receptor Expression on Intestinal Epithelia


Name Session Poster # Title SB Matthews

A 21 A Putative Role for Glutathione in the Cancer Stem Cell Phenotype in Luminal Breast Cancer

MK McCarthy

B 22 Disrupted Architecture and Function of Draining Lymphoid Tissue During Chronic Chikungunya Virus Infection

DS McDermott

A 23 Impact of Interferons on Listeria monocytogenes cell-to-cell spread

EK Meredith B 24 An RNA matchmaker protein regulates the activity of the long noncoding RNA HOTAIR

VC Minarchick

A 25 Indirect and Direct Nanomaterial Interaction Influences Vascular Reactivity

BV Murphy B 26 Enhanced phenotypic analysis of islet antigen specific CD8+ T cells

H O'Leary B 06 Acute effects of kainic acid induced early life seizures EV Otterloo A 35 Tissue specific redundancy of TFAP2 family members during

craniofacial patterning CT Raghavan

B 34 TGFb2-mediated Epithelial to Mesenchymal Transition of lens epithelial cells: Role of AGE-RAGE interaction

MJ Reichlen A 27 The role of the electron transport chain in promoting bacterial susceptibility and tolerance to cellular antibiotics

B Ross B 28 Reconstructing Chromosome Conformation by Fluorescence Microscopy

A Sang A 29 T cell Dependent Signals are Required for the Enrichment of Dual-Reactive B cells in Autoimmune Mice

TR Sippel B 30 PPAR-gamma Induced TGF-beta1 Expression in Myeloid Cells Contributes to Lung Cancer Progression and Metastasis

JR St. Clair A 31 Inhibition Ser293 Phosphorylation of Connexin-36 Prevents Cytokine- -cells in the Islet of Langerhans

AL Steckelberg

B 32 A structural switch in the CCR5 mRNA induces ribosomal frameshifting

BM Stevens A 33 Characterization and Targeting of Myelodysplastic Syndrome Stem Cells

MM Walker B 36 Cyclic-di-Nucleotides as STING-dependent Adjuvants and B cell Activators

S Wie A 37 Targeting Tyrosine Phosphorylation of PKC delta for Salivary Gland Radioprotection

Z Zhao B 38 A multiplex assay for simultaneous screening type 1 diabetes and multiple autoimmune diseases


ORAL PRESENTATION ABSTRACTS

Effect Of Frequent Interruptions Of Prolonged Sitting On Energy Expenditure And Substrate Oxidation In Overweight Adults A Bergouignan1, N DeJong1, CA Rynders1, EL Melanson1, DHBessesen1,2 1 Division of Endocrinology2 Denver Health Purpose: In overweight adults, to compare the metabolic effects of 4 days of activity microbursts (MICRO: 5-min of moderate-intensity walking every hour for 9 hrs) to a isocaloric single 45-min bout of moderate-intensity walking (ONE) and a sedentary control condition (SIT). Methods: Ten (7F/3M, 31.7±7.1 yo, BMI=31.0±1.9 kg/m2) overweight subjects have completed all 3 conditions (MICRO, ONE, SIT) of an ongoing randomized crossover study. Each condition consisted of 3 days in a free-living state during which sedentary time was measured by inclinometry followed by a 24hr stay in a room calorimeter to measure total energy expenditure (TEE) and substrate utilization. Protein oxidation was estimated by urinary nitrogen excretion. Energy intake was matched across days and conditions. Results: 24hr TEE increased in MICRO and ONE leading to energy deficit compared to SIT (p<0.05 for both). 24hr respiratory quotient (RQ) decreased in both active conditions compared to SIT (p<0.05) with the most pronounced drop in RQ observed at night (p<0.03 for both). Although 24hr fat oxidation increased in both ONE and MICRO (p<0.05 for both), carbohydrate and protein oxidation did not change. Both ONE and MICRO doubled nocturnal fat oxidation (p=0.08 for both), while MICRO only decreased nocturnal carbohydrate oxidation (-24%, p=0.04). In free-living, sedentary time decreased during MICRO (p=0.07). Conclusion: While both active conditions increase TEE and the proportion of fat oxidized, the underlying mechanisms may be different between one and multiple bouts of physical activity. Frequently interrupting sitting may be a promising strategy to reduce sedentary time in overweight adults. This work was funded by NIH 1K99DK100465-01

Redox-Active Sensing by Bacterial DksA Transcription Factors is Determined by Cysteine and Zinc Content MA Crawford1, T Tapscott2, LF Fitzsimmons1, L Liu1, AM Reyes3, SJ Libby4, M Trujillo3, FC Fang4,5, R Radi3, and A Vázquez-Torres1,2,5 1Department of Immunology and Microbiology, University of Colorado School of Medicine; 2Molecular Biology Program, University of Colorado School of Medicine;3Departamento de Bioquimica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República; 4Department of Laboratory Medicine, University of Washington School of Medicine; 5Department of Microbiology, University of Washington School of Medicine; 6Veterans Affairs Eastern Colorado Health Care System. The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of reactive oxygen and nitrogen species. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here we have addressed this challenge by complementing delta-dksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA orthologues are able to mediate appropriate stringent control in Salmonella, and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an


antioxidant, dampening cysteine reactivity and raising the threshold of post-translational thiol modification. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by C4 orthologues. Aberrant transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our observations highlight the importance of metabolic adaptation in bacterial pathogenesis and show the mechanism by which DksA orthologues are finely tuned to sense levels of oxidative and nitrosative stress encountered by bacteria in their natural and host environments.

The Autophagy Machinery Controls Cell Death Switching between Apoptosis and Necroptosis Megan L. Goodall1, Brent Fitzwalter1, Shadi Zahedi2, Min Wu1,3, Jean M. Mulcahy-Levy2, Michael Morgan1, Scott D. Cramer1, and Andrew Thorburn1 [1]Department of Pharmacology, University of Colorado Denver, Aurora, CO 80045, USA.2Department of Pediatrics, University of Colorado Denver, Aurora, CO 80045, USA.3Department of Molecular and Cellular Oncology, MD Anderson Cancer Center, Houston TX 77030, USA. Although autophagy controls cell death and survival, the underlying mechanisms are poorly understood. MAP3K7 is a tumor suppressor gene, which is associated with poor disease-free survival in prostate cancer. Consistent with previous reports, Map3k7 deletion in murine prostate cells confers cell death sensitivity to TNF-related apoptosis inducing ligand (TRAIL). Surprisingly, this death occurs through necroptosis, not apoptosis, due to assembly of the necrosome in association with the autophagy machinery by p62/Sqstm1 recruitment of Ripk1. However, the mechanism of cell death is switched to apoptosis if p62-dependent recruitment of necrosome components to the autophagy machinery is blocked. These data show that the autophagy machinery modulates both the amount and mode of programmed cell death. Importantly, this activity requires the formation but not degradation of autophagosomal structures, implying that different autophagy inhibitors may promote or inhibit tumor cell death depending on which step of autophagy is blocked.

L-type voltage-gated calcium channels regulate activity-induced fusion of postsynaptic recycling endosomes B. Hiester1, A. Bourke1, B. Sinnen1, E. Gibson1 and M. Kennedy1. 1Pharmacology The number and identity of ion channels and receptors displayed on the dendritic surface determines fundamental cellular properties of neurons and is tuned by neuronal activity. Activity-coupled surface expression of important channels and receptors is mediated by fusion of recycling endosomes (REs) with the dendritic plasma membrane (PM). Despite the established importance of RE trafficking for neuronal function, how synaptic activity is coupled to RE fusion with the PM remains largely unexplored. Here we demonstrate that RE fusion in dendrites and spines depends on NMDA receptor activation and a rise in intracellular calcium (Ca2+). Surprisingly, NMDA receptors were not the sole source of postsynaptic Ca2+ driving RE fusion. We found that L-type voltage-gated Ca2+ channels (L-VGCCs) play a critical role in RE trafficking by regulating the mode of vesicle fusion. When L-VGCCs were blocked, REs partially fused with the plasma membrane but failed to deliver their membrane-bound protein cargo to the dendritic surface. Conversely, potentiating L-VGCC activity enhanced RE fusion in response to synaptic activation. Together, these experiments reveal an unexpected cooperation between NMDA receptors and L-VGCCs in governing the mode of activity-triggered RE fusion in dendrites and spines.


Apoptosis during Extreme Cold Ischemia and Rewarming Involves a Caspase Independent Pathway S Jain1 and A Jani1 1 Renal Purpose of Study: Cold ischemia (CS) followed by warm reperfusion (REW) during hibernation is a natural model we have used to understand Delayed graft function (DGF). We have shown that hibernating ground squirrel kidneys and tubular cells (RTEC) survive CS for several days in torpor followed by REW in Arousal without RTEC apoptosis. In contrast mouse & human kidneys demonstrate significant tubular apoptosis after CS/REW. We have previously shown that apoptosis during CS/REW in mouse RTECs is mediated in part by a caspase dependent pathway. We hypothesized an alternate possibilty that caspase independent pathways also trigger apoptosis in mouse RTECs and play a role in DGF. Methods: Squirrel and mouse RTECs were subjected to cold storage (CS) in UW solution followed by rewarming (REW) in normal media. Apoptosis was quantified by TUNEL assay. AIF and EndoG expression was examined by immunoblot in mitochondrial and cytosolic fractions. Immunocytochemistry of AIF and EndoG was observed by confocal microscopy. Results: Mouse RTECs exposed to CS/REW had significantly increased apoptosis vs. squirrel RTECs. Furthermore, mouse RTECs subjected to CS/REW had significantly increased mitochondrial AIF and EndoG translocation to the cytosolic fraction vs. squirrel RTECs. Confocal imaging shows nuclear localization of AIF and EndoG in Mouse RTECs subjected to CS/REW vs. squirrel RTECs. Conclusions: The data provide evidence of mitochondrial disruption in mouse RTECs during CS/REW, resulting in caspase independent apoptosis mediated by AIF and EndoG. Understanding caspase independent pathways during CS/REW may lead to improved organ preservation, and novel therapies for DGF.

ETV6 Represses Pax5 in Early B Cell Development Courtney L. Jones1, Courtney J. Fleenor2, Seth Welsh2, Leila Noetzli1, Susan Fosmire1, Dmitry Baturin1, Lori Gardner1, Jorge Di Paola1, James R. Hagman2, and Christopher C. Porter1 1Department of Pediatrics, University of Colorado Denver, Aurora, CO2Department of Biomedical Research, National Jewish Health, Denver, CO Recent studies have revealed germline ETV6 mutations (P214L) predispose to acute lymphoblastic leukemia (ALL) development and impair the transcriptional activity of ETV6 in a dominant negative fashion. Our goal is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia. Here, we demonstrate Etv6 expression is inversely correlated to Pax5 and Ebf1 expression during mouse B cell development (r2=.9993; P= 0.0167), and overexpression of ETV6, but not ETV6 P214L, significantly decreased Pax5 expression in the murine lymphoid line Ba/F3 (Pproximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We then found ETV6, SIN3A and HDAC3 associated with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L, suggesting ETV6 is responsible for the recruitment of this co-repressor complex to the Pax5 locus. Furthermore, inhibition of HDACs with Vorinostat restored Pax5 expression in cells expressing WT ETV6. Finally, we have demonstrated ETV6, SIN3A, and HDAC3 are associated with the Pax5 locus in mouse lineage negative hematopoietic cells (do not express Pax5) but not in CD19- expressing cells (express Pax5). In conclusion, ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 is essential for commitment to the B cell lineage and aberrant expression ofPax5 has been shown to increase B cell progenitors, which likely increases the risk for leukemia development.


