Browsing by Author "Witzmann, Frank"
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Item Bioinformatic Analysis of Proteomic Changes That Occur in an Airway Epithelial Cell Line in Response to Exposure to Physiologically Relevant Concentrations of Carbon Nanotubes(Office of the Vice Chancellor for Research, 2013-04-05) Li, Pin; Lewis, Shanta; Witzmann, Frank; Blazer-Yost, Bonnie L.Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of acute exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate potential alterations in cellular function of airway epithelia after 24 hours exposure to different concentrations of two common carbon nanoparticles, single- and multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study the differential protein expression in Calu-3 cells. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatic analysis of proteins identified in LFQMS. Changes in protein abundance generated in response to 100 ng/ml exposure of both MWCNT and SWCNT suggest that cell functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis are predicted to be effected. The majority of the protein changes represent a decrease in amount suggesting a shut down of metabolism to protect cells. The STRING database was used to analyze the protein networks in different functions. Interestingly some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functions and tend to be in the center of the networks, which suggest they may play important roles in the cell function and activity.Item Calcium/Calmodulin-Dependent Protein Kinase II Regulation of IKs during Sustained Beta-Adrenergic Receptor Stimulation(Elsevier, 2018) Shugg, Tyler; Johnson, Derrick E.; Shao, Minghai; Lai, Xianyin; Witzmann, Frank; Cummins, Theodore R.; Rubart-Von der Lohe, Michael; Hudmon, Andy; Overholser, Brian R.; Biochemistry and Molecular Biology, School of MedicineBackground Sustained β-adrenergic receptor (β-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. Objective The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and associated functional implications on IKs. Methods KCNQ1 phosphorylation was assessed using LCMS/MS after sustained β-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral δCaMKII overexpression. Results Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained β-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest δCaMKII phosphorylation. Sustained β-AR stimulation reduced IKs activation (P = .02 vs control) similar to the phosphorylated mimic (P = .62 vs sustained β-AR). Individual phosphorylated mimics at S484 (P = .04) but not at T482 (P = .17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P < .01). δCaMKII overexpression reduced IKs similar to ISO treatment in wild type (P < .01) but not in the dephosphorylated S484 mimic (P = .99). Conclusion CaMKII regulates KCNQ1 at S484 during sustained β-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.Item Characterization of the Mitochondrial Proteome in Pyruvate Dehydrogenase Kinase 4 Wild-Type and Knockout Mice(2009-06-24T12:51:58Z) Ringham, Heather Nicole; Wang, Mu; Harris, Robert; Witzmann, FrankThe goal of this study was to determine the effect of a PDK4 (pyruvate dehydrogenase kinase isoenzyme 4) knock-out on mitochondrial protein expression. A 2-D gel based mass spectrometry approach was used to analyze the mitochondrial proteomes of PDK4 wild-type and knockout mice. Mitochondria were isolated from the kidneys of mice in both well-fed and starved states. Previous studies show PDK4 increases greatly in the kidney in response to starvation and diabetes suggesting its significance in glucose homeostasis. The mitochondrial fractions of the four experimental groups (PDK4+/+ fed, PDK4+/+ starved, PDK4-/- fed, and PDK4-/- starved) were separated via large- format, high resolution two-dimensional gel electrophoresis. Gels were scanned, image analyzed, and ANOVA performed followed by a pair-wise multiple comparison procedure (Holm-Sidak method) for statistical analysis. The abundance of a total of 87 unique protein spots was deemed significantly different (p<0.01). 22 spots were up- or down-regulated in the fed knockout vs. fed wild-type; 26 spots in the starved knockout vs. starved wild-type; 61 spots in the fed vs. starved wild-types; and 44 in the fed vs. starved knockouts. Altered protein spots were excised from the gel, trypsinized, and identified via tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins identified with high confidence include ATP synthase proteins, fatty acid metabolism proteins, components of the citric acid cycle and electron transport chain. Proteins of interest were analyzed with Ingenuity Pathway Analysis (IPA) to examine relationships among the proteins and analyze biological pathways, as well as ontological analysis with Generic Gene Ontology (GO) Term Mapper. IPA found a number of canonical pathways, biological functions, and functional networks associated with the 87 proteins. Oxidative phosphorylation was the pathway associated with a majority of the proteins, while the largest network of proteins involved carbohydrate metabolism