Medicine and Health Sciences Commons^™

Intravital Imaging Of Cellular Response Due To Traumatic Brain Injury Using Confocal Microscopy, Enoch G. Kim, Jeffrey Horbatiuk, Carolyn Harris

Medical Student Research Symposium

Introduction: Cellular reaction to traumatic brain injury is complex and involves considerable interactions between cells and reactivity to foreign bodies. Our objective was to assess neurons, microglia, astrocytes, and intracellular Ca²⁺ signaling by creating a novel confocal microscopy technique involving an air immersed lens that does not sacrifice resolution and limits signal attenuation. This study aimed to create a consistent dynamic methodology to observe the cortical cellular response using real-time intravital imaging as trauma is being induced.

Methods: Once surgical plane was achieved, rodent cortices were exposed via craniotomy and blunt insertion with a silicone shunt catheter into the …

The Development And Characterization Of Nanobodies Specific To Protein Tyrosine Phosphatase 4a3 (Ptp4a3/Prl-3) To Dissect And Target Its Role In Cancer., Caroline Smith

Theses and Dissertations--Molecular and Cellular Biochemistry

Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, …

Cellular Distribution Of The Prion Protein In Palatine Tonsils Of Mule Deer (Odocoileus Hemionus) And Rocky Mountain Elk (Cervus Elaphus Nelsoni), Matthew M. Hille, Jean E. Jewell, E. Lee Belden

School of Veterinary and Biomedical Sciences: Faculty Publications

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that affects members of the Cervidae family, including deer (Odocoileus spp.), elk (Cervus Canadensis spp.), and moose (Alces alces spp.). While CWD is a neurodegenerative disease, lymphoid accumulation of the abnormal isoform of the prion protein (PrPSc) is detectable early in the course of infection. It has been shown that a large portion of the PrPSc lymphoid accumulation in infected mule deer takes place on the surface of follicular dendritic cells (FDCs). In mice, FDC expression of PrPC has been shown to be essential for PrPSc accumulation. …

P5l Mutation In Ank Results In An Increase In Extracellular Inorganic Pyrophosphate During Proliferation And Nonmineralizing Hypertrophy In Stably Transduced Atdc5 Cells, Raihana Zaka, David Stokes, Arnold S. Dion, Anna Kusnierz, Fei Han, Charlene J. Williams

Charlene Williams

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank …

The Drosophila Melanogaster Model Of Human Uric Acid Nephrolithiasis As A Novel In Vivo Drug Screening Platform, Aymon N. Ali

Electronic Thesis and Dissertation Repository

Nephrolithiasis involves the supersaturation of a stone-forming solute in urine leading to the formation of a calculus. The development of novel therapeutic agents for this multifactorial urological disorder has been hindered by lack of a practical pre-clinical model. Currently established medical treatments can possess unfavorable side effect profiles and inconsistent efficacies in certain metabolic milieus. Here, Drosophila melanogaster ̶ an emerging model for calcium oxalate nephrolithiasis ̶ was investigated as a potential disease model and high throughput drug discovery platform for human uric acid nephrolithiasis. Through disruption of the Uro gene and purine-rich dietary manipulation, we successfully demonstrate the formation …

Multiple Ubxm Family Members Inhibit Retrovirus And Lentivirus Production And Canonical Nfkappabeta Signaling By Stabilizing Ikappabalpha, Y Hu, K O'Boyle, J Auer, S Raju, F You, Penghua Wang, E Fikrig, R Sutton

NYMC Faculty Publications

UBXN proteins likely participate in the global regulation of protein turnover, and we have shown that UBXN1 interferes with RIG-I-like receptor (RLR) signaling by interacting with MAVS and impeding its downstream effector functions. Here we demonstrate that over-expression of multiple UBXN family members decreased lentivirus and retrovirus production by several orders-of-magnitude in single cycle assays, at the level of long terminal repeat-driven transcription, and three family members, UBXN1, N9, and N11 blocked the canonical NFkappaB pathway by binding to Cullin1 (Cul1), inhibiting IkappaBalpha degradation. Multiple regions of UBXN1, including its UBA domain, were critical for its activity. Elimination of UBXN1 …

Multimodal Theranostic Nanoformulations Permit Magnetic Resonance Bioimaging Of Antiretroviral Drug Particle Tissue-Cell Biodistribution, Bhavesh D. Kevadiya, Christopher J. Woldstad, Brendan M. Ottemann, Prasanta Dash, Balasrinivasa R. Sajja, Benjamin G. Lamberty, Brenda M. Morsey, Ted Kocher, Rinku Dutta, Aditya N. Bade, Yutong Liu, Shannon E. Callen, Howard S. Fox, Siddappa N. Byrareddy, Joellyn M. Mcmillan, Tatiana K. Bronich, Benson J. Edagwa, Michael D. Boska, Howard Gendelman

