Browsing by Subject "Basic Research and Disease Models"
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Item AB1-42 Antibody Producing Plasma Cells in DNA AB42 Trimer Immunized Mice Reside Predominantly in the Bone Marrow(2013-01-22) Zacharias, Tresa; Langworthy, Suzanna; Fu, Min; Anderson, Larry; Stuve, Olaf; Rosenburg, Roger; Lambracht-Washington, DorisAlzheimer's disease (AD) is the most common form of age-related dementia and affects nearly 40 million people worldwide. Immunotherapy provides a possible avenue for prophylaxis of AD, but a clinical trial (AN1792) in which patients with early AD were immunized with Aβ1-42 peptide was halted after the occurrence of meningoencephalitis in 6% of the immunized people which was attributed to a T cell autoimmune response. DNA vaccination has been shown to have a polarized Th2 immune response that lacks many of the features responsible for inflammation seen in peptide immunizations. In this study, we show a new feature of the DNA Aβ42 trimer elicited B cell immune response and present data for the presence of a long lived plasma cell pool residing within the bone marrow in DNA immunized mice but not in peptide immunized mice. Two groups of mice were analyzed: one group of B6C3F1 mice (n=20) were studied 4 months after the last DNA vaccination, and a second group of BALB/c mice (n=14), which received DNA or peptide immunizations, were analyzed 10 days following the last immunization. The comparison of antibody producing cells in bone marrow and spleen for the DNA and peptide immunized mice with an Antibody Forming Cell (AFC) ELISPOT assay and subsequent ELISAs showed that bone marrow plasma cells from DNA immunized mice produced more anti-Aβ42 IgG producing cells and higher levels of secreted IgG antibodies. In peptide immunized mice, more IgG antibody producing cells were found to reside in the spleen. These data indicate that the bone marrow may be an important reservoir for B cells following DNA Aβ42 immunization and is in line with studies showing that the bone marrow represents an excellent niche for the survival of long lived plasma cells and a lifetime source for antibody producing B cells which are independent of continuous antigen specific stimulation. Further studies are needed to show whether it is possible to define additional phenotypic characteristics for the antigen specific B cell immune response in DNA Aβ42 trimer immunized mice or differences in the TH subsets directly involved in initial signaling events to B cells in the germinal center reactions.Item Administration of Fatty Acid Emulsions to Reduce Secondary Brain Injury in Mice(2018-01-23) Rodgers, Clifford; Chowdary, Ashish; Liu, Ming-Mei; Carlson, Deborah; Wolf, Steven E.; Minei, Joseph P.; Gatson, JoshuaBACKGROUND: Mild traumatic brain injuries are the most common type of injury to the head. Seventy-five to eighty percent of all traumatic brain injuries (TBI) are considered a mild TBI, or concussions, and involve only a short interruption of mental state and consciousness. Although the FDA reports no nutrition supplements for TBI therapy and/or symptom prevention, preclinical data has suggested that omega-3 poly unsaturated fatty acid (PUFAs) treatment decreases apoptosis, inflammation, and neurodegeneration following brain trauma. In this study, we hypothesized that Smoflipid® reduces inflammation in the brain of adult mice that have suffered a mild-to-moderate brain injury. Smoflipid® is an injectable liquid emulsion solution that contains omega-3, omega-6, omega-9, and medium chain triglycerides. METHODS: In this study, mice were subjected to a moderate brain injury using the controlled skull impact device (Leica microsystems) and we administered Smoflipid® intraperitoneally at day 1 and 3 after injury. At Day 14 after injury and treatment the mouse brains were harvested, processed, and stained using immunohistochemistry for the inflammatory markers, glial fibrillary acidic protein (GFAP) and Iba1. RESULTS: In this study after TBI, within the corpus callosum (C.C.) and cerebral cortex there was a significant increase in the levels of activated microglia (Day 14 p=0.05) compared to the control