and energy production. Overall, the effects of starvation were more extensive on mitochondrial protein expression than the PDK4 knockout.Item Effects of Carbon Nanotubes in Barrier Epithelial Cells via Effects on Lipid Bilayers(Office of the Vice Chancellor for Research, 2013-04-05) Lewis, Shanta; Petrache, Horia I.; Blazer-Yost, Bonnie L.; Witzmann, FrankCarbon nanotubes (CNTs) are one of many nanoparticles (NP) which are being developed as part of the burgeoning nanotechnology. The tubes have similar physical properties to known toxic materials, such as asbestos; yet there is a lack of evidence showing that they may be hazardous to humans, specifically to our barrier epithelial cells. We measured the effects of CNTs on human airway epithelial cells (Calu-3 cell line) using electrophysiology. This is a technique which measures transepithelial electrical resistance (TEER), a measure of monolayer integrity; and short circuit current (SCC) a measure of net ion transport across the cell. Exposed cells showed significant decreases in TEER when incubated for 48 hours with physiologically relevant concentrations of 4μg/cm2 - 0.4ng/cm2 of multi-wall (MW) and 4μg/cm2 - 0.04ng/cm2 single-wall (SW) CNT. TEER is a measure of barrier function which is important in cells that maintain separate compartments in the body. The impaired barrier function, despite sustained cell viability, led us to investigate the mechanism by which the CNT were interacting with the cell when applied topically. Model lipid membranes connected to an ion channel amplifier, Planar Bilayer Workstation (BLM), were used. Membranes were formed using the neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoyl phosphatidylserine (DPhPS) lipids. CNTs caused random, transient currents ranging from 0pA to 6479pA to traverse the membrane. In the presence of Gramicidin A, an ion channel reporter protein, the tubes induced increased gramicidin channel formation in the membrane to saturation level and then membrane lysis. This CNT- lipid interaction indicated that short MWCNTs permits unregulated ion movement across the lipid membrane. Disruption in the selective permeability of the plasmalemma may impact the tissue’s barrier function.Item Integrated Nanosystems Development Institute(Office of the Vice Chancellor for Research, 2011-04-08) El-Mounayri, Hazim; Witzmann, Frank; Agarwal, Mangilal; Naumann, Christoph; Rizkalla, Maher; Decca, RicardoIntegrated Nanosystems Development Institute (INDI) has been recognized and sponsored as a center under the IUPUI Signature Centers Initiative (SCI). INDI is a multidisciplinary institute dedicated to micro/nanoscale systems research, education, and commercialization while providing cluster of analytical equipment and labs serving over 30 faculty members from different departments and schools in support of their research. Specifically, the vision of the INDI is to be a world-class resource for the realization of nanotechnology-based systems that contribute to the economic growth and social advancement of Indiana and the nation and benefit humanity as a whole. The mission of the center is to: 1) Advance nanotechnology research at IUPUI by promoting innovative interdisciplinary research efforts that will lead to external funding; 2) Enhance IUPUI’s research reputation in nanotechnology, nationally and internationally, by providing an identifiable entity that draws in a diverse group of researchers and promotes the combined strength of the group; and 3) Be a leader in translating bionanotechnology and nanoenergy research into innovations that will contribute to the social well being and economic growth of central Indiana and the nation. INDI builds on an excellent research infrastructure at IUPUI. The core facilities of the institute include cleanroom, nano/microfabrication & characterization facilities, and high power simulation and computational resources. Currently, faculty from the Schools of Science, Engineering & Technology, Dentistry, and Medicine, are associated with INDI. The given faculty have expertise in a wide range of fields, including chemistry, physics, biology, material science, electrical and computer engineering, mechanical engineering, orthopaedics, and pathology & laboratory medicine. The research focus of the faculty ranges from nanostructured materials fabrication, study of properties, applications in sensors, energy, and biomedicine, and integration of the devices resulting in realization of nanosystems. As part of the INDI initiatives to developing new undergraduate and graduate track in nanotechnology, center members have been instrumental in the recent development of two interdisciplinary courses, Nanosystems Principles and Integrated Nanosystems Process & Devices which are now being offered by various departments. Moreover, INDI associated faculty members were recently awarded $200,000 from NSF Nanotechnology Undergraduate Education Program for integrating nanotechnology in engineering curricula at IUPUI. To increase the awareness in the community and promote recruitment of future students in nanotechnology, INDI