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Optimization of Liposomal Encapsulation Efficiency
Get PDFIntroduction:
My project was a continuation of the Vascular Research Lab’s (VRL) ongoing research at the University of Tennessee Medical Center Knoxville (UTMCK) aimed at optimizing liposomal encapsulation efficiency of small interfering RNA (siRNA) which can be used to silence genes to prevent a variety of disease pathologies.
Methods: Assay siRNA loading capacity of liposomes based on lipid concentration Development of a method for liposome purification: HPLC & HiTRAP Column
Results & Conclusion: siRNA loading capacity Higher lipid:siRNA resulted in increased encapsulation efficiency HPLC – did not work as expected HiTRAP Column – currently being optimized to be used as part of standard operating procedure
Molecular Response of Retinal Pigment Epithelial Cells to Oxidized Lipoproteins: Global and Targeted Studies
Get PDFGlobal-scale examinations of biological systems at the molecular level complement targeted approaches to scientific inquiry that focus on specific subsets of biomolecules, or on a single molecule of interest. In this dissertation, we utilized both the discovery-based approach to evaluate the proteomics workflows centered around mass spectrometry as the key technology, and the targeted approach to examine the molecular response of RPE due to oxidized lipoproteins (oxLDL) treatments. A crucial aspect in proteomics studies is the design of bioanalytical strategies that maximize coverage of the complex repertoire of a proteome. A comprehensive, unbiased examination of the proteome represents a powerful approach toward system-level insights into disease mechanisms. We evaluated the performance of bioanalytical platforms for profiling of the proteome in a biological system. We applied a discovery-based approach to evaluate the global transcriptome and proteome changes due to oxLDL treatment in ARPE-19 cells.
We studied the role of scavenger receptors CD36 and CD5L/AIM in ARPE-19 cells when induced with oxLDL. We compared three different multidimensional proteome fractionation platforms: polymeric reversed-phase liquid chromatography at high pH (PLRP), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF) separations. We applied a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) in a data-dependent setting and used bioinformatics for protein identification. The three platforms identified a total of 1043 proteins altogether. Among the three bioanalytical strategies, SDS-PAGE followed by LC-MS/MS provided the best coverage. We also evaluated another bioanalytical platform which consists of a highresolution mass spectrometer combined with nano-UPLC in a data-independent setting without pre-fractionation for oxLDL mediated proteome alteration in ARPE-19 cells.
This platform outperformed the SDS-PAGE based analytical platform in terms of proteome coverage as it identified around 2500 proteins, ca. 3-fold more proteins than the latter. Most importantly, this platform was able to perform label free quantification of differentially expressed proteome alteration. The platforms identified proteins with diverse physicochemical characteristics involved in various functional roles within the biological system.
Furthermore, we carried out the first comparative transcriptomic and proteomic study for the evaluation of oxLDL effects on ARPE-19 cells after a 4 h exposure. The treatment with oxLDL affected the regulation of more than 700 genes that were involved in regulation of cell cycle, oxidative stress, cholesterol efflux, circadian rhythm, NRF-2 pathways. However, LDL treatment alone did not induce the regulation of these pathways. The differential proteomic analysis found 41 proteins affected due to the oxLDL treatment. This study provided a foundation for a bioanalytical platform for identification and label-free quantification in the human retinal pigment epithelial cells (ARPE-19) proteome. The list of differentially expressed proteins due to oxLDL treatment identified in this study gives insights to the change in proteins that might be interrogated for their roles in pathogenesis of macular degeneration. These findings could give us targets to intervene in the pathogenesis of AMD progression in human for the development of better treatment and prevention against this degenerative disease.
Lastly, we studied the mechanistic role of scavenger receptors CD36 and CD5L/AIM in oxLDL uptake by ARPE-19 cells. We, for the first time, demonstrated the presence of scavenger receptor CD5L in ARPE-19 cell. The oxLDL uptake was primarily dependent on CD36, and both the CD5L/AIM and CD36 were seen to co-localize in the presence of oxLDL. Our results suggest a new dynamics on CD5L/AIM on the oxLDL uptake that was not seen in macrophages. The reduction in intracellular accumulation of oxLDL in the presence of extracellular recombinant CD5L/AIM is an interesting phenomenon as it has been recently shown the involvement of CD5L/AIM in autophagy
Molecular Interplay of Chromatin Remodeling Factor BRG1 and Transcription Factor STAT3 Regulates Stemness, Chemosensitivity and Tumorigenicity of Glioma Tumor Initiating Cells
Get PDFGlioblastoma Multiforme (GBM) is an aggressive brain tumor, characterized by high cellular heterogeneity, is refractory to treatment and has dismal prognosis. These characteristics of GBM have suggested the presence of stem-like cells that have the ability to initiate and maintain tumors of a heterogeneous nature, and bestow resistance to current therapeutic regimens. It is therefore imperative to identify the dysregulated molecular pathways which enable the maintenance of these cells in a stem-like state in order to inform strategies to therapeutically target them.