animals. Treatment with Smoflipid® shortly after injury, resulted in a significant decrease in the number of active microglia within these brain regions. CONCLUSIONS: Chronic activation of microglia and heightened inflammation in the cerebral cortex/C.C. after TBI, results in cognitive decline and long-term memory deficits. As a therapeutic strategy, by targeting these pro-inflammatory cells with Smoflipid®, we hypothesize that a reduction in the activity of microglia will improve results in better neurological outcomes. More definitive studies will be conducted to test the efficacy of Smoflipid® at reducing secondary brain injury after TBI.Item Alterations in Neural Stem Cell Fate Following Focal Ischemia(2014-02-04) Nguyen, Derek; Vemireddy, Vamsidhara; Mashimo, Tomoyuki; Battiste, James; Maher, Elizabeth; Bachoo, RobertINTRODUCTION: The purpose of this experiment is to determine the differentiation identity of the neural stem cells (NSC) in the subventricular zone (SVZ) of adult mouse brain after a middle cerebral artery occlusion (MCAO). Injury to the brain causes a large number of changes including inflammation and apoptosis, but the reaction of NSC's has been more difficult to characterize because of the transient nature of their response. Previously, adult neural stem cells (NSC) in the SVZ have been observed to differentiate predominantly into cells with neuronal characteristics. This theory is questioned via a tamoxifen-inducible cre-recombinase (Cre-ERT2) expression mouse model system. METHOD: The Cre-ERT2 expression mouse model system is driven by the Cystatin-C promoter to label NSC's in a time specific manner and track their cell fate after MCAO. After the ischemia, these brain sections were stained with different immunohistochemicals at three separate time points. One set was co-labeled with GFAP, an astrocyte marker, and BrdU, a proliferation marker. Another set was co-labeled with DCX, a neuronal marker, and BrdU. This was used to differentiate between latent NSCs and proliferating NSCs by comparing the ipsilateral side (ischemic) with the contralateral side (control) of the brain. RESULTS: Compared to the contralateral, the ipsilateral side had a significant increase in GFAP/BrdU positive cells between day 3 and day 7 time points. The cell quantity dropped between day 7 to day 14 time points. Compared to the contralateral, the ipsilateral side had a decrease in DCX/BrdU positive cells between day 3 and day 7 time points. The cell quantity significantly increased between day 7 to day 14 time points, and the quantity at day 14 was about twice to that of the day 3 time point. DISCUSSION: This data demonstrated that after the MCAO, the stem cells are not just undergoing neurogenesis, but are for certain period of time, also differentiating into astrocytes that are migrating towards the site of injury. This phenomenon is only witnessed in the NSCs towards the day 7 time point. Afterwards and leading up to day 14, the NSCs seem to be changing their cell fate programming from the astrocyte pathway back to the intended neuronal pathway. Thus, the staining results verify that after an ischemia, NSCs within the SVZ regions of the brain undergo a constant change of programmed cell fate, alternating between immature neurons and astrocytes implicating future aims for "programmed" neurogenesis in the development of therapeutic strategies for the treatment of brain damage and disease.Item Analyses of the Link Between Amyloid and Tau Pathology in an AD Mouse Model (3xtg-AD): Disease Progression with Increased Levels of Abeta and Tau Peptides(2018-01-23) Zapata, Lucio, Jr.; Ismail, Hannah; Lambracht-Washington, DorisINTRODUCTION: Pathological features of Alzheimer's disease (AD) include the accumulation of extracellular amyloid plaques composed of aggregated amyloid-β (Aβ) peptide and intracellular neurofibrillary tangles consisting of phosphorylated tau protein. Mutations in the genes that encode amyloid precursor protein (APP), and presenilin 1 and 2 (PS1/PS2) have been shown to cause familial AD in humans. Studies provided evidence that Aβ accumulation may initiate phosphorylation of tau protein, via the Ras/MEK/Extracellular