is organizing workshops, offering short courses for industrial employees, and hosting summer camps for high school teachers and students. Summer of 2010 attracted more than 30 high school students for the Nanotechnology Discovery Summer Camp hosted by INDI at IUPUI. Moreover, this program has been extended to include a session for high school teachers in summer of 2011. The poster will summarize the mission, vision, faculty and center collaboration, research projects, achievements, and future plans of INDI.Item Nanotechnology Research, Education, and Outreach by the Integrated Nanosystems Development Institute (INDI)(Office of the Vice Chancellor for Research, 2013-04-05) Naumann, Christoph A.; Rizkalla, Maher; Decca, Ricardo; El-Mounayri, Hazim; Witzmann, Frank; Agarwal, MangilalThrough multidisciplinary research and novel educational programing, the Integrated Nanosystems Development Institute (INDI) is sponsored under IUPUI’s Signature Center Initiative to advance nanotechnology-based systems research and spark student interest in this emerging STEM field. Innovation in the field of nanotechnology arises from interdisciplinary approaches and INDI draws on the expertise of faculty across departments and schools (including the School of Engineering and Technology, School of Science, School of Dentistry, and School of Medicine) in order to fuel research collaborations and offer nanosystems coursework to both science and engineering students. Current research efforts are focused in INDI’s thrust areas of bionanotechnology and sustainable nanoenergy, which build on the existing strengths of participating schools and span a range of critical issues in nanomaterials, nanodevices, nanosystems, energy, physics, and nanomedicine. Examples of research include the development of artificial biomaterials, toxicology of nanomaterials, and the development of nanomanufacturing techniques and educational tools. INDI facilitates research efforts by identifying funding opportunities, establishing research teams, offering seed funding, and providing a cluster of analytical equipment, characterization tools, and lab resources that support the work of faculty and student researchers. Aside from research, INDI plays a vital role in nanotechnology curriculum development on campus, in particular, the design and implementation of coursework offered within IUPUI’s newly developed Nanotechnology Track and Energy Engineering degree program. This academic track provides students with both theory and hands-on experiences involving the fabrication, characterization, and applications of nanomaterials, nanodevices and nanomedicine. Moreover, INDI’s community outreach activities, including its nanotechnology summer camps for K-12 students and teachers, aim to provide early student exposure and educate teachers in applying nanotechnology modules within their classrooms. These student experiences are designed to encourage higher education in an effort to generate the advanced nanotechnology workforce needed by Indiana and the nation.Item Nanotechnology Research, Education, and Outreach by the Integrated Nanosystems Development Institute (INDI)(Office of the Vice Chancellor for Research, 2012-04-13) Naumann, Christoph; Rizkalla, Maher; Decca, Ricardo; El-Mounayri, Hazim; Witzmann, Frank; Agarwal, MangilalAbstract: The Integrated Nanosystems Development Institute (INDI), sponsored under the IUPUI Signature Centers Initiative, with a vision of becoming a world-recognized resource for the realization of nanotechnology-based systems, is advancing both nanotechnology research and education on campus. Innovation in nanotechnology requires multidisciplinary approaches and INDI, a collective group of faculty members across departments and schools (including the School of Engineering and Technology, School of Science, School of Dentistry, and School of Medicine), enables interdisciplinary research collaborations and offers nanosystems coursework to students in science and engineering disciplines. Current research efforts span a range of critical issues in nanomaterials, nanodevices, nanosystems, energy, physics, and nanomedicine, and include projects such as the design and characterization of nanoarchitectures for biomedical applications, advancing fuel cell and energy storage technologies, and investigating nanoparticle toxicology. Several members of INDI have externally funded research and outreach projects. The nanotechnology research capabilities within INDI, including of a cluster of analytical equipment and lab resources for nanosystems development and characterization, support local industry needs as well as the research interests of over 30 faculty members and over 100 students (undergraduate, graduate and postdoctoral) on the IUPUI campus. INDI also provides, through the newly developed courses, students with both theory and hands-on experiences involving the fabrication, characterization, and applications of nanosystems. These courses are also part of IUPUI’s newly developed Nanotechnology Track in Mechanical Engineering and Electrical and Computer Engineering