Journal Articles: Radiology

RATIONALE: Long-acting slow effective release antiretroviral therapy (LASER ART) was developed to improve patient regimen adherence, prevent new infections, and facilitate drug delivery to human immunodeficiency virus cell and tissue reservoirs. In an effort to facilitate LASER ART development, “multimodal imaging theranostic nanoprobes” were created. These allow combined bioimaging, drug pharmacokinetics and tissue biodistribution tests in animal models.

METHODS: Europium (Eu³⁺)- doped cobalt ferrite (CF) dolutegravir (DTG)- loaded (EuCF-DTG) nanoparticles were synthesized then fully characterized based on their size, shape and stability. These were then used as platforms for nanoformulated drug biodistribution.

RESULTS: Folic acid (FA) decoration of …

Natural And Induced Mitochondrial Phosphate Carrier Loss: Differential Dependence Of Mitochondrial Metabolism And Dynamics And Cell Survival On The Extent Of Depletion., Erin L. Seifert, Aniko Gál, Michelle G. Acoba, Qipei Li, Lauren Anderson-Pullinger, Tünde Golenár, Cynthia Moffat, Neal Sondheimer, Steven M. Claypool, György Hajnóczky

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The relevance of mitochondrial phosphate carrier (PiC), encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3 First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the fifth matrix loop is incompatible with survival on non-fermentable substrate, whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants and HeLa cells with varying degrees of PiC depletion, PiC loss of …

Gene Delivery Using Calcium Phosphate Nanoparticles: Optimization Of The Transfection Process And The Effects Of Citrate And Poly(L-Lysine) As Additives, Mohammed A. Khan, Victoria M. Wu, Shreya Ghosh, Vuk Uskoković

Pharmacy Faculty Articles and Research

Despite the long history of nanoparticulate calcium phosphate (CaP) as a non-viral transfection agent, there has been limited success in attempts to optimize its properties for transfection comparable in efficiency to that of viral vectors. Here we focus on the optimization of: (a) CaP nanoparticle precipitation conditions, predominantly supersaturation and Ca/P molar ratios; (b) transfection conditions, mainly the concentrations of the carrier and plasmid DNA; (c) the presence of surface additives, including citrate anion and cationic poly(l-lysine) (PLL). CaP nanoparticles significantly improved transfection with plasmid DNA encoding enhanced green fluorescent protein (eGFP) in pre-osteoblastic MC3T3-E1 cells compared to a commercial …

The Rho Gtpases Rac1, Cdc42, And Rhoa Regulate App Transport To Lysosomes And Aβ Production, Justin K. Chiu .

Electronic Thesis and Dissertation Repository

Alzheimer’s Disease (AD) is characterized by Beta-Amyloid (Aβ) plaques within the brain. Aβ peptides are produced by the cleavage of Amyloid Precursor Protein (APP). Our lab has previously discovered a novel pathway for APP internalization mediated by ADP-ribosylation factor 6 (Arf6). This pathway resembles macropinocytosis, transporting cell surface APP directly to lysosomes, a possible site for Aβ production. We set out to characterize the effectors downstream of Arf6. In SN56 and N2A cells we co-transfected HA-tagged APP (to label cell-surface APP) with compartment markers, to visualize APP trafficking. We used dominant negative and constitutively active mutants, pharmacological inhibitors, and siRNA …

Viewing The Extracellular Matrix: An Imaging Method For Tissue Engineering, Michael Drakopoulos, Sarah Calve

The Summer Undergraduate Research Fellowship (SURF) Symposium

The field of regenerative medicine seeks to create replacement tissues and organs, both to repair deficiencies in biological function and to treat structural damage caused by injury. Scaffoldings mimicking extracellular matrix (ECM), the structure to which cells attach to form tissues, have been developed from synthetic polymers and also been prepared by decellularizing adult tissue. However, the structure of ECM undergoes significant remodeling during natural tissue repair, suggesting that ECM-replacement constructs that mirror developing tissues may promote better regeneration than those modeled on adult tissues. This work investigated the effectiveness of a method of viewing the extracellular matrix of developing …

Raman Micro Spectroscopy For In Vitro Drug Screening: Subcellular Localisation And Interactions Of Doxorubicin, Zeineb Farhane, Franck Bonnier, Alan Casey, Hugh Byrne

Articles

Vibrational spectroscopy, including Raman spectroscopy, has been widely used over the last few years to explore potential biomedical applications. Indeed, Raman spectroscopy has been demonstrated to be a powerful non-invasive tool in cancer diagnosis and monitoring. In confocal microscopic mode, the technique is also a molecularly specific analytical tool with optical resolution which has potential applications in subcellular analysis of biochemical processes, and therefore as an in vitro screening tool of the efficacy and mode of action of, for example, chemotherapeutic agents.