Signal-regulated Kinase (ERK) signaling cascade, activation of the mitogen-activated protein kinase (p38 MAPK), Cyclin dependent kinase 5 (CDK5) and/or glycogen synthase kinase-3β (GSK3β). We studied distribution of Aβ and tau oligomers, Erk activity in different brain lysate fractions from different age groups of a triple transgenic mouse model (3xTg-AD) and wild-type mice, and Erk activity in DNA Abeta42 immunized mice. METHODS: Brain lysates of 4-, 6-, 12-, and 20-month-old 3xTg-AD and wild-type mice were prepared via a 4-step extraction protocol in TBS (soluble), TBS-T, SDS, and formic acid. DNA Abeta42 vaccination administered via gene gun. Abeta and Tau concentrations and Tau phosphorylation levels were monitored by Dot blot, Semidenaturing detergent agarose gel electrophoresis (SDD-AGE), and ELISA using anti-Abeta and anti-Tau antibodies. Erk1/2 levels were monitored by Western blot using monoclonal antibodies. RESULTS: There was a significant increase of total tau concentrations with increasing age and we found also an increasing insolubility (more tau in the non-soluble brain lysate fractions). 4- and 20-month-old 3xTg-AD soluble brain lysates indicated the presence of aggregated tau peptide, which was not present in wild-type control mice. Kinases involved with tau phosphorylation were measured and showed an increase of activated/phosphorylated Erk1/2 with increasing age, with a drop in concentration at 12 months in half of the mice analyzed (n=5). Immunized 3xTg-AD mice showed a decrease in activated Erk1/2 when compared to non-immunized, age matched mice. DISCUSSION: The 3xTg-AD mouse model provides a good model to study pathologies and possible treatments for human Alzheimer's disease. Abeta 42 peptide and tau increase due to age in this mouse model. A link between the amyloid pathology is likely found in the wide spectrum of cellular kinases which are upregulated due to Abeta in Alzheimer's disease. Therefore, immunization against Abeta and generation of anti-Abeta antibody will indirectly reduce tau pathology.Item Anti-VEGF Induced Reduction in Microvessel Density Does Not Correlate with Anti-Tumor Repsonse in Lung Cancer Xenografts(2013-01-22) Jacob, Antonia J.; Sullivan, Laura A.; Toombs, Jason E.; Minna, John D.; Brekken, Rolf A.Vascular endothelial growth factor-A (VEGF) is a primary stimulant of angiogenesis in pathological conditions including tumor progression. Strategies to block VEGF activity prevent or slow tumor growth in preclinical settings; however, clinical studies with bevacizumab, a monoclonal antibody (mAb) specific for VEGF have resulted in only modest benefit to a subset of patients with lung cancer. Previous studies in our laboratory defined the therapeutic efficacy of bevacizumab and an alternative anti-VEGF mAb (r84) in 12 non-small cell lung cancer (NSCLC) xenografts. Three NSCLC xenografts (Calu-6, A549 and Calu-3) showed intrinsic resistance to bevacizumab therapy. In the present study we evaluated whether microvessel density (MVD) could be used to 1) demonstrate if the anti-VEGF mAbs were effective at reducing VEGF-driven angiogenesis and 2) if MVD changes induced by bevacizumab or r84 correlated with overall therapeutic efficacy as determined by tumor size after chronic therapy. 3-5 tumors from animals bearing NSCLC xenografts treated with a control mAb (XTLl, bevacizumab or r84 were evaluated by immunohistochemistry for endothelial cells as a measure of microvessel density. Two independent endothelial cell markers were used, endomucin and CD31. In 11 of the 12 xenografts treatment with bevaclzumab or r84 significantly reduced MVD compared to XTL treatment, suggesting that bevacizumab and r84 do reduce VEGF-driven angiogenesis. However, the reduction in MVD induced by anti-VEGF therapy did not correlate with overall tumor response to therapy. These results strongly implicate resistance to anti-VEGF therapy is not mediated by activation af alternative angiogenic programs to compensate for VEGF blockade. Further the results suggest that tumor cell adaptation to therapy-induced hypoxia underlies poor therapeutic response to anti-VEGF strategies. Microarray of gene expression