This work was funded by Pediatric Hematology-Oncology T32

The Role of T Cells in the Progression of Autosomal Dominant Polycystic Kidney Disease EK Kleczko1, KH Marsh1, K Hopp1, RA Nemenoff1 1Division of Renal Diseases and Hypertension, Department of Medicine, School of Medicine, University of Colorado Anshcutz Medical Campus Autosomal dominant polycystic kidney disease (ADPKD) is the third most common cause of end stage renal disease (ESRD) worldwide. Caused by a germ line mutation in either the PKD1 or PKD2 gene, the disease is characterized by an enlargement of the kidneys due to bilateral renal cysts, which results in ESRD. Currently, there is no treatment to slow the progression of ADPKD, and much of the research is focused on the effects of these mutations on signaling within affected epithelial cells. The role of the microenvironment in regulating progression has not been extensively examined; we aim to investigate the role of T lymphocytes in ADPKD. We have used a genetic mouse model with a point mutation in the pkd1 gene (R3277C) that mimics the human disease in both a fast progressing Balb/c strain and a slow progressing C57Bl/6 strain. Through the use of flow cytometry and immunofluorescence, we have observed an increase in CD4+ and CD8+ T cells in severe ADPKD mouse kidneys compared to wild-type. Furthermore, an increase in the Treg population was observed, as evidenced by an increase in FoxP3+ CD4 cells, as well as an increase in expression of PD1+ T cells. Immunoblot analyses of an immortalized human PKD cell line showed increased PDL1 expression in the PKD cells compared to normal renal cells, and human ADPKD ESRD kidneys showed positive PDL1 staining in the epithelium by immunohistochemistry. These data suggest that T cells play an important role in the disease progression of ADPKD, and we hypothesize that T cells recruited to the diseased kidney are being immunosuppressed, contributing to increased cyst progression. Preventing this immunosuppression, by inhibiting the PD1-PDL1 checkpoint pathway, could potentially slow the progression of ADPKD in patients.

Thrombospondin-1 Deficient Bone Marrow and Pharmacological Inhibition of Transforming Growth Factor-Beta1 Activation Protects against Schistosomiasis Pulmonary Hypertension R Kumar, C Mickael, B Kassa, L Gebreab, D Koyanangi, L Sanders, R Tuder, B Graham Program in Translational Lung Research, University of Colorado, Denver Purpose of study: TGF- histosomiasis-associated PH. We hypothesized that depletion of thrombospondin-1 (TSP-1) from bone marrow transplant (BMT) and its inhibition using the LSKL peptide mimetic of the TGF- LAP sequence that interacts with TSP-1, would be protective against Schistosoma-induced PH. Methods used: Wildtype (WT) mice with BMT from TSP-1-/- and converse (C57BL6/J background; N=6-8 per group) were first intraperitoneally (IP) sensitized to S. mansoni240 eggs/gram body weight (BW), and then intravenously (IV) challenged with the 175 eggs/gram BW of S. mansoni eggs. The IV egg challenged WT mice received every other day IP injections of either LSKL or SLLK scrambled/control peptide at a dose of 30mg/kg BW. One week after IV egg injection, right ventricular (RV) catheterization was performed. TGF-(MLEC) luciferase assay. Summary of results: TSP-1 mRNA increased by 1.6-fold (P=0.01) after S. mansoni exposure in lungs. S. mansoni-exposed WT mice with TSP-1-/- BM showed decreased RV systolic pressure (RVSP) as compared to TSP-1-/- mice with BMT from WT mice (mean pressure: 21.6 mmHg vs 31.3 mmHg, P<0.001). Further, LSKL treatment in S. mansoni exposed mice resulted into decreased RVSP


compared to SLLK treated mice (mean pressure: 24.9mmHg versus 29.1mmHg, P=0.01). Further, MLEC luciferase assay showed decreased active and total TGF-mice (P<0.05). Conclusions: TSP-1 deficient bone marrow and pharmacologic blockade of TSP-1 mediated TGF-activation may protect against Schistosoma-induced PH.

Molecular Analysis Identifies a Potential Epigenetic Role for AP- in Branchio-Oculo-Facial Syndrome H Li, T Williams Department of Craniofacial Biology and Cell and Developmental Biology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO Human Branchio-Oculo-Facial Syndrome (BOFS) is an autosomal-dominant trait caused by mutations in TFAP2A, the gene encoding transcription factor AP- al clefting, microophthalmia, and branchial skin anomalies, but expressivity is variable and there are no clear genotype:phenotype associations. We have previously demonstrated that the various BOFS mutations result in dominant negative versions of the AP-kinetics of DNA binding. We have now extended these studies by generating mouse models of human BOFS by conditionally knock-in of human patient mutations into the endogenous mouse tfap2a locus. The BOFS mouse models recapitulated the phenotypes seen in human patients, particularly variable cleft lip, cleft palate and nasolacrimal duct obstruction and revealed potential defects in thymus formation that have not been reported in human. These findings indicate that the TFAP2A mutations do act in a dominant negative manner in vivo, but also demonstrated that the same variable expressivity was also present in the mouse models as observed in human BOFS. We have now begun to probe the underlying cause of the variable clefting phenotype by examining the molecular and cellular behavior of AP- in vivo. Our results indicate that AP-gene expression and the cell cycle that is disrupted by the various human BOFS mutations.

Strategic use of BCL-2 inhibitors to target melanoma cells and melanoma initiating cells N Mukherjee, Shiau C, Lu Y, Almeida A, Luo, Y, Fujita M , Robinson S, Robinson W, Norris DA & Shellman YG Dermatology, University of Colorado AMC, Aurora, CO, USAInstitute of Biopharmaceutical Sciences, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Taipei, Taiwan Despite the recent advancement in treatment for melanoma, options are still limited for patients without BRAF mutations or in relapse. Melanoma’s resistance is known to be promoted by MCL-1, an anti-apoptotic protein, and/or Melanoma Initiating Cells (MICs). Targeting these two components may be a new alternative for melanoma treatment. We examined the effects of SC-2001, a MCL-1 inhibitor, alone or in combination, in treating melanoma, including MICs using melanoma lines and short-term cultures of patient samples. The tumors used were derived from patients who relapsed from BRAF-targeted therapy or immunotherapy. We used multiple in vitro and in vivoassays and the ground-breaking CRISPR/Cas9 genome-editing technique for this study. By itself, SC-2001 significantly killed the bulk of melanoma cells in vitro and in vivo (p<0.01). However, even at high doses, SC-2001 alone did not effectively eliminate MICs. In contrast, the combination of SC-2001 with ABT-737 (small molecule BCL-2 inhibitor) decreased ALDH+ cells, disrupted primary spheres, and inhibited the self-renewability of MICs (p<0.05). In addition, using a low-cell-number mouse xenograft model, we examined the effects of the combination on the tumor initiating ability of MIC-enriched cultures. The combination therapy reduced tumor formation of the relapsed patient sample significantly compared to either drug alone (P<0.05). Mechanistic studies demonstrated that the upregulation of the pro-apoptotic proteins NOXA


and BIM was a crucial factor in the combination-induced cell death. This suggests that the combination of MCL-1 and BCL-2 inhibitors is a promising treatment option for patients without BRAF mutations and those relapsed from current treatment.

Generation of TRPV1- and Chat-knock-in reporter mice to study urinary bladder pain syndrome Balachandar Nedumaran, Randall B. Meacham, and Anna P. Malykhina Division of Urology, Department of Surgery, University of Colorado Denver, Aurora, CO Introduction and objectives: Bladder pain syndrome (BPS) is a chronic condition characterized by bladder pain, urgency, frequency of micturition, detrusor overactivity and other symptoms. Our group provided some evidences that lower urinary tract dysfunction and pain associated with BPS could be associated with increased excitability in neural pathways innervating the lower urinary tract. Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) are novel class of designer drugs which are inert to endogenous targets, highly permeable across the blood-brain barrier. The objective of our study was to generate sensory and motor neuron-specific reporter mice and their pathways can be altered by DREADDs to study BPS. Methods: Transient receptor potential cation channel 1 (Trpv1)- and Choline acetyltransferase (Chat)-cre mice were bred to ROSA-CAG-LSL-ZsGreen (RG) mice to specifically express zsGreen in Trpv1- and Chat-positive cells. Two different Cre-driven DREADD-mCherry viruses has been produced to excite or silence the Cre positive neurons with the help of Clozapine N-oxide (CNO). Results: TRPV1- and Chat-positive heterozygous zsGreen (TG and CG) mice have been generated by setting up breeding TRPV1- and Chat-cre mice with RG mice. The expression of the mCherry reporter genes from DREADDs were confirmed in cells.Conclusions: Homozygous TG and CG mice will undergo intravesical instillation of lipopolysaccharide (LPS) and the sensory (Trpv1) and motor (Chat) nerve-mediated control of BPS will be investigated. This work was funded by This study was supported by Department of AEF Seed grant

Unraveling Non-Genetic Mechanisms of Asthma: Untargeted Metabolomics of Identical Twins Discordant for Asthma YM Nkrumah-Elie1,2, KD Quinn1,2, AF Faino2, SJ Walmsley1,2, CI Cruickshank-Quinn1,2, AH Liu1,2,3, AM Schiltz1,2,3, BC Haberstick4, CA Lozupone1, and NA Reisdorph1,2 1University of Colorado, Denver - Anschutz Medical Campus, Aurora, CO; 2National Jewish Health, Denver, CO; 3Children’s Hospital Colorado, Aurora, CO; 4University of Colorado, Boulder & Colorado Twin Registry Nearly one in ten children in the United States have been diagnosed with asthma. Concordance of asthma in identical twins is 50% and therefore suggests significant environmental causation. To evaluate the environmental mechanisms of asthma, plasma samples from ten sets of adolescent, (8-16 years old) monozygotic twins discordant for asthma were analyzed by untargeted LC/MS. Unpaired t-tests to compare the metabolites of asthmatic versus non asthmatic twins was conducted, and statistical significance was accepted at pcompounds, with 75 compounds increased in the asthmatic phenotype, and 28 compounds with at least a 2-fold change. 4-hydroxy-L-proline, an inhibitor of collagen synthesis and contributor to airway remodelling, a key feature in chronic asthma, was significantly increased (60.73 FC, p=0.022) in the asthmatic twins. 1-phosphatidyl-D-myo-inositol, the primary source of arachidonic acid for the biosynthesis of eicosanoids, the fatty acids involved in the regulation of airway inflammation and reactivity, was decreased by an average of 17% (p=0.029) in the asthmatic twins. Metabolite analysis using MetPA and Mbrole, revealed the recycling/salvage pathway of sphingolipid metabolism as the most significantly altered pathway (p=7.986E-6) resulting from the asthmatic phenotype. Metabolomic analysis of the plasma of identical twins discordant for asthma demonstrates significant small molecule


differences between the asthma phenotypes, and can lead to the elucidation of mechanisms associated with asthma pathobiology.

Identification of Novel Amino Acid Modifications in Human Lens Proteins S Rakete1, RH Nagaraj1 1Ophthalmology The formation of cataracts is accelerated by post-translational modifications of amino acids in lens proteins. We showed that erythrulose and kynoxazine were able to modify and crosslink proteins. The investigation of underlying pathways in vivo may help to understand how ascorbate degradation influences protein modification and therefore cataract formation. The 3-deoxythreosone-arginine-derived hydroimidazolinone and the respective lysine-arginine-hydroimidazolinone-crosslink were isolated and characterized by NMR and mass spectrometry. Purified

-crystallin was incubated under different conditions and analyzed by SDS-PAGE. Lens proteins were hydrolyzed by enzymatic digestion and analyzed by UPLC-MS2 –Dicarbonyls were trapped as stable quinoxalines and analyzed by UPLC-MS2.

- -–dicarbonyl quencher. Furthermore,

-crystallin also prevented crosslinking. We found that kynoxazine and erythrulose generate substantial amounts of 3-deoxythreosone at physiological conditions. Based on that, a 3-deoxythreosone-derived arginine modification and a 3-deoxythreosone-derived arginine-lysine crosslink were isolated, characterized and used for UPLC-MS2 analysis of enzymatically digested lens proteins. We were able to detect both modifications in lens proteins. The levels of the 3-deoxythreosone-derived arginine modification correlated positively with the age of the lenses.

-Crystallin is modified and crosslinked by kynoxazine and erythrulose via 3-deoxythreosone, which reacted with arginine and lysine residues. Those 3-deoxythreosone-derived amino acid modifications were identified in human lens proteins for the first time. They are likely to alter the solubility of lens proteins, cause protein aggregation and therefore promote cataract formation.

