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Dorothee Weihrauch, DVM, PhD “Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice”
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Brian Robert Hoffmann, PhD “Mechanisms of Mas1 Receptor-Mediated Signaling in the Vascular Endothelium”
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Jennifer Henningfeld, MD “Liberation from home mechanical ventilation and decannulation in children”
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Andres J. Yarur, MD “Higher Infliximab Trough Levels are Associated with Perianal Fistula Healing in Patients with Crohn's Disease”
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Jiaqing Tong, MS, BS “The Epac-Phospholipase C epsilon Pathway Regulates Endocannabinoid Signaling and CocaineInduced Disinhibition of Ventral Tegmental Area Dopamine Neurons”
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Ann Kolbach-Mandel “Guaifenesin stone matrix proteomics: a protocol for identifying proteins critical to stone formation”
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Agnes Keszler, PhD “Thiolate-based dinitrosyl iron complexes: Decomposition and detection and differentiation from Snitrosothiols”
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Nathan Skinner, PhD “Rapid in vivo detection of rat spinal cord injury with double-diffusion-encoded magnetic resonance spectroscopy”
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Matthew Faron, PhD “Automatic Digital Analysis of Chromogenic Media for Vancomycin-Resistant-Enterococcus Screens Using Copan WASPLab”
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Emily A. Kolyvas, BS “Mesothelin-targeted immunotoxin RG7787 has synergistic antitumor activity when combined with taxanes” All rights reserved. Contents are the property of the authors and/or journals cited.
Dorothee Weihrauch, DVM, PhD Associate Professor Departments of Anesthesiology and Cell Biology, Neurobiology & Anatomy Medical College of Wisconsin
I am a basic scientist and have been a part of the Division of Anesthesiology Research since 2000. My current research focuses on the changes in the extracellular matrix in chronic inflammatory diseases like scleroderma, diabetes and heart failure. My data demonstrates that the extracellular matrix contributes to the disease phenotype and is closely linked to cellular health. My approaches pursue two complementary paths: (1) I study the extracellular matrix in its role as a signaling component; (2) The role and impact of various cells (endothelial cell, cardiac fibroblast, cardiomyocytes, iPSCcardiomyocytes) residing in this environment. One of the overarching hypotheses is that a chronic inflammatory disease state with high levels of oxidative stress leads to a highly oxidized matrix, which has a detrimental influence on cellular health in development as well as in adulthood.
“Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice” Journal of Visualized Experiments. 2017;121. DOI: 10.3791/55036. IRF5 is crucial for cells to determine their response in a pro-inflammatory or anti-inflammatory fashion. IRF5 switches from one pathway to another. We designed a decoy peptide IRF5D. IRF5D inhibited IRF5, reduced alterations in extracellular matrix, improved endothelial vasodilatation in Tsk/+. Kd of IRF5D for recombinant IRF5 was determined by binding experiments. EC proliferation/ apoptosis were unchanged using increasing concentrations of IRF5D. Tsk/+ mice were treated with IRF5D. IRF5 and ICAM were decreased after IRF5D treatment. Endothelial function improved as assessed by vasodilatation after IRF5D treatment IRF5 translocation was assessed by immunohistochemistry on cardiac myocytes cultured on different cardiac extracellular matrices. IRF5D treatment of Tsk/+ reduced the number of IRF5 positive nuclei in comparison to no IRF5D treatment. IRF5 is crucial in inflammation and fibrosis in Tsk/+ mice. Figure 1. IRF5 Nuclear Translocation of IRF5 in Cultured Myocytes with IRF5 inhibition
IRF5 inhibition by IRF5D (50 µg/mL, 24 h) lead to fewer numbers of IRF5 positive nuclei in myocytes cultured on Tsk/+ cardiac matrix and C57Bl/6J cardiac matrix. The arrows depict IRF5 positive nuclei.
Figure 2. EC function and Vasodilation
IRF5D administration improved acetylcholine induced vasodilatation of facialis arteries of Tsk/+ mice.
Brian Robert Hoffmann, PhD Assistant Professor Department of Biomedical Engineering Department of Physiology Cardiovascular Center Medical College of Wisconsin Marquette University
My primary research program focuses on incorporating novel biochemical strategies aimed at determining causal factors that underlie impaired vascular function and regeneration during type 1 and 2 diabetes. However, my research also has a strong focus utilizing my expertise in innovative proteomic, metabolomic, and genomic technologies for deciphering the etiology behind disease states in numerous systems through collaboration. Through my primary research focus and collaboration, we aim to understand disease states at the molecular level with the ultimate goal of developing novel therapeutics and discover biomarkers for treatment and/or early diagnosis.