analysis of control treated tumors revealed several genes associated with metabolism, proliferation, and metastasis were significantly increased in tumors that displayed intrinsic resistant to bevacizumab. We conclude that response of tumor cells to therapy-induced hypoxia is a critical feature that drives the overall efficacy of anti-VEGF strategies.Item Antigen-Specific Natural Killer Cell Responses in Chronic Hepatitis C Virus Infection(2014-02-04) Bowman, Kathryn; Holz, Lauren; Rehermann, BarbaraChronic hepatitis C virus (HCV) infection results in an inflammatory liver disease leading to fibrosis and cirrhosis. The progression of liver disease is thought to be immune-mediated because HCV itself is non-cytopathic. Given that HCV-specific T cells are diminished in number and functionally exhausted in chronic HCV infection, it remains unclear which cell population drives disease pathogenesis. Here, we investigated the function of natural killer (NK) cells, the major innate immune cell population in the liver. The NK cell population increases further in the setting of chronic hepatitis C infection and have multiple mechanisms of cytotoxicity. We investigated whether NK cells could respond to HCV in an antigen-specific manner. PBMCs from 39 patients with chronic HCV infection (gt 1) not recently on medication (>2 years) were stimulated for 8 hours in a whole blood activation assay with pools of overlapping 18-mer peptides comprising HCV structural (E1, E2) and nonstructural (NS3) proteins. Cytokine production by NK cells and T cells was assessed by multicolor flow cytometry. The frequency of IFN-γ+ NK cells was 5 fold greater than the frequency of IFN-γ+ T cells. A minority of IFN-γ+ NK cells co-produced TNF-α. NK cell responses to HCV peptides varied between subjects, but did not correlate with T cell responses or viremia. This study demonstrates that NK cells are activated in an antigen-specific manner in chronic HCV infection and respond to both structural and nonstructural HCV proteins. Natural killer cell cytokine and cytotoxic responses were larger than corresponding T cell responses. The mechanism of antigen-specific NK cell activation is currently under investigation.Item Antisense Blockade of Efflux Systems in Gram-Negative Pathogens(2018-01-23) Subramanian, Naveen G.; Felder-Scott, Christina F.; Sturge, Carolyn R.; Greenberg, DavidAntibiotic resistant bacteria, aka "super bugs", are a critical threat to public health worldwide, as the medical community is running out of effective antibiotics against a growing number of bacteria. One of the ways that bacteria develop resistance to antibiotics is by utilizing efflux systems that are used to pump the antibiotic out. A strategy that is currently being investigated is to restore the susceptibility of these bacteria to antibiotics by using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to suppress genes within these bacteria that encode components of efflux pumps. This project studied the effectiveness of PPMOs that target the AcrAB-TolC efflux pump, which is a major component of the intrinsic antibiotic resistance mechanisms of E. coli and K. pneumoniae. Experiments tested for the effect of the PPMO targeting the acrA gene, specific sequences within the acrA gene, and the tolC gene. The effect of the PPMO was measured by a change in the minimum inhibitory concentration (MIC) of common antibiotics such as Piperacillin/Tazobactam (Pip/Tazo), Azithromycin, and Levofloxacin on strains of these two bacteria. The results show that PPMOs targeted to the acrA gene have a 4-8 fold effectiveness at lowering antibiotic MICs for the bacterial strains. PPMOs that targeted the tolC gene, on the other hand, have no synergistic effect in lowering antibiotic MICs for the bacterial strains. In addition, changing the sequence of the PPMOs targeting the acrA gene was shown to have an effect, albeit small, on susceptibility to antibiotics, which suggests that targeting specific regions of a gene of interest can induce more or less susceptibility in the bacteria to antibiotics.Item B-Catenin and K-ras Synergize to Form Wilm's Tumor with Concurrent p53 Pathways