Characterization of a Cardiomyopathy Using Cardiomyocytes Differentiated from Patient-Derived Induced Pluripotent Stem Cells TJ Rowland1, SI Hashem2, K Jones1, ED Adler2, L Mestroni1, MRG Taylor1 1Cardiovascular Institute and Adult Medical Genetics Program, University of Colorado Denver Anschutz Medical Campus; 2Department of Medicine, Division of Cardiology, University of California San Diego Danon disease is a rare but severe X-linked form of cardiomyopathy that leads to profound cardiac hypertrophy and causes death or a need for cardiac transplantation in males by the third decade. Danon disease is known to be caused by loss of the lysosome-associated membrane protein-2 (LAMP-2) protein, which is a membrane glycoprotein likely involved in lysosome-autophagosome fusion and vesicle trafficking during autophagy. However, it is unknown how LAMP-2 protein deficiency leads to the development of Danon disease. To better characterize molecular pathogenic mechanisms involved, human induced pluripotent stem cells (iPSCs) were generated from a patient with Danon disease and differentiated into cardiomyocytes. RNA transcription levels from the Danon iPSC-derived cardiomyocytes (iPSC-CMs) were analyzed using RNA-Sequencing (RNA-Seq). The data show significant upregulation of genes involved in cholesterol synthesis (but not cholesterol esterification) and cellular respiration in Danon iPSC-CMs compared to unaffected control iPSC-CMs. These preliminary findings suggest potential agreement with previous LAMP-2 mouse knock-out models that demonstrate impacted cholesterol trafficking and accumulation of unesterified cholesterol. Additionally, the upregulation of cellular respiration transcripts in our data align with published data suggesting mitochondrial function is affected in these cells, including increased levels of mitochondrial oxidative


stress. Overall, our RNA-Seq data point to molecular pathways that are potentially significantly impacted in the Danon iPSC-CM model and novel potential therapeutic targets to further explore, such as through high-throughput screening and drug discovery efforts.

Muc5b is an Endogenous Ligand of Siglec-F expressed on Alveolar Macrophage and Siglec-F Activation Modulates Cellular Function AL Stefanski1, AA Fletcher1, DS Rawclawska1, L Barthel2, WJ Janssen1,2, CM Evans1 1University of Colorado, SOM - Pulmonary, 2National Jewish Health Macrophages are critical for the clearance of inhaled particles and pathogens from the lungs during homeostasis. Murine resident alveolar macrophages (AM) express the inhibitory transmembrane receptor sialic acid-binding immunoglobulin-type lectin receptor-F (Siglec-F). Structurally, Siglec-F is characterized by four extracellular immunoglobulin domains including a V-set Sia-binding domain and three C2-set immunoglobulin domains, as well as intracellular tyrosine-based signaling motifs. Siglec-F

-linked sialic acid-containing glycoconjugates. Currently, the function(s) and endogenous ligand(s) of Siglec-F on AMs remain poorly defined. Mucins are glycoproteins that are the predominant macromolecules in airway secretions. Although they are conserved across evolution and essential for defense, how specific glycans regulate mucin functions is poorly understood. The mucin glycoprotein Muc5b is required for innate airway defense, and we demonstrate that secreted Muc5b is present in the alveoli and in contact with AMs (Roy et al 2014). Using Maackia amurensis lectin-II

-sialylated. Recently, Siglec-F Fc fusion proteins were utilized to probe mouse tracheal tissues and to identify the localization of Siglec-F ligands. Ligands were abundant in the submucosal glands of wild-type mice and absent in Muc5b deficient mice. Taken together, these data suggest that Siglec- -sialylated Muc5b interact in murine alveoli. Accordingly, we hypothesize that AM expressed Siglec-F has intracellular signaling functions capable of regulating AM function, and that Siglec-F interaction with

-sialylated Muc5b modulates this response. Presently, we are studying the effects of Muc5b:Siglec-F signaling on AM function, intracellular signaling downstream of Siglec-F, and intracellular Siglec-F localization. This work was funded by T32 - Division of Pulmonary Science

Rapid Harvesting of Highly Concentrated Tumor Cells from Bulk Human Blood with Buoyant Immuno-microbubbles G Wang1, G Shi2, M Benchimol2, S Pingle3, S Kesari3, Y Yeh4, YT Liu4, D Simberg1 1Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 East Montview Blvd., Aurora, CO 80045, USA2Diagnologix, LLC, 5820 Oberlin Drive, Suite 104, San Diego, CA 92121, USA3Department of Translational Neuro-Oncology and Neurotherapeutics, John Wayne Cancer Institute at Providence Saint John’s Health Center, 2200 Santa Monica Blvd., Santa Monica, CA 90404, USA4Moores UCSD Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093, USA Circulating tumor cells (CTCs) are extremely rare cells shed from primary tumor. A technology to quickly and inexpensively isolate viable CTCs will enable widespread clinical utilization of CTCs for cancer prognosis and drug testing. We have previously shown that lipid-shelled microbubbles (MBs) could isolate rare cells from biological fluids by flotation. We investigated the role of MB composition and type of tumor surface marker on MB stability and binding efficiency to breast cancer cells. Optimized MBs showed 95% binding efficiency to human breast carcinoma SKBR-3 cells. Cells were able to proliferate in culture with MBs attached. We further developed an inverted vacuum-assisted


harvesting setup in order to harvest (skim) highly concentrated tumor cells in a microliter volume. We demonstrated a high efficiency (~80%) of isolation of rare tumor cells spiked in 7 ml blood by anti-EpCAM MBs. A blinded study using anti-EpCAM MBs detected epithelial CTCs in samples of metastatic breast and ovarian cancers with brain metastases, but not in samples from non-epithelial brain malignancies. The simple and robust approach can be developed into an inexpensive tool for batch CTC isolation in basic research and clinical applications. This work was funded by Innovative Molecular Analysis Program (IMAT) of the National Cancer Institute (grant R33CA174554) to DS and YTL and Small Busine

Metabolic Adaptation of Leukemia Stem Cells to an Adipose Tissue Microenvironmental Niche H Ye1,2, B Adane1, N Khan1, M Gasparetto1, B Stevens1, M Mohammad1, S Pei1,M Balys4, JM Ashton3, CY Park5, CT Jordan1 1Division of Hematology, University of Colorado2Department of Pathology3Functional Genomics Center4James P. Wilmot Cancer CenterUniversity of Rochester Medical Center5Memorial Sloan Kettering Cancer Center Adipose tissue (AT) has previously been identified as an extra-medullary reservoir for normal hematopoietic stem cells (HSCs). In the present study, we evaluated the role of AT in supporting leukemia stem cells (LSCs). Using a murine model of blast crisis CML, we demonstrate AT serves as a reservoir for LSCs. Adipose-resident leukemia cells display a pro-inflammatory phenotype and lead to lipolysis in AT. Furthermore, lipolysis of AT fuels fatty acid oxidation in LSCs, especially within a LSC subpopulation expressing the fatty acid transporter CD36. Intriguingly, CD36+ LSCs show unique metabolic properties, and are strikingly enriched in AT, and are protected from chemotherapy by the AT microenvironment. Finally, in human blast crisis CML cells, CD36 also marks a leukemic fraction with similar biological properties. Based on these findings, we propose a model wherein the interplay between leukemia cells and AT creates a unique microenvironment to support the metabolic demand and survival of a distinct LSC subpopulation.

Oral Ghrelin Receptor Facilitate Fat Taste Sensitivity in Mice T Yu1, A Calder2, N Dahir2, RH Eckel1, SC Kinnamon3 and TA Gilbertson2 1Division of Endocrinology, Metabolism, & Diabetes, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado2Biology Department, Utah State University, Logan, Utah3Department of Otolaryngology, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado Excessive caloric intake is a major contributor to the current obesity epidemic. While much research has focused on understanding the neurobiology of eating behavior, the mechanisms linking gustatory taste sensation to over-feeding have been largely overlooked. Eating behavior is regulated by a complex set of peripheral satiety hormones that act on the hypothalamus. Of these, ghrelin is the only appetite-stimulating peptide hormone found in the systemic circulation. Previous studies have revealed that mice lacking the ghrelin receptor, growth hormone secretagogue receptor (GHSR) globally are resistant to diet-induced obesity with less food intake. Although ghrelin is primarily produced in the stomach, new evidence has shown that GHSR is also expressed in taste buds, which suggest that ghrelin may profoundly influence eating behavior. In this study, we examined the linoleic acid (LA) taste sensitivity in GHSR-/- mice using brief-access (5s) behavioral assays following the formation of conditioned taste aversion (CTA assay) to LA (conditioned stimulus, 100 µM) after 7 weeks of high fat diet (60% fat) feeding. In the assays, combined genders WT mice showed significant aversion to LA from 10 µM, however, GHSR-/- mice only showed trending aversion from 30 µM. This indicates that GHSR-/- mice cannot detect LA as well as WT, or have lower preference to LA. Interestingly, female mice had aversion to even higher concentrations (30 µM in WT vs. 100 µM in GHSR-/-) of LA. In


summary, these preliminary data suggest that ghrelin-GHSR signaling may play a pivotal role in taste sensation, and assist regulating eating behavior.


POSTER PRESENTATION ABSTRACTS Structure and dynamics of a nuclease-resistant RNA from arthropod-borne Flaviviruses BM Akiyama1 and JS Kieft1 1Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Anschutz Medical Campus Many viruses use non-coding RNAs to manipulate host cell processes. An example of one such RNA is found in arthropod-borne flaviviruses (FVs), a class of virus responsible for serious public health threats including Dengue, Yellow Fever, West Nile Virus, and Zika virus. FVs produce subgenomic flavivirus RNAs (sfRNAs), RNA fragments ~200-500 nts in length which have been found to be responsible for the pathology of FV infection in model infection systems. sfRNAs are generated by cleavage of the flaviviral genome by the 5’resist Xrn1 degradation, halting Xrn1 cleavage and leaving the sfRNA fragment behind. These RNA elements are known as Xrn1-resistant RNAs (xrRNAs), and mutations to xrRNAs eliminate the accumulation of sfRNA fragments during FV infection. Recently, a high-resolution structure demonstrated that xrRNAs resist Xrn1 decay through a unique fold which presents a mechanical unfolding problem to Xrn1. In order to better understand the manner in which this fold resists Xrn1 degradation, we have developed a single-molecule Forster resonance energy transfer (FRET) assay to characterize the stability of the xrRNA fold and determine the contribution of various conserved elements of the RNA. We will also determine whether the xrRNA achieves Xrn1 resistance through mechanical stability alone, or if there are regulated levels of RNA dynamics within the structure. In support of the single-molecule studies, we are also conducting high resolution structural studies in order to determine the structure of xrRNAs in an alternative folding state.

Microbiota-Derived Tryptophan Metabolites Activate Aryl Hydrocarbon Receptor and Induce IL-10 Receptor Expression in Intestinal Epithelia Erica E. Alexeev1, Jordi M. Lanis1, Douglas J. Kominsky2, Sean P. Colgan1 1Department of Medicine, University of Colorado, Anschutz Medical Campus, 2Department of Microbiology and Immunology, Montana State University Endogenous tryptophan metabolites play an important role in gut immune homeostasis, yet the role of tryptophan metabolites from host microbiota have not been extensively explored. Indole-3-aldehyde (IAld), a tryptophan metabolite produced by anaerobic intestinal bacteria, is known for its intercellular signal activity and serves as an aryl hydrocarbon receptor (AHR) ligand. The physiological function of AHR includes roles in immune regulation and mucosal barrier function. Further, IL-10 is an anti-inflammatory cytokine that inhibits production of numerous pro-inflammatory mediators in various cell types, including intestinal epithelial cells (IECs). This cytokine functions through binding to the IL-10 receptor alpha subunit (IL-10R1). An unbiased metabolomic profile of whole colon tissue from healthy and colitic mice identified significant decreases in several microbial-derived indole derivatives. Guided by these results, we hypothesized that indole-containing tryptophan metabolites regulate epithelial IL-10R1 expression. Human IECs were exposed to IAld and examined for expression of IL-10R1. These results revealed a prominent induction of IL-10R1 mRNA and protein expression following IAld treatment. IL-10R1 expression was diminished when IAld was administered to AHR nuclear translocator (ARNT) knockdown cells. Likewise, the AHR inhibitor, CH-223191, significantly reduced IL-10R1 expression in cells exposed to IAld. Together, these results provide strong evidence for the role of tryptophan-indole derivatives in anti-inflammatory pathways mediated by IL-10 signaling in IECs. The discovery of tryptophan metabolites as AHR ligands provides new insight to our understanding of the host-microbial axis during inflammatory processes. These studies also identify possible avenues for incorporating the indole-AHR


pathway as novel therapies for mucosal disease.