In this study, we investigated the role of the Y705 and S727 phosphorylation domains of STAT3, a multifunctional transcription factor that is constitutively activated in the glioma stem like cells or glioma-initiating cells (GIC). We demonstrate that STAT3 is critical for GIC tumorigenesis. Furthermore, we show that the Y705 phosphorylation of STAT3 is necessary for GIC-induced tumor formation. We demonstrate that in GICs Y705 and S727 phosphorylation of STAT3 is sequential, such that Y705 phosphorylation precedes S727 phosphorylation. In addition, we also observed that S727 phosphorylation is dependent on Y705 phosphorylation.
Furthermore, by targeted microarrays and RNA Sequencing we have identified ~160 genes that are STAT3-regulated. These genes are involved in several critical biological processes including the cell cycle, hypoxia, TGF- and the extracellular matrix. We further show that a subset of these STAT3-regulated genes are also associated with GBM disease stratification.
Additionally, we demonstrate that BRG1, the catalytic component of the chromatin remodeling complex SWI/SNF, plays a critical role in the maintenance of the GICs and in the drug resistance of these cells. We show that BRG1 promotes the expression of key stem cell markers, Oct4, Nanog and CD44, while loss of BRG1 led to upregulation of astrocyte differentiation marker GFAP and S100, as well as the oligodendroglial marker Olig2. These findings indicate that BRG1 loss in GICs resulted in a transition towards a more differentiated fate. Targeted microarrays identified several genes that are dependent on BRG1 in GICs. One of these genes is TXNIP, a major redox regulator that is also a negative regulator of glycolysis. We found that TXNIP expression in the GICs is regulated by both BRG1 and STAT3. Furthermore, using genetic and pharmacological means we demonstrate that BRG1 plays a critical role in the chemo sensitivity of the GICs
Pathogenicity and Protection Mediated by a Single TCRβ in Experimental Autoimmune Encephalomyelitis
Get PDFHow the TCR repertoire, together with risk-associated major histocompatibility complex (MHC), imposes susceptibility for autoimmune disease is not fully understood. A small fraction of TCR α or β chains are “public”, and are shared by most individuals.High-throughput sequencing of the mouse TCRβ repertoire during myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmuneencephalomyelitis (EAE) identified a public TCRβ chain, TCRβ1, which was highly shared among individual mice and preferentially deployed during EAE. Retrogenic expression of TCRβ1 resulted in spontaneous early-onset EAE in mice with high penetrance and severity, despite being paired with a diverse endogenous TCRα repertoire. To further study autoimmunity conferred by this highly reactive beta chain, we generated TCRβ1 transgenic mice. Unexpectedly, TCRβ1 transgenic mice failed to develop spontaneous disease and were also resistant to standard EAE induction by MOG immunization. Despite the absence of disease, ~5% of TCRβ1 transgenic CD4+ T cells from unimmunized mice were MOG-specific, and these further expanded in response to MOG immunization. Three independent transfer models, including transfer of in vitro activated TCRβ1 splenocytes, transfer of TCRβ1 bone marrow, and a mock retrogenic system, all resulted in EAE in recipient mice, indicating there is no T cell-intrinsic blockade to pathogenesis. MOG-responsive transgenic T cells also expressed higher levels of PD-1 and Lag3 in comparison to those from WT and 2D2 mice, suggesting that they might be more prone to exhaustion. TCRβ1 transgenic T cells secreted higher levels of inflammatory cytokines IFN-γ and IL-17 relative to WT T cells, however these levels were comparable to those in pathogenic 2D2 MOG-responsive T cells. These results suggest that tolerance mechanisms in TCRβ1 transgenic mice prevent T cell pathogenicity and disease. Further studies are needed to fully resolve the mechanisms responsible for protection
The Role of BiP Co-chaperone SIL1 in Marinesco-Sjögren Syndrome Pathogenesis
No full textMarinesco-Sjögren syndrome (MSS) is a rare, autosomal recessive, multisystem disorder, which is characterized by cerebellar ataxia, early-onset bilateral cataracts, and progressive myopathy amongst other symptoms. MSS has been attributed to mutations in the SIL1 gene, which encodes a nucleotide exchange factor for the endoplasmicreticulum- resident Hsp70 chaperone, BiP. To date, there are 46 MSS-associated mutations that have been reported in SIL1, which occur throughout this gene and are predicted to result in a loss of SIL1’s function. The large majority of these mutations cause deletions of large fractions of the SIL1 protein. Nine MSS-associated mutations are particularly interesting because they alter less than six amino acids, yet are associated with a phenotype indistinguishable from a near-full length deletion of SIL1. The mechanisms by which these nine mutations lead to a loss of SIL1’s function are not well understood. On the other hand, it remains unclear how the loss of SIL1 leads to the multisystem defects observed in MSS, selectively affecting certain tissues while sparing others. Our goal was to answer these two questions.
