BiomedicalPostdocSymposium
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PLATFORM PRESENTATIONS I

10:30 A.M.

A genome-wide screen in zebrafish reveals CYFIP2 as a novel regulator of startle sensitivity

Kurt C. Marsden1, Roshan Jain1, Marc Wolman2, Hannah Bell2, Katharina Hayer3, John Hogenesch3, and Michael Granato1

1Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania; 2Zoology Department, University of Wisconsin, Madison, WI; 3Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania

A critical function of the nervous system is to determine if a stimulus requires a behavioral response. For example, only intense auditory stimuli that indicate danger should induce a startle response. While an inability to regulate startle sensitivity is a hallmark of several neuropsychiatric disorders such as schizophrenia, anxiety and autism, the molecular-genetic basis of startle threshold regulation is not well defined. Because the startle response and its underlying neuronal circuits are well conserved across vertebrates, we have established larval zebrafish as a model in which to study several aspects of the acoustic startle response, including threshold modulation. In a genome-wide, forward genetic screen, we isolated eight mutants with heightened startle sensitivity. Analysis of these mutants reveals that the startle threshold can be regulated at multiple points within the circuit. We built a powerful setup to simultaneously monitor neuronal activity in identified neurons of the startle circuit in response to acoustic stimulation while recording the behavioral response at millisecond resolution. Using this setup, we find that in wild-type larvae the site of threshold regulation likely resides within a single hindbrain command neuron, the Mauthner cell, and relies on a mechanism that restricts neuronal activity to the site of synaptic input. We have identified the causative mutation in the hypersensitive mutant triggerhappy as a premature stop codon in the gene CYFIP2 (cytoplasmic FMR1 interacting protein 2). CYFIP2 is dysregulated in Fragile X syndrome and is known to be critical for axon guidance and synaptogenesis. We will present ongoing work on the cellular and molecular mechanisms by which CYFIP2 regulates the acoustic startle threshold.

10:45 A.M.

Adenovirus protein VII acts as a pseudo-histone to alter cellular chromatin

Daphne C. Avgousti1,2, Rosalynn C. Molden3,4, Nikolina Sekulic3, Joana Petrescu1,5, Emigdio D. Reyes1,2, Ben E. Black3, Benjamin A. Garcia3, and Matthew D. Weitzman1,2

1Division of Cancer Pathobiology, Children’s Hospital of Philadelphia; 2Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania; 3Department of Biochemistry and Biophysics, Perlman School of Medicine, University of Pennsylvania; 4Department of Molecular Biology, Princeton University; 5Villanova University

Viruses rely on their ability to mimic the structure and function of host proteins to redirect cellular processes towards viral production. Viruses interact with cellular chromatin to promote viral replication and regulate host responses such as the DNA damage response (DDR), which is activated and manipulated by viruses such as adenoviruses. Adenovirus contains a highly basic protein called protein VII that bears remarkable similarity to cellular histones. When protein VII was first described as histone-like based on primary sequence in the late 1970s, there was little understanding of the functions of histone proteins. Advancements in the field of epigenetics have led to an increased appreciation for conserved post-translational modifications (PTMs) on histone proteins and their impact on cellular processes. Protein VII is suggested to protect viral genomes from the DDR but its impact on cellular chromatin is unknown. Here, we propose that in addition to its effects on the viral genome, protein VII controls host chromatin through interactions with histones at the nucleosome level. We show that expression of protein VII alone is sufficient to alter cellular chromatin, similarly to chromatin changes seen during adenovirus infection. Furthermore, we find that protein VII alone can down-regulate the DDR upon irradiation. We also find by mass spectrometry analysis that protein VII is modified analogously to histone PTMs. These PTMs may be involved in the mechanism of chromatin change or DDR down-regulation. Finally, we find that protein VII can bind to nucleosomes in vitro and may replace core histones to restructure cellular chromatin. This research is the first investigation into the function of a viral pseudo-histone in the context of cellular chromatin.

11:00 A.M.

Gold core polyphosphazene nanospheres as biodegradable contrast agents for computed tomography

Rabee Cheheltani1, Peter Chhour1,2, Rami Ezzibdeh2, Pratap Naha1, Victor Ferrari3, Harry Allcock4, David Cormode1,2

1Department of Radiology, 2Department of Bioenginering, and 3Department of Cardiology, University of Pennsylvania; 4Department of Chemistry, Pennsylvania State University, University Park, PA

Gold nanoparticles (AuNP) have been proposed as novel contrast agents for computed tomography (CT). Although large AuNP (>5 nm) are desirable for their longer circulation times and accumulation in diseased tissue, they cannot be excreted via the kidneys. Small AuNP are easily excreted, but do not possess the favorable properties of large AuNP. We propose a novel contrast agent platform where small AuNP are encapsulated into large biodegradable polydi(carboxylatophenoxy)phosphazene (PCPP) nanospheres (Au-PCPP) that will perform their function as contrast agents, subsequently break down into harmless byproducts and release the small AuNP for swift excretion. Here we present the synthesis, in vitro biocompatibility, and in vitro biodegradability of these nanoparticles.