RAD IS AN AGENT OF SKELETAL MUSCLE ATROPHY D. Beqollari1, C.F. Romberg1, S. Perni2, C. Franzini-Armstrong3 and R.A. Bannister1 1Department of Medicine-Cardiology Division University of Colorado School of Medicine Aurora, CO, USA 2Department of Physiology and Biophysics University of Colorado School of Medicine Aurora, CO, USA 3Department of Cell and Developmental BiologyUniversity of PennsylvaniaPhiladelphia, PA, USA Rad, a member of the RGK (Rad, Rem, Rem2, Gem/Kir) family of small GTP binding proteins, is expressed at very low levels in “normal” skeletal muscle. However, Rad expression is substantially upregulated in muscle of sporadic ALS patients and two established familial ALS mouse models just prior to presentation of symptoms. Rad is a potent, endogenous inhibitor of skeletal muscle L-type Ca2+ channels (CaV1.1). Recently, downregulation of CaV1.1 expression via exon skipping was reported to cause profound atrophy and fibrosis. In light of the increased Rad expression in ALS muscle, we investigated the effect(s) of long-term elevated Rad expression in otherwise normal skeletal muscle. To do so, we injected a serotype 1 Adeno-Associated Virus encoding a muscle-specific, tMCK promoter-driven Venus-Rad fusion construct into various mouse hindlimb muscles. Five months post-injection, we observed profound atrophy of V-Rad-expressing tibialis anterior (TA) muscles. These TA muscles displayed extensive fibrosis and considerable reductions in fiber diameter and area relative to naïve and GFP-expressing control TA muscles. Examination of V-Rad-expressingextensor digitorum longus fibers in thin sections revealed a general disruption of muscle ultrastructure. L-type currents and intramembrane charge movement were both reduced (~55% and ~34%, respectively) in V-Rad-expressing flexor digitorum brevis (FDB) fibers in comparison to naïve and GFP-expressing fibers. Likewise, depolarization-induced myoplasmic Ca2+ transients were reduced (~55%) in V-Rad–expressing FDB fibers. Taken together, our results indicate that Rad can engage atrophic signaling in skeletal muscle, perhaps as an effect of inactivity due to impaired EC coupling.

Metabolic Processes and Physiological Parameters Involved with the Ischemia/Reperfusion Protective Phenotype in Hibernators LK Bogren1, A D'alessandro2, T Nemkov2, KC Hansen2, SL Martin1 1 Cell and Developmental Biology, 2 Biochemistry and Molecular Genetics Hibernating mammals exhibit profound physiological plasticity. Hibernators cycle between summer homeothermic and winter heterothermic seasons bracketed by fall and spring transition periods in which physiological and environmental parameters are mixed. Winter heterothermy arises from cycles between long periods of cold torpor and brief arousals to euthermia. During torpor, organ blood perfusion decreases by ~90% (ischemia) and is rapidly re-established during arousal (reperfusion). Hibernators are resistant to ischemia/reperfusion (I/R) injury during the winter heterothermic season. Neither the molecular drivers underlying I/R resistance nor those in the fall transition that enable torpor are known. Here, GC/LC-MS plasma metabolomics data from 11 physiologically distinct points in the hibernator’s year were compared to biomarkers of I/R. Fall animals were used to: 1) identify changing physiological and environmental factors that correlate to the metabolome of the protective winter phenotype; and 2) determine if experiencing I/R alters the metabolome. We found that, during arousal, succinate concentrations changed consistent with its utilization during reperfusion but there were no increases in biomarkers of I/R injury. In fall animals, Tb was the only measured factor correlated with the percent winter protective metabolome. There was no difference in metabolome between fall animals that had or had not experienced I/R from torpor and arousal. Results suggest the I/R protective phenotype may be due in part to a combination of reduced glycolytic energy production and increases in oxidative stress mitigation that are independent of experiencing an initial torpor bout in the fall but are


instead related to changes in Tb.

Interleukin-19: A Novel Sexually Dimorphic Cardiac Cytokine DR Bruns, AR Ghincea, CV Ghincea, SF Thoemmes, LA Walker University of Colorado-Denver, Department of Medicine, Cardiology, 12700 E 19th Ave, Aurora, CO, 80045, USA. Pro-inflammatory cytokines contribute significantly to cardiac pathophysiology whereas anti-inflammatory cytokines have positive effects on cardiac remodeling. Interleukin-19 (IL-19) is a recently described member of the anti-inflammatory IL-10 cytokine family. There is significant controversy surrounding the role of IL-19 in cellular signaling as it has been shown to be both pro- and anti-inflammatory depending on cellular context. To date, the role of IL-19 in the heart has not been characterized. We show that IL-19 is expressed in the rodent and human heart. Further, we show that the relative expression of the two IL-19 receptor isoforms (IL-influences on cell phenotype, manifest differently in the heart by sex. Treatment of mouse cardiac myocytes and fibroblasts with recombinant IL-19 activates STAT3, a canonical downstream IL-19 signaling cascade, suggesting cardiac IL-19 signaling is intact. In a female-centric model of heart failure, the dominant-negative CREB (dnCREB) transgenic mouse, IL-19 expression is upregulated compared to control. Therefore, we generated a double transgenic (DTG) mouse of IL-19 knockout and dnCREB to test the hypothesis that IL-19 is a key regulator of cardiac dysfunction in female mice. Survival analyses suggest male DTG mice had accelerated, while female DTG showed similar mortality, as their corresponding dnCREB cohort. Together, these data suggest IL-19 is an important cytokine in sex-specific cardiac dysfunction. Ongoing investigations will elucidate the mechanism(s) of sex-specific IL-19 mediated cardiac remodeling.

MMTV-PyMT and Derived Met-1 Cells as Immunocompetent Models of Androgen Receptor-Positive Triple-Negative Breast Cancer JL Christenson1*, KT Butterfield1, NS Spoelstra1, JD Norris2, JS Josan3, JA Pollock4, BS Katzenellenbogen5, JA Katzenellenbogen5, JK Richer1 1Department of Pathology, University of Colorado Anschutz Medical Campus2Deparment of Pharmacology and Cancer Biology, Duke University3Department of Chemistry, Virginia Tech University4Department of Chemistry, University of Richmond5Deparment of Molecular and Integrative Physiology, 6Department of Chemistry, University of Illinois*Corresponding author Triple-negative breast cancer (TNBC) has a poorer prognosis and a faster rate of metastasis compared to other molecular subtypes of BC. Because TNBC lacks expression of the traditional BC biomarkers, no effective targeted therapies are currently available. Recent data indicate that the androgen receptor (AR), expressed in approximately 32% of TNBCs, promotes TNBC survival and may serve as a potential therapeutic target. Unfortunately investigations into the role of AR in TNBC progression are hindered by the lack of available in vivo models with functional immune systems. In this study we examined whether the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) model of BC and Met-1 cells derived from this model might serve as models for the study of AR+ TNBC. IHC was used to examine protein expression in late-stage MMTV-PyMT primary tumors and lung metastases. Tumors and metastases expressed high levels of AR, but little to no estrogen receptor (ER) and progesterone receptor (PR). Receptor expression in Met-1 cells showed similar expression patterns. Met-1 sensitivity to dihydrotestosterone and anti-androgen therapy confirmed a reliance on AR for growth. Furthermore, an orthotopic xenograft experiment with Met-1 cells was used to examine the effects of anti-androgens on tumor growth. Together, these data suggest that the MMTV-PyMT model and Met-1 cells may serve as in vivo models of AR+ TNBC.


Unique Requirements for Listeria Induction of Natural Killer Cell IL-10 Production SE Clark1, LL Lenz1 1Immunology and Microbiology Natural killer (NK) cells modulate immune responses through the production of both pro- and anti-inflammatory cytokines. Bacterial infections can induce NK cell activity, but the consequences of this activation on protective immunity are unclear. We find that infection with the bacterial pathogen Listeria monocytogenes (Lm) results in systemic, NK cell-dependent, cytokine responses. The cytokines we observe include early IFN-gamma followed by a later shift to IL-10. Lm infection of dendritic cells (DCs) in vitroinduces activation of purified NK cells in co-culture with similar kinetics. NK cell IL-10 is detrimental to host protection, and the goal of this project is to determine the requirements for Listeria activation of this response. We found that IL-18, STAT3, and STAT1 signaling are critical for Listeria-induced activation of NK cell IL-10 production. We also observed that IFN-gamma signaling suppresses NK cell IL-10 production. These data indicate that IL-18, which is required for Lm-induced NK cell IFN-gamma and IL-10 production, drives NK cell activation, while IFN-gamma limits secretion of IL-10 at early timepoints. We also found that cell-cell contact is not required for NK cell IL-10 production, in contrast to IFN-gamma production. These data suggest that extrinsic factors drive IL-10 secretion by NK cells in response to Listeria infection. Ongoing work aims to identify these extrinsic factors and further define how NK cells are induced to switch from pro- to anti-inflammatory cytokine production. This work was funded by NIAID of the NIH under award number F32AI114075

The Influence of Neddylation and the NF- pathway on the Mucosal Inflammatory Response VF Curtis1, SF Ehrentraut2, J Onyiah1, EL Campbell1, LE Glover1, and SP Colgan1 1 Mucosal Inflammation Program, University of Colorado, Aurora, CO, USA, 2 Department of Anesthesiology, University Hospital Bonn, Germany Recent work has revealed a central role for neddylation (the conjugation of a Nedd8 protein to Cullin proteins) in the induction of the NF- -1) and stabilization of hypoxia-inducible factor (HIF, via Cullin-2) during inflammatory responses. Our recent work indicated that pharmacological stabilization of HIF with the neddylation inhibitor MLN4924 significantly abrogated colitis endpoints in mice. Here, we addressed the contribution of Cullin-1 neddylation and NF-signaling to mucosal inflammatory responses. Initial in vitro studies using western blot analysis and luciferase reporter assays were performed on Caco-2 cells. Barrier function of T84 cells treated with cytokines and MLN4924 were measured by transepithelial resistance (TER) and permeability assays. Finally, in vivo administration of MLN4924 (3 mg/kg/day) in a TNBS colitis model was performed to evaluate its influence on disease severity. In vitro assays revealed that MLN4924 prominently induces the deneddylation of Cullin-1 and inhibited NF-function as demonstrated by decreased TER and increased. Increased caspase-3 activity contributed to these observations and was attenuated in the presence of a caspase-3 inhibitor. In vivo treatment with MLN4924 significantly increased disease severity as measured by increased colon shortening and mortality early in the inflammatory response (day 2). Histologic analysis of the colon revealed that neddylation inhibition results in increased tissue damage and significantly increased mucosal apoptosis as determined by TUNEL and cleaved caspase-3 staining, particularly within the epithelium. These studies reveal that Cullin-1 neddylation, and most particularly NF- g, is protective during mucosal inflammatory responses.

The L-arginine Metabolic Axis in Macrophages Regulates Pathogenicity of Cutaneous Leishmaniasis BJ Davenport1, N May1, M McCarthy1, L Liu1, J Jones-Carson1, A Vazquez-Torres1, TE Morrison1


1Immunology & Microbiology The causative agents of cutaneous and visceral leishmaniasis are vector-borne Leishmania parasites. Leishmania parasites infect macrophages and can establish persistent infections in mammalian hosts. Inducible nitric oxide synthase (iNOS), an enzyme that utilizes L-arginine to produce the reactive nitrogen species nitric oxide (NO), and arginase 1 (Arg1), an enzyme that converts L-arginine into polyamines, are critical host determinants of Leishmania pathogenesis. Mice deficient in iNOS are highly susceptible to leishmania infection presumably due to the direct anti-parasitic activities of NO. In contrast, pharmacological inhibition of Arg1 activity reduces the severity of infection. Using mice deficient in iNOS, Arg1, or both enzymes, we investigated how shifting L-arginine metabolic pathways influences progression of Leishmania major (L. major) infection. In contrast to iNOS-deficient mice, L. major infection of mice deficient in Arg1 resulted in less severe lesions and reduced parasite loads in comparison withL. major infected WT mice, suggesting that Arg1 promotes Leishmania infection. To determine if Arg1 protentiates disease severity in the absence of iNOS and NO production, we assessed L. major infection in control mice and mice lacking both Arg1 and iNOS. Remarkably, the increased disease severity in the absence of iNOS is reversed when macrophages lack Arg1 activity. These data suggest that iNOS-mediated production of NO is not directly anti-parasitic. Instead, our findings suggest that the utilization of L-arginine by iNOS restricts Arg1-mediated enhancement of Leishmania infection.