“Mechanisms of Mas1 Receptor-Mediated Signaling in the Vascular Endothelium” Arteriosclerosis, Thrombosis, and Vascular Biology. 2017;37(3):433-445. Our manuscript entitled “Mechanisms of Mas1 Receptor-Mediated Signaling in the Vascular Endothelium” was recently published in Arteriosclerosis, Thrombosis, & Vascular Biology and is the first comprehensive overview of Ang-(1-7) stimulated Mas1 receptor signaling in the vascular endothelium. This study shows that low-dose Ang-(1–7), acting through the Mas1 receptor, promotes angiogenesis and vasodilation similar to a low, subpressor dose of AngII acting through AngII receptor type 1. The data in this manuscript significantly expands the known Ang-(1–7)/Mas1 receptor signaling pathway and demonstrates an important distinction between the pathological effects of elevated and suppressed AngII compared with the beneficial effects of AngII normalization and Ang-(1–7) administration.
Visual Overview
Jennifer Henningfeld, MD Assistant Professor Department of Pediatrics Pulmonary Division Assistant Director Tracheostomy/Home Ventilator Program Medical College of Wisconsin
I am a pediatric pulmonologist and Assistant Director of the Tracheostomy and Home Ventilator Program at Children’s Hospital of Wisconsin. Our team cares for around 200 children per year who are tracheostomy dependent, with about half requiring home long-term mechanical ventilation. My research focuses on liberation from respiratory technology and decannulation. I am also currently involved in two pediatric tracheostomy research studies: Feeding Delays in Pediatric Tracheostomy and Use of Polysomnography in Liberating Children from Respiratory Technology. We are also working to introduce the use of highly realistic, enhanced simulation program to train caregivers of tracheostomy dependent children for potential outpatient emergencies.
“Liberation from home mechanical ventilation and decannulation in children� Pediatric Pulmonology. 2016;51(8):838-849. The prevalence of children requiring outpatient invasive long-term mechanical ventilation is increasing. For some children, liberation from home mechanical ventilation (HMV) and decannulation is the desired outcome. This study showed that 46 children achieved HMV independence and decannulation between July 1999 and December 2011. The median ages at tracheostomy, initiation of HMV, initiation of tracheostomy collar trials, HMV independence and decannulation were 3.5, 6.0, 12.0, 25.5 and 40.5 months, respectively. Common procedures prior to decannulation were airway surgery, bronchoscopy and polysomnography (n=30, 46, and 46 children, respectively). HMV was primarily weaned in an outpatient setting. Based on these findings we showed that liberation from respiratory technology is a complex, multistep process that can be accomplished in medically complex children with varying underlying disease processes at relatively young ages. Figure 1. Median ages at major weaning steps. IQR = Interquartile Range
Figure 3. Frequency of airway surgeries in children prior to decannulation.
My name is Andres Yarur. I am an Assistant Professor of Medicine at the Medical College of Wisconsin. I completed my residency at the University of Miami and began research in gastroenterology during fellowships at UM and at the University of Chicago.
Andres J. Yarur, MD Assistant Professor Department of Medicine Division of Gastroenterology & Hepatology
My research is on Crohn’s disease and ulcerative colitis, focusing on therapy, including the use of novel pharmacological agents and optimization of current drugs.
“Higher Infliximab Trough Levels are Associated with Perianal Fistula Healing in Patients with Crohn's Disease” Alimentary Pharmacology & Therapeutics. 2017;45(7)933-940. In the study we recently published, we looked into how dosing and drug levels affect response to therapy of fistulas in patients with Crohn’s disease. Unfortunately, many patients with Crohn’s develop fistulas from the intestine to either other internal organs or the skin. This can dramatically affect the quality of life of patients and lead to complications requiring surgery. Fistulas are also very hard to treat. While infliximab (a monoclonal antibody that targets tumor necrosis factor) has been useful in the treatment of Crohn’s disease and fistulas, many patients do not respond to treatment. In this study, we found that a significant amount of patients need higher doses of infliximab and achieve higher serum levels of the drug in order to respond. Until now, many physicians would discontinue infliximab if there was no response to the standard doses. However, after this study, we know that we can induce remission in many of the patients that do not respond by optimizing the drug. Figure 3. Rates of mucosal healing, fistula healing and fistula closure by quartiles of trough level of infliximab (P values for analysis of variance for each group)
“The Epac-Phospholipase C epsilon Pathway Regulates Endocannabinoid Signaling and Cocaine-Induced Disinhibition of Ventral Tegmental Area Dopamine Neurons” Journal of Neuroscience. 2017;37(11):3030-3044. 2-arachidonoylglycerol (2-AG) is an endogenous cannabinoid that depresses synaptic transmission through stimulation of CB1 receptors. Among the six isoforms of phospholipase C (PLC; PLCβ, PLCγ, PLCδ, PLCε, PLCζ, PLCη), only PLCβ has been linked to 2-AG synthesis. We demonstrated that 8-CPT-2Me-cAMP, a selective agonist of the cAMP sensor protein Epac, enhances 2-AG-mediated synaptic depression in ventral tegmental area (VTA) dopamine neurons via activation of PLCε. These results identify a novel mechanism for 2-AG synthesis via activation of the Epac-PLCε pathway. Furthermore, we showed that cocaine-induced conditioned place preference and disinhibition of VTA dopamine neurons were impaired in mice lacking Epac or PLCε. Thus, the Epac-PLCε signaling pathway contributes to cocaine-induced disinhibition of VTA dopamine neurons and formation of drug-associated memories.