AuNP were synthesized via reduction of gold chloride with sodium borohydride and capped with glutathione for stabilization in biological media and purification by centrifugation. This results in glutathione coated AuNP that are stable in PBS and have diameters tunable between 2-5 nm. Incubation of these AuNP with macrophage, endothelial, kidney and liver cells revealed no reduction in cell viability at concentrations as high as 1 mg/ml, indicating very high biocompatibility. AuNP cores were entrapped in PCPP by mixing the polymer and AuNP, cross-linking with spermine, hardening in calcium chloride and purification by centrifugation. Encapsulation has been confirmed using transmission electron microscopy (TEM). These Au-PCPP nanoparticles produce strong CT contrast. Their incubation with cells does not affect cell viability when measured by MTS assay. TEM and elemental analysis of the Au-PCPP in serum and in cells support the hypothesis that Au-PCPP are biodegradable.

11:15 A.M.

Biological super-resolution imaging by using microspheres

Arash Darafsheh, Consuelo Guardiola, Avery Palovack, Jarod C. Finlay, and Alejandro Carabe-Fernández

Department of Radiation Oncology, University of Pennsylvania

Optical microscopy is one of the most important non-destructive real-time imaging techniques in life sciences; however, its spatial resolution is limited by the diffraction of light waves. Optical super-resolution imaging would have a profound impact on many interdisciplinary areas, such as medical sciences, microfluidics, and nanophotonics. In this work, we demonstrate feasibility of super-resolution imaging through high-index microspheres embedded in a transparent film or immersed in a liquid. We performed imaging through implanted barium titanate glass microspheres (Diameters ~40-150 μm and refractive index ~1.9-2.1) in silicon-based transparent films placed on top of the specimen under study. Each microsphere acts as a complimentary lens that forms a magnified virtual image underneath the specimen’s surface that is captured by the objective lens. Our proposed imaging method is a promising candidate to improve the spatial resolution for applications in biomedical microscopy. As a proof-of-principle, we used this microsphere-assisted imaging method for observation of double-strand DNA breaks in U87 glioblastoma cells irradiated by X-ray and proton beams.

 

PLATFORM PRESENTATIONS II

1:15 P.M.

Selective vulnerability of spinal and bulbar motor neuron subpopulations in a mouse model of TDP-43 proteinopathy

Krista J. Spiller1, Claudia Cheung1, Adam K. Walker1, Linda Kwong1, John Q. Trojanowski1,2, Virginia M-Y Lee1,2

1Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine; 2Institute on Aging, Perelman School of Medicine, University of Pennsylvania

Over 90% of ALS patients have an accumulation of cytoplasmic TDP-43 aggregates in postmortem brain and spinal cord and it has been suggested that the accumulation of TDP-43 inclusions may represent the final common pathway of the disease. Furthermore, TDP-43 deposition patterns at the time of death are suggestive of spread, but the mechanisms of disease progression are poorly understood. To investigate disease progression with regard to TDP-43 accumulation, we have generated new lines of TDP-43 bigenic mice with doxycycline (dox)-inducible expression of human TDP-43 (h-TDP-43) and a defective nuclear localization signal (designated as hTDP-43∆NLS) under the control of the human neurofilament heavy chain (NEFH) promoter. Following the removal of dox chow, the mice have widespread neuronal expression of the transgene, yet we find that there is a striking difference in how individual neuronal populations handle the accumulation of cytoplasmic human (hu) TDP-43. Though lower motor neurons (MNs) are generally susceptible, there are intrinsic differences in disease vulnerability in individual MN pools and subtypes. We show that despite comparable levels of huTDP-43 expression, by disease end-stage in these mice, a significant number of MNs have been lost in the hypoglossal nucleus in the brainstem and at the lumbar level L4-L5 of the spinal cord, whereas other pools remain completely intact (for example, oculomotor, trochlear and trigeminal nuclei). Furthermore, even within the generally vulnerable L4-L5 spinal cord, putative slow (S) α-MNs survive to end-stage, whereas fast fatigable (FF) MNs are lost. Correspondingly, in the hind limbs of NEFH8 mice, symmetrical axonal dieback occurs first from fast-twitch muscle fibers, whereas slow fibers remain innervated even at terminal disease stages. Therefore, though pathological TDP-43 does indeed initiate degeneration, the process is not stochastic, but is characterized by temporally defined selective vulnerabilities of distinct synapses or axons.

1:30 P.M.