In order to demonstrate and explore the potential in this field, established, model chemotherapeutic agents can be valuable. …

Mcnamara 2014 Mmb1075 - Low Magnification Confocal Microscopy Of Tumor Angiogenesis, George Mcnamara

George McNamara

McNamara 2014 MMB1075 - Low Magnification Confocal Microscopy of Tumor Angiogenesis

Book chapter from Paddock 2014 Confocal Microscopy

Book is online at http://www.springer.com/us/book/9781588293510

Three Dimensional Dual Labelled Dna Fluorescent In Situ Hybridization Analysis In Fixed Tissue Sections, Kristin D Kernohan, Nathalie G. Bérubé

Paediatrics Publications

Emerging studies demonstrate that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. DNA fluorescent in situ hybridization (FISH) is a common molecular technique used to visualize the location of DNA sequences. The vast majority of DNA FISH studies are conducted on cultured cells due to the technical difficulties encountered using fixed tissue sections. However, the use of cultured cells poses important limitations that could yield misleading results, making in vivo analysis a far superior approach. Here we present a protocol for multiplexed three dimensional DNA FISH in mouse brain sections, which is also …

In Situ Detection Of Autoreactive Cd4 T Cells In Brain And Heart Using Major Histocompatibility Complex Class Ii Dextramers, Chandirasegaran Massilamany, Arunakumar Gangaplara, Ting Jia, Christian Elowsky, Qingsheng Li, You Zhou, Jay Reddy

School of Veterinary and Biomedical Sciences: Faculty Publications

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-α (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti- CD4 and IAs/Theiler’s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/ anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of …

Responsiveness Of Voltage-Gated Calcium Channels In Sh-Sy5y Human Neuroblastoma Cells On Quasi-Three-Dimensional Micropatterns Formed With Poly (L-Lactic Acid), Ze-Zhi Wu, Zheng-Wei Wang, Li-Guang Zhang, Zhi-Xing An, Dong-Huo Zhong, Qi-Ping Huang, Mei-Rong Luo, Yan-Jian Liao, Liang Jin, Chen-Zhong Li, William S. Kisaalita

Department of Biomedical Engineering Faculty Publications

Introduction:

In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs).

Methods and materials:

SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells’ location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under …

Targeting Apoptosis For Optical Imaging Of Infection, Mathew L Thakur, Kaijun Zhang, Bishnuhari Paudyal, Devadhas Devakumar, Maria Y Covarrubias, Changpo Cheng, Brian D Gray, Eric Wickstrom, Koon Y Pak

Department of Radiology Faculty Papers

PURPOSE: Infection is ubiquitous and a major cause of morbidity and mortality. The most reliable method for localizing infection requires radiolabeling autologous white blood cells ex vivo. A compound that can be injected directly into a patient and can selectively image infectious foci will eliminate the drawbacks. The resolution of infection is associated with neutrophil apoptosis and necrosis presenting phosphatidylserine (PS) on the neutrophil outer leaflet. Targeting PS with intravenous administration of a PS-specific, near-infrared (NIR) fluorophore will permit localization of infectious foci by optical imaging.

METHODS: Bacterial infection and sterile inflammation were induced in separate groups (n = 5) …

Detection And Imaging Of The Free Radical Dna In Cells—Site-Specific Radical Formation Induced By Fenton Chemistry And Its Repair In Cellular Dna As Seen By Electron Spin Resonance, Immuno-Spin Trapping And Confocal Microscopy, Suchandra Bhattacharjee, Saurabh Chatterjee, Jinjie Jiang, Birandra K. Sinha, Ronald P. Mason

Faculty Publications

Oxidative stress-related damage to the DNA macromolecule produces lesions that are implicated in various diseases. To understand damage to DNA, it is important to study the free radical reactions causing the damage. Measurement of DNA damage has been a matter of debate as most of the available methods measure the end product of a sequence of events and provide limited information on the initial free radical formation. We report a measurement of free radical damage in DNA induced by a Cu(II)-H2O2 oxidizing system using immuno-spin trapping supplemented with electron paramagnetic resonance. In this investigation, the short-lived radical generated is trapped …

Tegument Protein Subcellular Localization Of Human Cytomegalovirus, John Paul Tomtishen Iii

Honors Theses

To determine the subcellular localization of the tegument proteins pp65, pp71, pp150, and pp28 as fusions to one of several fluorescent proteins. Since these tegument proteins play pivotal roles in several stages of the viral life cycle, knowledge of where and the mechanism of how these proteins localize upon release could result in a better understanding of their function during a lytic infection as well as assist in the development of an effective, novel antiviral treatment.