Jiaqing Tong, MS, BS Research Assistant Graduate School
Ann Kolbach-Mandel Research Associate II Department of Medicine: Nephrology Zablocki VA Medical Center
“Guaifenesin stone matrix proteomics: a protocol for identifying proteins critical to stone formation” Urolithiasis. 2017;45(2):139-149. Drug related kidney stones are difficult to diagnose. A poorly-defined stone study patient provided three urine samples; one containing heavy stone-sediment. These solids were characterized with Fourier transform infrared spectroscopy as 60% protein and 40% guaifenesin (GF; a common cold medication). Proteomic studies, both immunoblotting and mass spectrometry, defined proteins in urine and stone-matrix. These studies identified 49 proteins in the stone-matrix, half of those proteins also in urine. Many of the GF stone-matrix proteins have been reported in uric-acid and calcium stones. Protein enrichment (matrix vs. urine) revealed 17 proteins that suggest a critical role in GF stone formation. This study defines a protocol for identifying proteins critical to stone formation.
“Thiolate-based dinitrosyl iron complexes: Decomposition and detection and differentiation from S-nitrosothiols” Nitric Oxcide. 2017;65(1):1-9. Dinitrosyl iron complexes (DNIC) spontaneously form in aqueous solutions of Fe(II), nitric oxide (NO), and various anions. They exist as an equilibrium between diamagnetic, dimeric (bi-DNIC) and paramagnetic, monomeric (mono-DNIC) forms. Thiolate groups are the most biologically relevant anions to coordinate to Fe(II). Low molecular weight DNIC have been suggested to be important mediators of NO biology, and they play role in the control of iron-dependent cellular processes. In this work, we examined the stability of low molecular weight DNIC; and their detection in the presence of S-nitrosothiols. We established that at neutral pH, bi-DNIC remain stable for hours, whereas excess thiol results in decomposition to form nitrite, but not S-nitrosothiol. Importantly, mercury chloride accelerated the degradation of DNIC; thus, the implications of this finding for the diagnostic use of mercury chloride in the detection of S-nitrosothiols were determined in simple and complex biological systems. We conclude S-nitrosothiol levels may have been substantially overestimated in all methods where mercury chloride has been used.
Agnes Keszler, PhD Research Scientist Department of Medicine: Cardiology
Nathan Skinner, PhD Medical Student Medical Scientist Training Program
“Rapid in vivo detection of rat spinal cord injury with double-diffusionencoded magnetic resonance spectroscopy” Magnetic Resonance in Medicine. 2017;77(4):1639-1649. Spinal cord injury is a devastating condition that relies on prompt evaluation for treatment and rehabilitation. However, current imaging techniques cannot reliably measure acute injury severity. This study focused on the development of a new magnetic resonance imaging technique, known as double diffusion encoding (DDE) for more sensitive assessment of spinal cord injury. In a rat model at two days post-injury, DDE showed a stronger relationship with injury severity and substantially faster acquisition time than traditional techniques, with subsequent analysis remaining entirely automated. These findings show substantial promise for future pre-clinical and clinical applications in spinal cord injury.
“Automatic Digital Analysis of Chromogenic Media for VancomycinResistant-Enterococcus Screens Using Copan WASPLab” Journal of Clinical Microbiology. 2016;54(10):2464-2469. Clinical laboratories are needed to perform more testing with quicker results all while experiencing a shortage in technologist. Our laboratory in collaboration with Copan Diagnostics has developed software to alleviate work burden by using digital software analysis to interpret chromogenic agar for the screening of VancomycinResistant Enterococcus (VRE), a major cause of healthcare-acquired infections. The software compares images before and after incubation and detects changes in pixels that correspond to VRE growth, separating plates into negative and non-negative. Compare to manual reading, the software was 100% sensitive and 89.5% specific. In practice this software could remove the need of a technologist to review 84% of all plates sent for VRE screening.
Matthew Faron, PhD Instructor, Clinical Microbiology Department of Pathology
Emily A. Kolyvas, BS MSTP Student (M1) MD/PhD Candidate
“Mesothelin-targeted immunotoxin RG7787 has synergistic antitumor activity when combined with taxanes” Oncotarget. 2017;8(6):9189-9199. Recombinant immunotoxins (RITs) are antibody-based therapeutics that carry a toxin payload. The RG7787 RIT targets the cancer antigen mesothelin to deliver a recombinantly-engineered, reduced immunogenicity variant of Pseudomonas exotoxin A (PE) to the cytosol where it inhibits protein synthesis. We demonstrate that maximal doses of RG7787 halt growth of pancreatic cancer tumor xenografts, similar to the approved drugs gemcitabine and nab-paclitaxel, however, combination of the RIT with nab-paclitaxel produces durable complete regressions in most mice. Our data suggest that PE increases cell sensitivity to taxane-mediated killing by increasing taxane-mediated microtubule stability and priming cells for apoptosis by decreasing levels of the pro-survival factor Mcl-1.
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