Whole exome sequencing to identify novel breast cancer susceptibility genes

Kara N. Maxwell MD PhD1, Joseph Vijai PhD2, Kasmintan Schrader MBBS2, Lucia Guidugli PhD3, Steven Hart PhD3,
Tinu Thomas2, Xianshu Wang PhD3, Bradley Wubbenhorst MS1, Susan M. Domchek MD1, Mark E. Robson4,
Susan Neuhausen PhD5, Kenneth Offit MD2, Csilla Szabo PhD6, Jeffrey Weitzel MD5, Katherine L. Nathanson MD7,
Fergus J. Couch PhD3

1Department of Medicine Division of Hematology-Oncology, University of Pennsylvania, PA, 2Clinical Genetics Research Lab, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, 3Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 4Clinical Genetics Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, 5Beckman Research Institute of City of Hope, Duarte, CA, 6National Institutes of Health, 7Department of Medicine Division of Translational Medicine and Genetics, University of Pennsylvania, PA

Over 50% of the familial relative risk of breast cancer is unexplained by currently identified common low risk polymorphisms and rare moderate to high-risk alleles in genes such as BRCA1/2. To further define the genetic basis for inherited risk to breast cancer, whole exome sequencing was undertaken in 333 BRCA1/2 negative individuals with at least one primary breast and ovarian cancer or with high-risk familial breast cancer, defined as a proband and at least two first or second degree relatives with breast cancer. Samples included 256 individuals from 109 families and seventy-seven unrelated individuals, for a total of 186 independent cases. Data were first analyzed for rare mutation identification in forty-nine known cancer susceptibility genes, and likely causative mutations were identified in 15% of cases. For novel gene discovery, whole exome data was harmonized from three separate sites into a single vcf file for variant filtering and analysis. Two major methodologies were used to select genes for further study, a candidate gene approach and an agnostic gene ranking approach. In the candidate gene approach, genes matching either specific gene ontologies or those identified by published somatic tumor studies were queried for the presence of rare deleterious mutations in the dataset. In the agnostic gene ranking approach, genes were ranked based on the frequency of specific types of variants in the dataset versus population estimates and the level of segregation of these rare variants in families. These methodologies identified 636 candidate cancer susceptibility genes in the discovery exome set. These genes are being evaluated by a targeted sequencing approach in a validation set of over 2000 individuals with familial breast cancer.

1:45 P.M.

4D dynamic MR image construction and quantification in young children with Thoracic Insufficiency Syndrome

Yubing Tong1, Jayaram K. Udupa1, Joseph M. McDonough2, David A. Mong2, Robert M. Campbell Jr.2

1Medical Image Processing Group, Department of Radiology, University of Pennsylvania; 2Center for Thoracic Insufficiency Syndrome, Children’s Hospital of Philadelphia

Purpose: Outcome assessment methods for thoracic and spinal surgery for Thoracic Insufficiency Syndrome (TIS) are currently outdated and limited to static radiographs. We propose a novel approach based on 4D dynamic MR images to develop a fundamental understanding of the TIS phenomenon and transform current methods of treating TIS.

Methods: The approach consists of constructing a consistent 4D dynamic MR image, delineating dynamic objects, and quantifying excursion volumes and motion of objects. A novel solution is proposed for 4D MR image construction from free-breathing MRI slice acquisitions without any restrictions on the patients. A graph-based path optimization algorithm is developed for finding a globally optimal 4D volume from several thousands of acquired slices. Segmentation of the lungs and diaphragm can be done efficiently with iterative relative fuzzy connectedness method in a time propagated manner. Key quantitative measurements include dynamic left and right lung volumes, chest wall and diaphragm excursion volumes.

Results: Preliminary quantitative measures from TIS patients pre- and post-VEPTR surgery demonstrate that the dynamic volume components increase significantly post surgery and allow us insights into previously unobtainable outcomes, such as improved mobility of the kidney on the affected side, different manners in which volume excursions change post surgery on the concave and convex side of the affected lungs, and varying degrees of improvement achieved in chest wall and diaphragm excursion depending on the deformity.

Conclusions: Key motion parameters of the thoracic and abdominal contents derivable only through dynamic MRI have the potential to become new standard outcome assessment metrics for a slew of pediatric thoracic ailments.

2:00 P.M.

De novo formation of insulin-producing “neo-ß-cell islets” from intestinal crypts

Yi-Ju Chen1,2, Stacy R. Finkbeiner4, Changhong Li3, Jason R. Spence4,5,6, and Ben Z. Stanger1,2

1Gastroenterology Division, Department of Medicine, 2Abramson Family Cancer Research Institute, and 3Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, 4Gastroenterology Division, Department of Internal Medicine, 5Department of Cell and Developmental Biology, 6Center for Organogenesis, Department of Medicine, University of Michigan, Ann Arbor, MI

The ability to interconvert terminally differentiated cells could serve as a powerful tool for cell-based treatment of degenerative diseases, including diabetes mellitus. To determine if any adult tissues are competent to activate an islet β-cell program, we performed an in vivo screen by expressing three β-cell “reprogramming factors” in a wide spectrum of tissues. We report that transient intestinal expression of these factors – Pdx1, MafA, and Ngn3 (PMN) – promoted rapid conversion of intestinal crypt cells into endocrine cells, which coalesced into “neo-islets” below the crypt base. Neo-islet cells expressed insulin and had ultrastructural features of β-cells. Importantly, intestinal neo-islets were glucose responsive and able to ameliorate hyperglycemia in diabetic mice. Moreover, PMN expression in human ES-derived intestinal “organoids” stimulated the conversion of intestinal epithelial cells into β-like cells. Our results thus demonstrate that the intestine is an accessible and abundant source of functional insulin-producing cells.