Biochemical, Biophysical, and Infection-Based Studies of the ciRNA-RNase L Complex DR Eiler1 and JS Kieft1 1Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA Many viruses use structured non-coding RNA elements to manipulate cellular machinery for infection and replication. An example is the competitive inhibitor RNA (ciRNA) within the C3 protease coding region of group C enteroviruses, including coxsackievirus (CV) and poliovirus (PV). This 303 nt element competitively inhibits activated RNase L (RL), suggesting pressure to retain the ciRNA sequence to reduce the antiviral response. Understanding the detailed molecular events during infection is key for the advance of new therapies against PV and CV infection and a novel therapy with CV strain A21 against melanoma and prostate cancers. The molecular, biophysical and in vivo features of the ciRNA that inhibits RL are unknown; understanding the interactions between RL and ciRNA is vital in understanding how an RNA is able to directly inhibit an enzyme made to ensure its destruction. We hypothesize that the ciRNA works by occlusion to bind and envelope the area around the RL active site and during infection the ciRNA sequesters and RL. We aim to define the ciRNA elements needed for binding RL and the cellular localization of the RL-ciRNA complex during viral infection. We have identified ciRNA mutations that result in a range of RL inhibition including key regions. We have shown that CVA21 acts akin to PV infection and have used RNA FISH during CVA21 infection of HeLa and melanoma cells to show punctate features consistent with viral replication complexes and stress granules

Connexin36 Gap Junction Coupling Protects Against Cytokine-Induced -Cell Death and Dysfunction with Ca2+ Dependence NL Farnsworth1,2, A Heintz2, RKP Benninger1,2 1Barbara Davis Center for Childhood Diabetes, 2Department of Bioengineering Pro-inflammatory cytokines mediate the decline in islet mass and function in type 1 diabetes.


-cell Ca2+ signaling and insulin secretion across the islet. Gap junction coupling is decreased upon treatment with pro-inflammatory cytokines, leading to disrupted Ca2+ signaling. Gap junctions have been shown to protect against cytokine-mediated apoptosis. The goal of this study is to understand the protection of gap junctions against cytokine-induced apoptosis and disruption to insulin secretion and the role Ca2+ plays in this protection. Apoptosis and insulin secretion were measured in isolated islets from Cx36(+/+) or Cx36(-/-) mice and human islets. Islets were treated for 24h with 0, 0.1, or 1 relative concentration of a cocktail of cytokines (10ng/mL TNF- - -

Native levels of connexin36 protected against cytokine-induced apoptosis and altered insulin secretion in mouse islets. Regulating gap junction coupling with Modafinil in human islets showed similar protection against apoptosis. Decreasing levels of gap junction coupling also correlates with decreased insulin secretion in cytokine treated human islets. Modulating intracellular Ca2+ in the absence of gap junction coupling recovered cytokine-induced cell death and altered insulin secretion. In conclusion, gap junctions protect against cytokine-induced cell death and dysfunction through Ca2+ dependent mechanisms in both mouse and human islets. Our data suggests that modulating connexin36 may reduce the decline -cell mass and function in type 1 diabetes, thereby delaying or preventing the onset of disease. This work was funded by NIH Grants R00 DK085145, R01 DK102950, R01 DK106412, F32 DK102276, and a JDRF Grant 5-CDA-20140198-A-N

Targeting Epigenetic regulator WD-Repeat Containing protein 5 (WDR5) in Myc-driven Medulloblastoma S Ghosh, S Venkataraman, D Birks, N Foreman, R Vibhakar Department of Pediatrics and Section of Pediatric Hematology/Oncology/BMT, Children’s Hospital Colorado and University of Colorado Denver, Anschutz Medical Campus, Aurora, CO. Medulloblastoma (MB) is the most common type of malignant brain tumor that afflicts children. It is classified into 4 distinct molecular subgroups based on genomic and clinical features. Among these, Group 3 MB is poorly understood. Thus, there is a critical need for more effective therapies to combat group 3 MB. Recent studies have identified several epigenetic mechanisms that control MYC driven MB. However, their functions in tumor cell self-renewal and proliferation are poorly understood. To address this, we performed an epigenome wide RNAi screen targeting 406 epigenetic molecules associated with chromatin remodeling and histone modification. Unbiased analysis identified several key regulators of long-term viability in these MYC amplified MB cells. WDR5 was found to be one of the top hits and is critical for methylation of histone-3-lysine-4-mediated transcriptional activation. We established that WDR5 is important for MYC driven MB cell survival. Importantly knockdown of WDR5 attenuates clonogenic growth of tumor cells and prevents formation of neurospheres suggesting an inhibition of self-renewal of the MB cells. Co-immunoprecipitation assay revealed the interaction between cMYC and WDR5. Confocal microscopy using high cMYC medulloblastoma cells also demonstrated that WDR5 co-localizes with cMYC in nucleus. Immunofluorescence shows that the cMYC-overexpressed neural stem cells differentiate upon WDR5 knockdown as a result of an increase in TUJ1 expression. We are now performing more detailed studies on the activity of WDR5 in vitro and in vivo in MYC-driven MB. Further characterization of WDR5 will provide deeper insight into understanding of group3 MB and its therapeutic intervention.


Targeting the long noncoding RNA MALAT1 in ovarian cancer MA Gordon1, DR Cochrane1, JK Richer1 1Pathology Background: Ovarian cancer metastasizes via a distinct mechanism, termed direct seeding, whereby cells must resist anoikis (detachment-induced cell death) in the peritoneal cavity before invading through the mesothelium to metastasize into peritoneal organs. The molecular mechanisms that facilitate these processes are poorly understood. Methods: Modified anti-sense oligonucleotides targeting MALAT1 (metastasis associated lung adenocarcinoma transcript 1) were used to inhibit MALAT1 expression in ovarian cancer cells in vitro. Poly-HEMA coated plates were used to grow cells in forced suspension and microarray profiling was performed on Affymetrix arrays. Migration and invasion assays were performed on an XCelligence Cell Analyzer. Anoikis was measured using Caspase 3/7 assay. Gene expression was measured by qRT-PCR. Datasets on oncomine.org were analyzed for association of MALAT1 gene expression with clinicopathological variables. Results: Expression of the long non-coding RNA (lncRNA) MALAT1 significantly increased after 24 hours in forced suspension culture versus attached culture. MALAT1 knockdown decreased proliferation, migration, invasion, and increased anoikis. MALAT1 knockdown also resulted in decreased expression of the splicing factor RBFOX2, which is associated with epithelial-to-mesenchymal transition, as well as alternative splicing of the pro-apoptotic tumor suppressor gene KIF1B, which contains binding sites for RBFOX2 located within the spliced region of KIF1B. High MALAT1 expression is associated with higher stage of ovarian cancer, increased risk of recurrence, and reduced survival. Conclusions: The lncRNA MALAT1 facilitates a pro-metastatic phenotype in ovarian cancer cells, and it is possible to reverse these characteristics by targeting MALAT1.

Characterizing the Mutational Landscape of 418 Melanoma Tumors using Whole Exome Sequencing Jennifer D. Hintzsche1, Carol M. Amato1, Eric Seelenfreund1, Steven E. Robinson1, Jacqueline A. Turner1, Allison J Applegate1, Rita Gonzalez1, Kasey L. Couts1, Jihye Kim1, Yiqun G. Shellman2,6, Joshua Wisell3,6, Martin D. McCarter4,6, Neil F. Box2,6, John J. Tentler1,6, Aik Choon Tan1,5,6, William A. Robinson1,6 1Division of Medical Oncology, Department of Medicine, School of Medicine, 2Department of Dermatology, School of Medicine, 3Department of Pathology, School of Medicine, 4Department of Surgery, School of Medicine, 5Department of Biostatistics and Informatics, Colorado School of Public Health, 6University of Colorado Cancer Center, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA. The International Melanoma Biorepository and Research Laboratory have collected over 3,300 samples from melanoma patients complemented by extensive clinical data. Whole exome sequencing was completed on 418 tumors and 199 matched normal samples from 330 patients. This cohort is enriched with brain metastases and rare melanoma subtypes. Multiple patient samples before and after targeted or immunotherapy treatment were also included in this cohort. We have sequenced 88 primary tumors, 145 brain metastases, 103 subcutaneous tissue, 38 lymph nodes, 16 gastrointestinal tract metastases, and 28 other metastatic sites. Subtypes of melanoma samples consisted of superficial spreading melanoma (106), nodular (57), unknown primary (56), mucosal (41), and acral (29). From the sequencing results, lymph node metastases had the highest mutational burden (3,959), followed by primary (2,764), brain metastases (2,570), and subcutaneous (2,474) tissues. Mucosal melanoma carried the highest mutational burden (6,256), followed by nodular (3,907), SSM (3,579), acral (3,484) and unknown primary (2,319). Based on the TCGA genomic classification of melanoma subtypes, this cohort could be classified into 36% BRAF mutant, 11% RAS mutant, 19% NF1 mutant and 34% triple-


wild-type. We observed that the NF1 mutant subtype is enriched with TP53 and RB1 mutations, suggesting dysregulation of cell cycle for this subtype. We found that the BRAF mutant subtype is enriched in brain metastases (p < 0.05, chi-square). We also observed that the triple-wild-type subtype is enriched with primary acral melanoma. This study provides the foundation for further investigations into the mutational landscape of various subtypes of melanoma, common metastatic sites, and discovery of potential new novel driver mutations in rare subtypes of melanoma. This work was funded by The Barbra Davis Foundation, The Cancer League of Colorado, The Moore Family Foundation

Deletion of Integrin Linked Kinase (ILK) in neural progenitor cells disturbs neuron glia balance during early development Rashad Hussain and Wendy B Macklin Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, Colorado 80045 Oligodendrocyte precursor cell (OPC) proliferation, maturation and differentiation is resulted by a very organized orchestration of events during early development. Any membranous, sub cellular or nuclear factor dysregulation may cause a major developmental deficit. Therefore, proper characterization of these membrane associated proteins is of utmost importance. Integrin linked kinase (ILK) is a serine/threonine protein kinase, localized within inner margin of cell membrane, thus serves as scaffolding protein/focal adhesion mediator. Its presence has been reported in neurons, astrocytes and oligodendrocytes in the central nervous system. But its exact role during the embryonic development of glial - neuronal profile of brain and spinal cord remain unknown. We selectively knockdown ILK in neural progenitor cell population identified by expression of transcription factor olig1 and 2’3 Cyclic nucleotide phosphodiesterase (CNP). We observed that deletion of ILK severely impairs the number of proliferating (Ki67+) oligodendrocyte precursor cells (OPC) which ultimately results low cell count within different regions of embryonic spinal cord. Deletion of ILK not only reduces the proliferation potential of OPC but also increase the number of apoptotic OPCs, identified by TUNEL staining. Significantly high death rate of embryos has been found Olig1cre-cILK fl/fl mice over CNPcre-cILK fl/fl mice. Furthermore, we demonstrate through different immunohistological markers i.e. NeuN, Pax6, GABA, somatostatin, Mash1, ISLET1, HuCD, expressed by early neuronal cell lineage that the normal neuron-glia balance shift towards more production of neuronal cells in the absence of ILK. In conclusion, ILK is of utmost importance for the normal neuron-glia profile development of CNS. This work was funded by National Multiple Sclerosis Society

Serum Circulating Factors in Children with Dilated Cardiomyopathy Are Important In Modulating Pathological Responses In Cadiomyocytes Xuan Jiang1, Anis Karimpou-Fard55, Armin W. Korst1, Joseph C. Wu 4, Brian L. Stauffer1,3, Shelley D. Miyamoto2, Carmen C. Sucharov1 1 Division of Cardiology, Department of Medicine, University of Colorado Denver, Aurora, CO2 Department of Pediatrics, University of Colorado School of Medicine, Children’s Hospital, Aurora, CO3 Division of Cardiology, Denver Health and Hospital Authority, Denver, CO4 Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA5 Division of Pharmocology, Department of Medicine, University of Colorado Denver, Aurora, CO Stimulation of the renin-angiotensin- -adrenergic receptors plays an important role in adult heart failure (HF). Despite the demonstrated benefits of RAAS inhibition and b-


adrenergic receptor blockade in adult HF patients, no substantial improvement in survival rate has been observed in children with HF. This suggests that the underlying mechanism is uniquely regulated in pediatric HF, which hinders our ability to develop new drug therapies that can prevent or reverse the progression of the disease. The myocardium of children with IDC demonstrates a gene expression pattern consistent with myocardial remodeling: increased atrial natiuretic factor (ANF) an -myosin

- -MHC. Here, we show that treatment of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) with serum from pediatric IDC patients induces pathologic changes in gene expression similar to changes in explanted heart tissues, and serum-

-adrenergic systems. We also show that treatment with serum from pediatric IDC patients promotes cellular hypertrophy in NRVMs. Microarray analysis of circulating miRNA suggests that miRNAs are differentially regulated in pediatric IDC patients, and the aptamer-based screening of serum proteins showed up-regulation of proteins involved in the activation of maladaptive inflammatory signaling pathways. Therefore, it is reasonable to propose that serum circulating factors are important in cardiomyocyte remodeling in pediatric IDC patients.