The Efficacy Of Topical Antibiofilm Agents In A Sheep Model Of Rhinosinusitis, Peter-John Wormald

Peter-John Wormald

Background: Biofilms have been shown to be resistant to conventional antibiotic treatment. This study uses a sheep biofilm model developed by our department to investigate several novel topical anti-biofilm treatments. Methods: Staphylococcal biofilms were grown in 54 sheep frontal sinuses over 8 days: Each sinus was randomized to (1) no intervention, (2) single mupirocin flush, (3) regular 12-hourly mupirocin flushes for 5 days, (4) Citric Acid Zwitterionic Surfactant (CAZS) via hydrodebrider, (5) gallium nitrate, (6) CAZS with gallium nitrate, (7) CAZS with mupirocin, and (8) saline regular flushes. Sheep were sacrificed and the sinus mucosa harvested 1 or 8 days …

Antitumour And Antimalarial Activity Of Artemisinin–Acridine Hybrids, Michael Jones, Amy Mercer, Paul Stocks, Louise La Pensee, Rick Cosstick, B. Kevin Park, Miriam Kennedy, Ivo Piantanida, Stephen Ward, Jill Davies, Patrick Bray, Sarah Rawe, Jonathon Baird, Tafadzwa Charidza, Omar Janneh, Paul O'Neill

Articles

Artemisinin–acridine hybrids were prepared and evaluated for their in vitro activity against tumour cell lines and a chloroquine sensitive strain of Plasmodium falciparum. They showed a 2–4-fold increase in activity against HL60, MDA-MB-231 and MCF-7 cells in comparison with dihydroartemisinin (DHA) and moderate antimalarial activity. Strong evidence that the compounds induce apoptosis in HL60 cells was obtained by flow cytometry, which indicated accumulation of cells in the G1 phase of the cell cycle.

In Vivo Trafficking Of Endogenous Opioid Receptors, Yulin Wang, Elisabeth J. Van Bockstaele, Lee-Yuan Liu-Chen

Department of Neurosurgery Faculty Papers

Studies on trafficking of endogenous opioid receptors in vivo are subject of the present review. In many of the in vivo studies, the use of semi-quantitative immuno-electron microscopy is the approach of choice. Endogenous opioid receptors display differential subcellular distributions with μ opioid receptor (MOPR) being mostly present on the plasma membrane and δ- and κ-opioid receptors (DOPR and KOPR, respectively) having a significant intracellular pool. Etorphine and DAMGO cause endocytosis of the MOPR, but morphine does not, except in some dendrites. Interestingly, chronic inflammatory pain and morphine treatment promote trafficking of intracellular DOPR to the cell surface which may …

P5l Mutation In Ank Results In An Increase In Extracellular Inorganic Pyrophosphate During Proliferation And Nonmineralizing Hypertrophy In Stably Transduced Atdc5 Cells, Raihana Zaka, David Stokes, Arnold S. Dion, Anna Kusnierz, Fei Han, Charlene J. Williams

Center for Translational Medicine Faculty Papers

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank …

The Tight Skin Mouse: Demonstration Of Mutant Fibrillin-1 Production And Assembly Into Abnormal Microfibrils, Cay M. Kielty, Michael Raghunath, Linda D. Siracusa, Michael J. Sherratt, Reiner Peters, C. Adrian Shuttleworth, Sergio A. Jimenez

Selected Works of Sergio Jiménez, MD, MACR

Mice carrying the Tight skin (Tsk) mutation harbor a genomic duplication within the fibrillin-1 (Fbn 1) gene that results in a larger than normal in-frame Fbn 1 transcript. In this study, the consequences of the Tsk mutation for fibrillin-containing microfibrils have been examined. Dermal fibroblasts from Tsk/+ mice synthesized and secreted both normal fibrillin (approximately 330 kD) and the mutant oversized Tsk fibrillin-1 (approximately 450 kD) in comparable amounts, and Tsk fibrillin-1 was stably incorporated into cell layers. Immunohistochemical and ultrastructural analyses of normal and Tsk/+ mouse skin highlighted differences in the gross organization and distribution of microfibrillar arrays. Rotary …