degree programs, and the Energy Engineering degree program. In addition, INDI’s active community outreach activities, including its nanotechnology summer camps for K-12 students and teachers, provide early exposure to nanofabrication techniques and research. These classroom and lab-based experiences are designed to encourage higher education and involvement in academic research in an effort to generate the advanced workforce needed by Indiana and the nation.Item Nanotechnology Research, Education, and Outreach by the Integrated Nanosystems Development Institute (INDI)(Office of the Vice Chancellor for Research, 2014-04-11) Agarwal, Mangilal; Rizkalla, Maher; Naumann, Christoph A.; Decca, Ricardo; El-Mounayri, Hazim; Witzmann, FrankThrough multidisciplinary research and novel educational programing, the Integrated Nanosystems Development Institute (INDI) is sponsored under IUPUI’s Signature Center Initiative to advance nanotechnology-based systems research and spark student interest in this emerging STEM field. Innovation in the field of nanotechnology arises from interdisciplinary approaches and INDI draws on the expertise of faculty across departments and schools (including the School of Engineering and Technology, School of Science, School of Dentistry, and School of Medicine) in order to fuel research collaborations and offer nanosystems coursework to both science and engineering students. Current research efforts are focused in INDI’s thrust areas of bionanotechnology and sustainable nanoenergy, which build on the existing strengths of participating schools and span a range of critical issues in nanomaterials, nanodevices, nanosystems, energy, physics, and nanomedicine. Examples of research include the development of artificial biomaterials, nanosensor arrays for disease detection, and the development of nanomanufacturing techniques and educational tools. INDI facilitates research efforts by identifying funding opportunities, establishing research teams, offering seed funding, and providing a cluster of analytical equipment, characterization tools, and lab resources that support the work of faculty and student researchers. INDI continues to expand its shared resources through the acquisition on new cutting edge instrumentation, including a new field emission scanning electron microscopy facility, which is now open to researchers across campus. Aside from research, INDI plays a vital role in nanotechnology curriculum development on campus, in particular, the design and implementation of coursework offered within IUPUI’s newly developed Nanotechnology Track and Energy Engineering degree program. This academic track provides students with both theory and hands-on experiences involving the fabrication, characterization, and applications of nanomaterials, nanodevices and nanomedicine. Moreover, INDI’s community outreach activities, including its nanotechnology summer camps for K-12 students and teachers, aim to provide early student exposure and educate teachers in applying nanotechnology modules within their classrooms. These student experiences are designed to encourage higher education in an effort to generate the advanced nanotechnology workforce needed by Indiana and the nation.Item Protein expression profiles in hematopoietic stem/progenitor cells after exposure of mice to silicon (28Si) ions(Oxford University Press, 2014) Rithidech, Kanokporn Noy; Tungjai, Montree; Honikel, Louise; Gordon, Chris; Lai, Xianyin; Witzmann, Frank; Biochemistry and Molecular Biology, School of MedicineIt has been well recognized that exposure to space radiation is a major challenge to space exploration. To protect astronauts in space environments, improvement in our knowledge of radiation-induced changes in specific target cells that may affect the health of astronauts is required. Cancer of blood cells, in particular myeloid leukemia (ML), is one of the major health concerns from exposure to space radiation. However, the predictions of risks for developing ML post-exposure to space radiation are unsatisfactory. To increase the reliability of predicting risk for ML, a much improved understanding of space radiation-induced changes in the target cells, i.e. hematopoietic stem/progenitor cells (HSPCs), is critically important. In vivo studies of humans are not possible. Thus, controlled and randomized animal experiments are critically important. Most proteomic applications mentioned above have used 2-DE or stable isotope-tagged mass spectrometry. Although the 2-DE has several advantages (e.g. the detection of potential post-translational modifications of proteins which can be readily visualized on the gel, although the exact type of modification requires determination by mass spectrometry), such technology is simply not as comprehensive and reliable as desired. To overcome these limitations, we recently developed a unique label-free quantitative mass spectrometry (LFQMS) platform [ 1]. This is an innovative, experimentally based method that accurately determines peptide peak retention-time and uses multiple filters for exclusion of unqualified peptides