Dysbiosis alters mucosal immunity towards development of rheumatoid arthritis WK Jubair1, JD Hendrickson1, S Acharya1, NK Banda1, CE Robertson2, DN Frank2, and KA Kuhn1 1 Divisions of Rheumatology, Department of Medicine, University of Colorado School of Medicine, Aurora, CO 2 Divisions of Infectious Diseases, Department of Medicine, University of Colorado School of Medicine, Aurora, CO Rheumatoid arthritis (RA) is an autoimmune disease that is associated with variable degrees of morbidity. Identifying preclinical immune changes are crucial to minimize and control RA chronic manifestations. Multiple studies suggest that RA may result from microbial-mucosal interactions: microbial dysbiosis occurs in new onset seropositive RA as does an expansion of mucosal autoantibodies and plasmablasts. Using the murine collagen-induced arthritis (CIA) model of RA, we found that microbiome diversity changes quickly after immunization with bovine type II collagen, at the time when disease-related autoantibodies develop, but before clinical CIA is evident. Furthermore, during the development of murine CIA, increased intestinal permeability and altered cytokine profiles paralleled disease development and microbiota alterations. Taken together, these data suggest a model in which intestinal dysbiosis stimulates mucosal immune responses that drive the development of pathogenic autoantibodies. Future studies are aimed at identifying specific bacterial species and elucidating the pathway by which they stimulate autoantibody production.

Manipulation of NOS activity in the context of glucose stress alters AMPK and mitochondrial oxphos protein content AC Keller1,2, LA Knaub1,2, CA Connon1,2, JEB Reusch1,2 1Department of Endocrinology, Metabolism & Diabetes, 2Denver VA Medical Center Diabetes (DM), a worldwide health threat, confers an excess risk of cardiovascular disease, preceded by dysfunction in vascular contractility. Nitric oxide (NO), produced by nitric oxide synthase (NOS), regulates vascular contractility and mitochondrial function. In DM, NOS may become uncoupled, decreasing NO production. It is unknown whether restoration of NOS activity and/or mitochondrial function will improve vascular function in DM. We hypothesized that Sepiapterin, a NOS enzyme cofactor, and/or -(-) epicatechin (EPICAT), a flavonoid plant compound, would recouple NOS and clarify the consequences of NOS uncoupling by elevated glucose. We incubated endothelial cells with either 1 mM of Sepiapterin or 0.1 and 1.0 µM of EPICAT in control (7 mM, LG) or high glucose (30 mM, HG) environments for 30 minutes and 4 hours. As expected, Sepiapterin increased NO production 2.26 and


1.44 fold over control cells at 30 minutes and 4 hours, respectively, with decreased AMPK activity (p<0.08 and <0.05, respectively). EPICAT incubation decreased AMPK and eNOS activity, increased SIRT3 and decreased MnSOD expression (p<0.05 for all) after 30 minutes. After 4 hours, EPICAT decreased eNOS activity and mitochondrial complexes II, III, IV, and V (p<0.05 for all). Manipulation of eNOS and NO production in cells exposed to glucose mediated stress results in inhibition of the AMPK/eNOS/PGC-compounds may modulate the cells' adaptation to nutrient stress by supporting NOS function or by restoring multiple points in a particular signaling pathway. Current work investigates further cellular signaling and alterations in mitochondrial respiration. This work was funded by Colorado Nutrition Obesity Research Center Pilot Project Program

Tryptophan Metabolite Activation of the Aryl Hydrocarbon Receptor Regulates IL-10 Receptor Expression on Intestinal Epithelia JM Lanis1,2, EE Alexeev1,2, VF Curtis1,2, DA Kitzenberg1,2, DJ Kao1,2, ME Gerich1,2, DJ Kominsky1,3#, SP Colgan1,2# 1Mucosal Inflammation Program, 2Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO; 3Department of Microbiology and Immunology, Montana State University, Bozeman, MT. IL-10 is an anti-inflammatory cytokine that inhibits the production of pro-inflammatory mediators in various cell types. Studies in animals and humans have demonstrated a critical role of IL-10 signaling in chronic inflammatory conditions, including inflammatory bowel disease. Signaling by IL-10 occurs through the IL-10 receptor (IL-10R), which is expressed in numerous cell types, including intestinal epithelial cells (IEC), where it is associated with mucosal barrier function. Our recent studies revealed that colonic IEC express IL-10R1 on the apical cell surface in response to IFN-understood mechanisms. Guided by an unbiased metabolomics screen, we identified tryptophan (Trp) metabolism as a major modifying pathway in IFN- - dominant murine colitis. In parallel, we demonstrated that IFN-(Kyn). Based on these findings, we hypothesized that IL-10R1 expression on IEC is regulated by Trp metabolites. Analysis of the promoter region of IL-10R1 revealed an aryl hydrocarbon response element (AHRE), which can be induced by endogenous Kyn. Luciferase-based IL-10R1 promoter assays showed an increase in gene expression in response to the AHR agonist FICZ. Additionally, western blot analysis confirmed that IL-10R1 protein levels were increased in response to FICZ in IEC in vitro. Importantly, studies utilizing an in vitro wound model demonstrated that FICZ accelerates cell proliferation and wound closure in the presence of IL-10. Together, these results provide compelling evidence on the importance of IL-10 signaling in intestinal epithelia and implicate AHR in the regulation of IL-10R1 expression in the colon. This work was funded by This work was supported by NIH grants DK099452, DK50189, DK095491, VA Merit BX002182 and by the Crohn’s and Colitis Foundation o

A Putative Role for Glutathione in the Cancer Stem Cell Phenotype in Luminal Breast Cancer SB Matthews1, J Finlay-Schultz1, and CA Sartorius1 1Department of Pathology, University of Colorado Denver - Anschutz Medical Campus Breast cancer is the most commonly diagnosed cancer in women and the second most common cause of cancer death. Most breast tumors are diagnosed in postmenopausal women, express the estrogen receptor (ER+), and are initially treatable with endocrine-targeting drugs. However, most breast tumors contain populations of cancer cells that exist in a state of prolonged dormancy, which display resistance to most conventional chemotherapeutic or endocrine-targeted drugs. Expansion of these quiescent populations, called cancer stem cells (CSCs), favors breast cancer recurrence >5-10 years after initial


diagnosis and primary tumor removal, at which time to survival is <2 years. Using a GFP-based promoter-reporter to identify and isolate CSCs with a basal-like phenotype (cytokeratin 5 (CK5+)) within ER+ breast cancer cell line T47D and ER+ patient-derived xenografts (PDX), gene-set enrichment analysis of metabolomics data revealed that CK5+ cell populations from cultured cells and PDX are enriched in the glutathione antioxidant pathway, a pathway that has been associated with a benefit in breast cancer progression. Compared to CK5- cells, CK5+ cells display elevated reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG), and CK5+ cells display

-glutamyl-cysteine ligase (GCL), the rate-limiting enzyme for glutathione synthesis. These data suggest that the glutathione pathway may be involved in enabling or maintaining the cancer stem cell phenotype, and that perturbation of the glutathione pathway may be a strategy for targeting CSC populations in luminal breast cancer. This work was funded by NIH 5T32007446-34

Disrupted Architecture and Function of Draining Lymphoid Tissue During Chronic Chikungunya Virus Infection MK McCarthy1, DW Hawman1, SA Elmore2, TE Morrison1 1Department of Immunology & Microbiology, University of Colorado School of Medicine and 2The National Institute of Environmental Health Sciences Chikungunya virus (CHIKV) causes large outbreaks of rheumatologic disease, resulting in chronic disease symptoms in up to two-thirds of infected individuals. Studies in humans and animal models suggest that CHIKV infection persists in musculoskeletal tissues, although the mechanisms are unclear. The pathogenic clinical isolate AF15561 persists in joint tissue of WT mice, while the attenuated strain 181/25 is cleared. In contrast, 181/25 persists in joint tissue of Rag1-/- mice at levels similar to AF15561, suggesting that adaptive immune responses are critical for clearance of 181/25 but are impaired during AF15561 infection. We hypothesized that early events in lymphoid tissue during persistent or acute CHIKV infection are distinct and these differences contribute to CHIKV persistence or clearance in joint tissue. To test this hypothesis, we performed functional and histological analyses of the draining lymph node (dLN) during acute and persistent CHIKV infection. In mice infected with 181/25, we detected activation of CD21+CD23+ conventional follicular B cells and increased numbers of Tfh cells. In contrast, activated B cells in the dLN of AF15561-infected mice were CD21lo/-CD23- and Tfh cell numbers were reduced. Acute 181/25 infection also resulted in increased cortex area, plasma cells, and germinal center (GC) formation in the dLN while the dLN of AF15561-infected mice was depleted of lymphocytes and became fibrotic. Following NP-OVA immunization, development of NP-specific GC B cells was decreased during AF15561, but not 181/25 infection, suggesting that persistent CHIKV infection impairs dLN function. These data provide new insight into the immunologic mechanisms that facilitate CHIKV persistence in joint tissue.

Impact of Interferons on Listeria monocytogenes cell-to-cell spread DS McDermott1 and LL Lenz1 1Department of Immunology and Microbiology, Anschutz Medical Campus, University of Colorado-Denver, Denver, CO Microbes have developed diverse strategies to circumvent or co-opt immune mechanisms in order to infect and persist within macrophages and other host cells. Listeria monocytogenes utilizes the virulence factor ActA to polymerize actin, consequently forming actin tails that propel the bacterium from its host cell into an adjacent cell in a process termed cell-to-cell spread. Previous studies from our laboratory have demonstrated that the addition of Toll-like receptor-agonists such as lipopolysaccharide (LPS) enhances cell-to-cell spread in a nitric oxide (NO)-dependent manner. However, the mechanism of LPS-enhanced cell-to-cell spread is unknown. Initial studies determined that STAT1 is required for


LPS-enhanced cell-to-cell spread. Given the role of STAT1 in type I interferon (IFN) and IFN-gamma signaling, we sought to examine the role of these cytokines in LPS-enhanced cell-to-cell spread. Macrophages lacking the IFN-alpha receptor fail to induce NO upon LPS stimulation, and do not exhibit LPS-enhanced cell-to-cell spread. This indicates a vital role for type I IFNs in mediating LPS-induced NO therefore driving the LPS-enhanced cell-to-cell spread. In contrast to type I IFNs, IFN-gamma enhances NO production from macrophages when given prior to or at the same time as LPS. Interestingly, despite the enhanced NO production IFN-gamma inhibits the LPS-enhanced cell-to-cell spread. Furthermore, IFN-gamma inhibits NO donor-enhanced cell-to-cell spread, suggesting that IFN-gamma overrides the NO effects during LPS-enhanced cell-to-cell spread. Together these data demonstrate that IFNs play a contrasting role, with type I IFNs facilitating LPS-enhanced cell-to-cell spread and IFN-gamma inhibiting LPS-enhanced cell-to-cell spread.