We have shown that the selected nine MSS-associated SIL1 mutations may dramatically alter the protein microenvironment and disrupt intramolecular interactions, such that it alters the folding properties of SIL1 and renders it aggregation-prone. This offers a potential mechanism by which mutations in SIL1 cause a loss of its function. We validated that the C57BL/6 Sil1Gt mouse model, which harbors a genetic disruption of Sil1, phenocopies numerous aspects of the MSS-phenotype and represents a valid preclinical model system to investigate the MSS-associated pathology and explore pharmacotherapeutic strategies. Using a combination of the Sil1Gt mice and SIL1- deficient MSS-patient-derived lymphoblastoid cell lines, we explored the biosynthesis and secretion of immunoglobulins (Ig), which are the best characterized substrate of BiP to date. In vivo antigen-specific immunizations and ex vivo LPS stimulation of splenic B cells revealed that the Sil1Gt mouse was indistinguishable from wild-type age-matched controls, in terms of both the kinetics and magnitude of antigen-specific antibody responses. There was no significant accumulation of BiP-associated Ig assembly intermediates or evidence that another molecular chaperone system was used for antibody production in the LPS-stimulated splenic B cells from Sil1Gt mice. ER chaperones were expressed at the same level in wild-type and Sil1Gt mice, indicating that there was no evident compensation for the disruption of Sil1. These results were confirmed and extended in lymphoblastoid cell lines from individuals with MSS, leading us to conclude that, surprisingly, the SIL1 was dispensable for antibody production.
Using Sil1Gt mice, we next characterized the molecular aspects of progressive myopathy associated with MSS. Proteomic-profiling of quadriceps at the onset of myopathy revealed that SIL1 deficiency affected multiple pathways critical to muscle physiology. We observed an increase in ER chaperones prior to the onset of muscle weakness, which was complemented by up-regulation of multiple protein degradation pathways. These responses were inadequate to maintain normal expression of secretory pathway proteins, including insulin and IGF-1 receptors. There was a paradoxical downstream PI3K-AKT signaling and glucose uptake in Sil1-disrupted skeletal muscles, all of which were insufficient to maintain systemic glucose homeostasis and muscle mass. Together, these data reveal defects in maintaining ER homeostasis upon SIL1 loss, which are countered by multiple compensatory responses that are ultimately unsuccessful, leading to trans-organellar proteostasis collapse and myopathy
Can You Credit This? A Credit-Bearing Information Literacy Course for Graduate Health Science Students
Get PDFObjective: University of Florida HSCL librarians have long offered information-related instruction through guest lectures in the programs they serve and stand-alone workshops; however, librarians had not taught any credit-bearing courses focused on information literacy prior to 2016. To more fully integrate information-related skills into curricula, librarians developed a one-credit course for graduate students, initially targeting those in basic science programs but expecting that its modular format would allow easy adaptation for other health science programs. After two successful semesters teaching this course, librarians adapted the instructional content to an audience of graduate students in the College of Public Health and Health Professions. Methods: The basic science version of the course covered literature searching, bibliographic citation software, basic NCBI resources, funding sources, data management, and information ethics. In transforming the class for health professional students, librarians replaced the genetic and genomic resources sessions with introductory sessions on systematic reviews and grey literature. Conceptual material fit mapped well to the Association of College and Research Libraries (ACRL) Information Literacy Framework. Health professions PhD program coordinators provided feedback on the syllabus and helped inform students about the course. Results: Formal student evaluation data is not yet available; however, informal feedback indicated that the course was incredibly valuable for doctoral students; one student even suggested making the course required. Student quiz scores and class discussion revealed that the choice of topics resonated with students, captured their interests, and fulfilled a need not met by their other courses. Conclusions: Developing and teaching a credit-bearing graduate course on information-related topics is one mechanism for librarians to further integrate into the curricula of their programs and broaden their reach. A course that has been developed and approved by a specific campus unit may prove relevant to other units and easily customized to fit their needs, thus increasing its impact