by peptide frequency, retention time, intensity coefficient of variation and intensity correlation to enhance protein quantification of qualified peptides and proteins. In this study, we used the LFQMS platform to examine protein expression-profiles in the colonies of HSPCs (the best population of cells for studying in vivo biological effects of radiation on hematopoietic stem cells) obtained at 6 months after exposure (at which radiation-induced genomic instability and chronic inflammation have been detected [ 2]) of CBA/CaJ mice whole-body to a total dose of 0, 0.1, 0.25 or 0.5 Gy of 300 MeV/nucleon 28Si ions, using a fractionated schedule (two exposures, 15 days apart that totaled each selected dose). These doses of 300 MeV/nucleon 28Si ions are comparable to what astronauts encounter in space. Mice exposed to 0 Gy of 28Si ions served as non-irradiated sham controls. The colonies of HSPCs were obtained from BM cells of five mice per treatment group, by means of an in vitro colony forming unit assay (CFU-A) using methylcellulose-based medium. Proteins were extracted from HSPC colonies and protein concentrations were determined by the Bradford Protein Assay. The trans-proteomic pipeline (TPP) was used to assign a probability of a particular protein being in the sample. A total of 1344 unique, non-redundant proteins were identified with ≥90% confidence from 3254 peptides, quantified and their abundances were compared statistically. Among the 1344 proteins, differential expression of 198 proteins was found to be statistically significant in HSPC colonies obtained from treated groups, in relation to those found in non-irradiated sham controls. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 198 identified proteins. The majority of these proteins are cancer-related (P < 0.0001). Biochemical analyses of the molecular and cellular functions of these proteins reveal association with perturbation in cell survival, free radical scavenging, cell cycle, DNA repair, cellular assembly, hematological system development and inflammatory responses. These proteins are linked to two major molecular networks that are linked to cancer and inflammatory responses (i.e. nuclear factor-κ B and the protein phosphatase 2 A networks). Our results indicated 28Si ion-induced damage in HSPCs.Item Proteomic analysis of murine kidney proximal tubule sub-segment derived cell lines reveals preferences in mitochondrial pathway activity(Elsevier, 2023) Ferreira, Ricardo Melo; de Almeida, Rita; Culp, Clayton; Witzmann, Frank; Wang, Mu; Kher, Rajesh; Nagami, Glenn T.; Mohallem, Rodrigo; Andolino, Chaylen Jade; Aryal, Uma K.; Eadon, Michael T.; Bacallao, Robert L.; Medicine, School of MedicineThe proximal tubule (PT) is a nephron segment that is responsible for the majority of solute and water reabsorption in the kidney. Each of its sub-segments have specialized functions; however, little is known about the genes and proteins that determine the oxidative phosphorylation capacity of the PT sub-segments. This information is critical to understanding kidney function and will provide a comprehensive landscape of renal cell adaptations to injury, physiologic stressors, and development. This study analyzed three immortalized murine renal cell lines (PT S1, S2, and S3 segments) for protein content and compared them to a murine fibroblast cell line. All three proximal tubule cell lines generate ATP predominantly by oxidative phosphorylation while the fibroblast cell line is glycolytic. The proteomic data demonstrates that the most significant difference in proteomic signatures between the cell lines are proteins known to be localized in the nucleus followed by mitochondrial proteins. Mitochondrial metabolic substrate utilization assays were performed using the proximal tubule cell lines to determine substrate utilization kinetics thereby providing a physiologic context to the proteomic dataset. This data will allow researchers to study differences in nephron-specific cell lines, between epithelial and fibroblast cells, and between actively respiring cells and glycolytic cells. SIGNIFICANCE: Proteomic analysis of proteins expressed in immortalized murine renal proximal tubule cells was compared to a murine fibroblast cell line proteome. The proximal tubule segment specific cell lines: S1, S2 and S3 are all grown under conditions whereby the cells generate ATP by oxidative phosphorylation while the fibroblast cell line utilizes anaerobic glycolysis for ATP generation. The proteomic studies allow for the following queries: 1) comparisons between the proximal tubule segment specific cell lines, 2) comparisons between polarized epithelia and fibroblasts, 3) comparison between cells employing oxidative phosphorylation versus anaerobic glycolysis and 4) comparisons between cells grown on clear versus opaque membrane supports. The data finds major differences in nuclear protein expression and mitochondrial proteins. This proteomic data set will be an important baseline dataset for investigators who need immortalized renal proximal tubule epithelial cells for their research.