An RNA matchmaker protein regulates the activity of the long noncoding RNA HOTAIR EK Meredith1, MM Balas1,2, K Sindy1, K Haislop1, and AM Johnson1,2 1Department of Biochemistry and Molecular Genetics,2Molecular Biology Program The human long noncoding RNA (lncRNA) HOTAIR acts in trans to recruit the Polycomb Repressive Complex 2 (PRC2) to the HOXD gene cluster and promote gene silencing during development. In breast cancers, overexpression of HOTAIR increases metastatic potential via the repression of many additional genes; yet, it has remained unclear what factors determine HOTAIR-dependent gene silencing at specific genomic loci. To identify additional proteins that contribute to the specific action of HOTAIR, we performed a quantitative proteomic analysis of the HOTAIR interactome. We identified a strikingly specific interaction between HOTAIR and the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, a member of a family of proteins involved in nascent mRNA processing and RNA matchmaking. Our data suggest that A2/B1 are key contributors to HOTAIR-mediated chromatin regulation in breast cancer cells: A2/B1 knockdown reduces HOTAIR-dependent breast cancer cell invasion and decreases PRC2 activity at the majority of HOTAIR-dependent loci. We found that the B1 isoform, which differs from A2 by 12 additional amino acids, binds with highest specificity to HOTAIR. B1 also binds chromatin and associates preferentially with RNA transcripts of HOTAIR gene targets. We furthermore demonstrate a direct RNA-RNA interaction between HOTAIR and a target transcript that is enhanced by B1 binding. Together, these results suggest a model in which B1 matches HOTAIR with transcripts of target genes on chromatin, leading to repression by PRC2. This work was funded by DOD Postdoctoral Fellowship to EM

Indirect and Direct Nanomaterial Interaction Influences Vascular Reactivity VC Minarchick1, PA Stapleton2, JH Shannahan1, EM Sabolsky3, TR Nurkiewicz2, JM Brown1 1 UC Denver, Dept. of Pharmaceutical Sciences, Aurora, CO2 WVU Center for Cardiovascular and Respiratory Sciences, Morgantown, WV3 WVU Department of Mechanical Engineering, Morgantown, WV Cerium dioxide nanoparticles (CeO2 NP) have been shown to cause vascular dysfunction. However, it is unknown if this dysfunction is due to circulating factors and/or direct nanomaterial interaction. We hypothesized that the vascular dysfunction is due to endothelial changes, which may interrupt cellular signaling, and these alterations (i.e. oxidative stress, inflammatory markers) will be dependent on the physicochemical properties of the nanomaterial. Rat aortic endothelial cells (RAEC) were exposed to CeO2NP (0, 6.25, 12.5, 25, and 100 µg/ml) for 24 hours. Cytotoxicity, alterations in inflammatory and surface adhesion markers (IL-6, VCAM-1, and ICAM-1 via mRNA expression and western blot analysis), nitric oxide synthase (NOS) expression, and reactive oxygen species (ROS) generation (via mitochondrial superoxide release and cellular hydrogen peroxide production) were assessed following


exposure. Additionally, isolated naïve arterioles were intraluminally exposed to CeO2 NP (0, 6.25, 12.5, 25, and 100 µg/ml) or the supernatant from exposed RAEC. Endothelium-dependent and –independent reactivity was assessed with acetylcholine (ACh, 10-9-10-4 M), and sodium nitroprusside (SNP, 10-9-10-4

M). Finally, enhanced dark field microscopy was used to determine CeO2 NP cellular uptake. Following exposure, RAEC had no change in cellular viability and NOS expression. There was a significant increase in IL-6 production and approximately a 20% decrease in ROS generation at the lowest concentration. Furthermore, CeO2 NP exposure significantly impaired ACh (75±8% vs. naïve control) and SNP (58±5% vs. naïve control) induced dilation. Taken together, these results indicate that both direct and indirect CeO2 NP interactions likely alter cell-cell and/or intracellular signaling within the vascular wall. This work was funded by R01-ES019311 (JMB), R01-ES015022 (TRN), K99-ES024783 (PAS), K99-ES024392 (JHS)

Enhanced phenotypic analysis of islet antigen specific CD8+ T cells BV Murphy1,3, AC Hohenstein1,3, M Dang1,3, T Sosinowski1,3 & HW Davidson1,2,3. University of Colorado Anschutz Medical Campus, Dept. of Pediatrics1, Immunology2 and Barbara Davis Center for Childhood Diabetes3. T1D results from T cell mediated destruction of pancreatic b-cells. However, at present biomarkers based on monitoring islet-reactive T cells in humans are generally considered unreliable. While many assays have been developed over the years that can discriminate disease specific T cells from those in healthy controls, most have high degrees of variation and cannot accurately predict disease progression or therapeutic efficacy in longitudinal studies. Thus, there is a critical need to develop improved assays that are robust, reproducible and can accurately reflect disease progression and be used clinically to stratify subjects and measure therapeutic efficacy. We propose that functional assays using panels of “Antigen Presenting Cell Avitars” (aAPCs) can provide a solution to this problem. To test this hypothesis we have created a set of ~30 standardized aAPCs (based on a parental K562 cell line engineered to constitutively express CD80 and CD83) that each express up to 4 defined single chain peptide:b2 microglobulin:MHC class I heavy chain trimers (SCTs). Such aAPCs can provide strong and immediate stimulus to the T cells, through their expression of high levels of peptide-MHC (pMHC) with no necessary processing or competition for binding of the peptide. Our initial data demonstrate that this system can readily detect viral antigen-specific CD8+ T cells in cryopreserved blood from asymptomatic human HLA-A2+ subjects, with greater sensitivity than using endogenous APCs and soluble peptides. Similarly, we can detect preproinsulin specific CD8+ T cells in PBMCs from patients with type 1 diabetes that have been cryopreserved for over 2y, suggesting that our assay may be suitable for use in future clinical trials.

Acute effects of kainic acid induced early life seizures H O'Leary, Bernard PB, Castano AM, Benke TA Departments of Pediatrics, Neurology, Pharmacology Neonatal seizures are associated with long term disabilities including epilepsy and cognitive deficits. Using a neonatal seizure rat model that does not develop epilepsy, but develops a phenotype consistent with other models of intellectual disability (ID) and autism spectrum disorders (ASD), we sought to isolate the acute effects of a single episode of early life seizure on hippocampal CA1 synaptic development and plasticity. In the adult rat, we have previously shown chronic changes in glutamatergic synapses, loss of long term potentiation (LTP) and enhanced long term depression (LTD), ~50 days following kainic acid (KA) induced early life seizure (KA-ELS) in post-natal (P) 7 day old male Sprague-Dawley rats. In the present work, we examined the electrophysiological properties


and expression levels of glutamate receptors in the acute period, 2 and 7 days, post KA-ELS. Our results show for the first time enhanced LTP 7 days after KA-ELS, but no change 2 days post KA-ELS. Additionally, we report that ionotrop -amino-3-hydroxy-5-methyl-isoxazole-propionic acid type glutamate receptor (AMPAR) desensitization is decreased in the same time frame, with no changes in AMPAR expression, phosphorylation, or membrane insertion. Inappropriate enhancement of the synaptic connections in the acute period after the seizure could alter the normal patterning of synaptic development in the hippocampus during this critical period and contribute to learning deficits. Thus, this study demonstrates a novel mechanism by which KA-ELS alters early network properties that potentially lead to adverse outcomes.

Tissue specific redundancy of TFAP2 family members during craniofacial patterning E Van Otterloo1, KL Jones2, T Williams1, 3, 4 1 Craniofacial Biology, 2 Biochemistry and Molecular Genetics, 3 Cell and Developmental Biology, 4 Department of Pediatrics Transcription factor activator protein 2 (TFAP2) is a key regulator of craniofacial development, highlighted by the fact that mutations to two family members, TFAP2A and TFAP2B, result in syndromic disorders in humans that include craniofacial dysmorphisms (BOFS and Char syndrome, respectively). In addition, TFAP2 proteins likely contributed to the emergence of neural crest (NC), a key stem-cell population that emerges from the non-neural ectoderm (NNE) during development and attributed to generating the “new head” observed in vertebrates. Although a variety of TFAP2 family members are expressed robustly in both the NC and NNE during development, tissue specific knockouts have resulted in subtle craniofacial defects in animal models. In an effort to better understand the potential for redundancy in masking the functional role of TFAP2 paralogs in these two tissues (i.e. the NNE and NC) we have systematically deleted two family members simultaneously in either the NC or NNE during craniofacial development. Focusing on TFAP2A and TFAP2B, we find striking perturbances in normal craniofacial development, including midface hypoplasia, mimicking to some extent frontonasal dysplasia, upon deletion in the NC, and major patterning defects upon deletion in the NNE. Molecular profiling of the facial prominences in both models identifies key genes and pathways presumably co-targeted either directly or indirectly by TFAP2A and TFAP2B during craniofacial development, including unique targets in either the upper or lower face. Currently, biochemical analysis is being utilized to further uncover this gene-regulatory-network guiding proper facial morphogenesis. Collectively, these findings should provide clues into the molecular pathology associated with BOFS and Char syndrome as well as the role of TFAP2 proteins in the evolution of the vertebrate face.

TGFb2-mediated Epithelial to Mesenchymal Transition of lens epithelial cells: Role of AGE-RAGE interaction Cibin T. Raghavan and Ram H. Nagaraj Department of Ophthalmology, University of Colorado, School of Medicine, Aurora, CO 80045. Purpose. Proteins in basement membrane accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). Our previous study has shown that AGEs in human lens capsule promote TGFb2- mediated epithelial to mesenchymal transition (EMT) of lens epithelial cells. In this study we have investigated the role of a receptor for AGEs (RAGE) in TGFb2-mediated EMT of lens epithelial cells. Methods. Tissue culture plates were coated with a basement membrane extract (BME; 50 mg/ml) overnight and AGE-modified with a glycating mixture (2 mM ascorbate, methylglyoxal) for 7 days at 37°C. FHL 124 cells were cultured on unmodified or AGE-modified BME and treated with 10 ng/ml of TGFb2 for 24 h at 37°C. RNA was isolated, cDNA generated and real time PCR analysis was carried for EMT-associated proteins. RAGE overexpression studies were carried out by transient transfection of GFP-RAGE. Effect of RAGE blockade on TGFb2-mediated EMT response


was studied using a RAGE antibody (10 mg/ml) and EN-RAGE (10 mg/ml), an endogenous ligand for RAGE. Results. Similar to our previous observation in primary human lens epithelial cells, FHL124 cells also

-modified BME than on unmodified BME. RAGE was detected in FHL124 cells and its levels were unaltered in cells grown on either native or AGE-

-mediated EMT response. Moreover, blockade of RAGE with an antibody or EN-RAGE followed by TGFb2 treatment resulted a significant reduction in the EMT response. Conclusions. These results imply that the interaction of matrix AGEs with RAGE plays an important role in TGFb2-mediated EMT of lens epithelial cells and suggest that blockade of RAGE could be a strategy to prevent posterior capsule opacification. This work was funded by NIH Grant EY022061

The role of the electron transport chain in promoting bacterial susceptibility and tolerance to cellular antibiotics MJ Reichlen1, IL Bartek1, MS Scobey1, CR Covey1, and MI Voskuil1 1Immunology and Microbiology The emergence of extensively antibiotic resistant bacteria is a major health challenge of the 21st century. Thus gaining a complete understanding of the primary physiological consequences bacteria must overcome in the face of antibiotic treatment is of critical importance. Recent work from our lab has identified a common mechanism of antibiotic killing among diverse bacterial species wherein cell death from nearly all drugs ultimately stems from intracellular alkalization, that a drug’s bactericidal activity changes as a function of extracellular pH (i.e. increases with increasing pH), and that bactericidal activity is stopped by alleviating alkaline stress through collapse of the H+ gradient at the cell membrane. Here we investigated whether members of the electron transport chain (ETC) promoted antibiotic susceptibility or tolerance using the model organismsMycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb). We tested drug susceptibility patterns of Msm and/or Mtb with individual components of the ETC either genetically deleted or chemically inhibited. We found that deletion or inhibition of ETC components with high H+ pumping capacity (e.g. aa3-type cytochrome c oxidoreductase complex) induced general antibiotic tolerance whereas ETC components that do not couple H+ translocation to electron transfer (e.g. succinate dehydrogenase) lead to a general increase in antibiotic susceptibility. These findings are consistent with our model of antibiotic killing by intracellular alkalization and provide mechanistic insight into how this occurs. Additional findings in this area will be highly beneficial in the search for novel therapeutics and in the rational design of new antibiotic combination therapies.

Reconstructing Chromosome Conformation by Fluorescence Microscopy Brian Ross1, Fabio Anaclerio2, and Andrew Laszlo3 1Pharmacology, 2University of Bari, 3University of Washington Despite the importance of chromosomes little is known about their in-vivo structure. If it were possible to identify and localize many genetic loci under a microscope despite the limited palette of distinguishable fluorophores, then the structure could be solved at some resolution by 'connecting the dots'. Here we report the results of an experiment that used relatively few types of fluorophore (3) to localize a larger number of loci (10) on one chromosome. The extra information that helped to identify each label was the known spacing and color-ordering of the labels along the chromosome, along with a calibration between genetic distance and spatial distance. Simulations indicate that our methodology


can reconstruct and validate much richer conformations based on hundreds or thousands of labeled loci. This work was funded by NHGRI grant R01HG005115 and NLM grant 5T15LM009451

T cell Dependent Signals are Required for the Enrichment of Dual-Reactive B cells in Autoimmune Mice A Sang1, EM Fournier1, R Torres1, and R Pelanda1 1Department of Immunology and Microbiology, University of Colorado, Aurora, CO, 80045. Dual-reactive B cells expressing two different immunoglobulin heavy or light chains are present in healthy humans and mice. However, some patients with systemic lupus erythematosus (SLE) display an enrichment in dual-reactive B cells. To study dual-reactive B cells in a lupus-prone autoimmune

identification and tracking of dual-that dual-and memory B cell compartment of lupus-prone MRL/lpr mice. Current studies showed that T cells play a major role in the enrichment of activated dual- -21 signaling was required for the maximal differentiation of dual- -cells were shown to significantly overexpress the antigen-presenting molecule MHCII in comparison to single- in vivo immunizations against a T-dependent antigen showed that dual- lpr mice preferentially respond to antigen compared to single-results show that T cells, and potentially TLR9 ligation, promote the enrichment of dual-reactive B cells in an autoimmune background.

PPAR-gamma Induced TGF-beta1 Expression in Myeloid Cells Contributes to Lung Cancer Progression and Metastasis TR Sippel1, H Li1,3, D Hanson1, TT Nguyen1, LI Greene1, BL Bullock1, JM Poczobutt1, MCM Weiser-Evans1,2, and RA Nemenoff1,2,4 1Departments of Medicine and 2Pharmacology, University of Colorado Denver, Aurora, CO 80045 and 3Veterans Affairs Medical Center, Denver, CO. Purpose: We have previously reported that targeted deletion of the nuclear receptor PPARg in myeloid cells inhibits tumor metastasis in an immunocompetent orthotopic model of lung cancer in which murine lung cancer cells are directly implanted into the lung. The goal of this study was to identify mechanisms mediating this effect and compare how these mechanisms function in a well-differentiated epithelial lung cancer cell line versus a mesenchymal lung cancer cell line. Experimental Design:

- oduction in tumors. The effects of TGF-epithelial (CMT167) or mesenchymal (Lewis Lung Carcinoma – LLC) lung cancer cells were analyzed in vitro and in vivo through silencing TGF-the small molecule inhibitor SB431542. Results: We detected high levels of TGF-b1 in CMT167 tumors that were dependent on PPARg expression in myeloid cells. Treatment of bone marrow-derived macrophages with tumor homogenate from CMT167 tumors increased TGF-b1 productblocked the ability of TGF-b1 to induce an epithelial-mesenchymal transition (EMT) in CMT167 cells in vitro and slowed tumor growth and metastasis in vivo. However, in mesenchymal LLC cells, TGFbR inhibition had no effect EMT in vitro or metastasis in vivo and decreased primary tumor size. Conclusion: PPARg-dependent production of TGF-b1 by macrophages promotes EMT and more rapid


progression of epithelial, differentiated lung tumors, but has no effect on the metastatic ability of mesenchymal tumors. We conclude that EMT is a critical event in epithelial lung tumor progression and

This work was funded by The University of Colorado Lung Head and Neck Cancer T32CA17468 Training Grant and the NCI

Inhibition Ser293 Phosphorylation of Connexin-36 Prevents Cytokine-Induced Dysfunction of -cells in the Islet of Langerhans JR St. Clair1 and RK Benninger1,2 1Department of Bioengineering, 2Department of Pediatrics During insulitis and the development of diabetes, pro- -cell apoptosis and dysfunction resulting in loss of insulin secretion from the islet of Langerhans. Connexin-36 (Cx36)-containing gap junctions (GJs) are essential for coordinated electrical coupling and pulsatile

-cells; and we have recently shown GJ function is disrupted upon treatment with pro-inflammatory cytokines. Thus, regul -cells may be an important

-cell dysfunction during the development of diabetes. Protein kinase A (PKA)-dependent phosphorylation of Cx36 reduces GJ coupling in the retina, yet its regulatory effect on GJ funct -cells is not understood. Here, we tested the hypothesis that cytokine-induced gap junction

-cells involves PKA-dependent phosphorylation of Cx36 at Serine-293. We used fluorescence recovery after photobleaching (FRAP) in isolated mouse and human islets to show that PKA activation resulted in a significant decrease in GJ coupling that was similar in magnitude to the decrease observed when islets were treated with the pro-inflammatory cytokines TNF- -IFN- the role of Serine-293, we developed an inhibitory peptide targeted against this residue on Cx36 (Ser293i) and found that it prevented the PKA- and cytokine-induced GJ uncoupling. Additionally, treatment of islets with S293i significantly reduced the number of apoptotic cells induced by cytokines. Finally, S293i prevented the cytokine-induced decreases in glucose-stimulated insulin release from isolated islets. These results suggest that regulation of serine-293 on Cx36, plays a role cytokine- -cell death and dysfunction during the development of diabetes. This work was funded by Juvenile Diabetes Research Foundation and the National Institutes of Health

A structural switch in the CCR5 mRNA induces ribosomal frameshifting AL Steckelberg1, MD Ruehle1, AR MacFadden1, JS Kieft1 1 Biochemistry and Molecular Genetics Programmed ribosomal frameshifting (PRF) enables the translating ribosome to change its reading frame at a specific location on a messenger RNA (mRNA). This process usually involves a cis-acting structured RNA element that causes the ribosome to pause downstream of a ‘slippery’ sequence. Although PRF has been mostly described in viruses, a recent study identified a -1 PRF signal in the transcript of the human C-C chemokine receptor 5 (CCR5). PRF is likely to play a role in fine-tuning CCR5 expression by generating a premature translation termination codon which leads to the degradation of the mRNA by nonsense-mediated mRNA decay (NMD). Interestingly, CCR5 frameshifting is increased in the presence of miRNA-1224, which directly binds to the frameshifting signal. However, the mechanism by which miR-1224 regulates CCR5 PRF is not well understood. Based on 2-dimensional chemical probing data, we propose that the structure of the CCR5 PRF signal consists of two consecutive stem loops. The chemical probing pattern changes in the presence of miR-1224, consistent with a structural reorganization of the RNA element. Moreover, we identified a point mutant within the CCR5 PRF signal that leads to a change in chemical reactivities analogous to that


observed upon miR-1224 binding. Of note, this point mutant displays increased frameshifting activity in an in vitro translation assay. Together, our results suggest that the CCR5 PRF signal comprises a structural switch. miR-1224 binding shifts the equilibrium towards the high-frameshifting state and thus regulates CCR5 levels by inducing frameshifting-dependent NMD.

Characterization and Targeting of Myelodysplastic Syndrome Stem Cells Brett M. Stevens1, Daniel Pollyea1, Nabilah Khan1, Craig T. Jordan1 1Hematology Myelodysplastic syndrome (MDS) is a malignant disorder of myeloid hematopoietic cells, resulting in ineffective hematopoiesis leading to bone marrow dysfunction and failure, and frequently evolves to acute myelogenous leukemia. While MDS is thought to arise from a malignant stem cell population, the properties of such cells are not well understood. Thus, we performed studies to better characterize candidate MDS stem cells and allow insights into the development of novel stem cell directed targeted therapies. One line of investigation examined the cytokine receptor CD123, which we have previously shown to be a marker of AML stem cells. Analysis of primary human specimens demonstrates that CD123 is not expressed in the normal stem cell population or in stem cells from low risk MDS patients, but is readily detected in high-risk patients. These data suggest that up-regulation of CD123 may be a hallmark of pathogenesis as MDS progresses toward AML. To better understand the properties of CD123+ vs. CD123- stem cells, we performed global expression studies (RNA-seq) on high-risk MDS patients. The data indicate strong up-regulation of protein synthesis machinery, which indicates a major change in underlying cellular physiology and metabolism. We tested the protein synthesis inhibitors and BCL-2 inhibitors both and demonstrated highly selective eradication of CD123+ stem cells. Taken together, these data show that we have identified two novel pharmacological approaches that may effectively target the MDS stem cell population. Further, these agents may function well in conjunction with commonly used agents used in the treatment of MDS.

Cyclic-di-Nucleotides as STING-dependent Adjuvants and B cell Activators MM Walker, Ph.D*.1,2, BW. Crute1, A Getahun, Ph.D1., and JC Cambier, Ph.D.1 UCD Department of Immunology and Microbiology1, National Jewish Health Center2 Generation of protective immune responses requires coordinate stimulation of innate and adaptive immune systems. An important mediator of innate immunity isSTimulator of INterferon Genes (STING), a ubiquitously expressed adaptor molecule that functions in the relay of signals initiated by sensing of cytosolic DNA and bacterial ci-di-nucleotides. Allelic variants of STING, HAQ-STING and R232H-STING, found in 15-20% of humans appear, based on in vitro studies, to be defective in signal relay, suggesting that carriers may be predisposed to certain infections. Our laboratory is engaged in studying the functions of STING, HAQ-STING and R232H-STING. Here we show that STING promotion of in vivo IgG and IgA antibody responses requires both systemic and B cell expression of STING. Analyses of the possible direct effects ci-di-GMP on B cells revealed that CDNs stimulate upregulation of activation markers (CD69 and CD86) as well as apoptotic death, and these responses are dependent on STING expression. Coordinate BCR signals synergize in induction of B cell activation but spare cells from apoptosis. Consistent with defective function of HAQ-STING, B cells from a newly-produced human HAQ and R231H knockin mouse were defective in mounting responses to ci-di-GMP. Taken together, these data indicate that STING and BCR function coordinately in B cells to promote cell activation and antibody responses. Human carriers of HAQ and R231H STING may be defective in


responses to certain immunogens.

Targeting Tyrosine Phosphorylation of PKC delta for Salivary Gland Radioprotection S Wie1, M Reyland1,2 1Craniofacial Biology, 2Cancer Biology Irradiation (IR) therapy in head and neck squamous cell carcinoma (HNSCC) patients often results in permanent moderate to severe damage to the salivary glands, resulting in xerostomia, chronic oral infections and severe discomfort. Current efforts to mitigate IR induced damage are partially successful yet many patients report experiencing a decline in saliva flow. Our investigatory efforts attempt to understand the underlying mechanisms contributing to functional loss of the salivary glands. We have identified Protein Kinase C-delta (PKCd) as an essential regulator of apoptosis in salivary gland acinar cells. Further, we demonstrated that phosphorylation of tyrosine residues in the regulatory domain of PKCd by c-Abl and c-Src is necessary for apoptotic signaling in response to IR. We set out to demonstrate that pharmacological inhibition of c-Abl and c-Src with FDA approved tyrosine kinase inhibitors (TKIs) can provide a novel therapeutic approach to protect against IR induced xerostomia. Our current study demonstrates that mice treated with TKIs alongside radiation experience a significant improvement in saliva flow rates. Additionally, we found that treatment with TKIs failed to enhance survival of HNSCC cell lines. We conclude that FDA approved TKIs with activity against c-Abl and c-Src offers a novel adjunct therapy for HNSCC patients. Our results support the advancement of these studies into a phase I/II clinical trial with HNSCC patients. This work was funded by NIH/NIDCRIrradiation

A multiplex assay for simultaneous screening type 1 diabetes and multiple autoimmune diseases Zhiyuan Zhao, Dongmei Miao, Aaron Michels, Peter Gottlieb, Marian Rewers, and Liping Yu Barbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine, Aurora, CO Presence of autoantibodies to insulin (IAA), GAD65 (GADA) or IA-2 (IA-2A) is the cornerstone of diagnosis of type 1 diabetes (T1D). In addition, one third of the patients present with autoimmune thyroiditis or celiac autoimmunity at diagnosis of T1D. Availability of a multiplex autoantibody assay could greatly simplify diagnosis of T1D and screening for the associated autoimmune conditions. We developed a 7-plex electrochemiluminescence (ECL) assay that combines, in a single well and using only 6µl of serum, testing for IAA, GADA and IA-2A, autoantibodies to thyroid peroxidase (TPOA),

respectively, markers of celiac disease and autoimmune polyglandular syndrome type 1. The 7-plex assay was validated in new-onset T1D patients (n=168) and healthy controls (n=118). The positive cut-offs for all autoantibodies were set at the 100th percentile of the 118 controls. Results from the 7-plex assay were compared with those from corresponding single-autoantibody ECL and RIA assays and a s -plex assay correlated well in levels with each corresponding single-antibody RIA (R2=0.74, 0.75, 0.75, 0.66, 0.64, 0.62, and 0.51 for IAA, GADA, IA- tively, p<0.0001 for all). The 7-plex assay retained 100% sensitivity for all autoantibodies and the positivity of 7-plex assay and their corresponding RIA or ELISA in 168 T1D patients is summarized in table 1. The few discordant samples were at low levels, close to the cut-offs. In conclusion, this novel multiplex assay can facilitate screening for T1D and associated autoimmune diseases at diagnosis of diabetes

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