Modulation(2014-02-04) Hembd, Austin; Clark, Peter; DeGraff, DavidHumans can develop pediatric kidney tumors called Wilm's tumors. If one identifies the specific genes that cause Wilm's tumor, or that concomitantly change expression levels in the tumor tissue, then diagnosis and eventually drug targets for therapy are expedited. Characterizing genetic determinants in the mouse model can help actualize these future therapies. When the genes Kras and βCatenin are overexpressed in a mouse, it develops a renal tumor histologically identical to a human Wilm's tumor. Microarray analysis on mouse tumor tissue showed modulated expression levels of gene targets in the p53 tumor suppressor pathway. Immunohistochemistry stained mice tissue specifically for p53. In tissue with Kras and βcatenin overexpression, p53 staining is positive surrounding the tumor. RT qPCR measured levels of gene expression of p53 pathway associated genes. Combination mutants βCatenin and Kras were compared with controls. This PCR array analysis identified genes, such as cJun, Traf1, and Dapk1, that had significant expression changes in the combination mutant when compared to either mutant individually. The expression is modulated in a nonadditive fashion in Kras + βcatenin mutant tissues, which can explain the phenotype of Wilm's tumor in only double mutant mice. These genes individually represent targets for therapy in the future, and together represent an identifying fingerprint for diagnosis and prediction.Item A Bacterial Cholesterol Sensor to Assess Cholesterol Accessibility in Red Blood Cells(2016-01-19) Chakrabarti, Rima Shah; Radhakrishnan, Arun; Cohen, Jonathan C.; Hobbs, Helen H.Mammals are able to gain cholesterol from two sources: diet and endogenous synthesis. However, the only means of cholesterol removal is reverse cholesterol transport (RCT), in which cholesterol is transported to the liver and exported into bile. While high density lipoprotein (HDL) is considered to be the major conduit for RCT, studies with HDL-deficient animals reveal no defect in tissue cholesterol balance. We hypothesize that red blood cells (RBCs), which contain 50% of blood cholesterol, also play a role in RCT. To measure accessible cholesterol in RBCs, we developed an assay that utilizes the cholesterol binding properties of the toxin Anthrolysin-O (ALO). We purified and fluorescently labeled domain 4 of ALO (fALOD4). We then incubated fALOD4 with RBCs from 164 subjects and measured fluorescence intensity using flow cytometry. Both intra-assay and intra-individual variability of the assay were less than 10%. In the test population, fALOD4 binding varied 10-fold. fALOD4 binding did not correlate with total RBC cholesterol but did correlate with RBC phosphatidylcholine (PC) (-0.42, p=6e-7) and lyso-phosphatidylcholine (LPC) (0.40, p=6e-6). Increasing the LPC:PC ratio in RBCs with phospholipase A2 (PLA2) increased fALOD4 binding by 3-fold. fALOD4 binding also correlated with plasma HDL (0.30, p=6e-4) and triglycerides (-0.57, p=2e-12). These data suggest that RBC accessible cholesterol varies in a population, is driven by intrinsic RBC phospholipid composition and interacts with known cholesterol transporters in the blood. Future studies will determine if variability in fALOD4 binding is driven by non-lipid RBC membrane components, is genetically determined, or contributes to atherosclerosis.Item Burn Serum Stimulated Mitochondrial Fission Was Decreased with IL-6 Antibody Treatment(2017-01-17) Sehat, Alvand; Song, Juquan; Kulangara, Rohan; Maredia, Navin; Maxwell, Christian; Panwar, Kunal; Huebinger, Ryan M.; Carlson, Deborah L.; Zang, Qun S.; Wolf, Steven E.INTRODUCTION: Burn patients suffer muscle mass loss associated with hyperinflammation and hypercatabolism. We previously observed that burn serum resulted in cell death with elevated mitochondrial fragmentation in C2C12 myoblast. IL-6 as the key cytokine response to thermal injury has been showed to increase mitochondrial fragmentation. We thus posit that inhibition of IL-6 expression in burn serum will alleviate mitochondrial fragmentation. The aim of this study is to investigate the neutralization effect of IL-6 antibody in burn serum stimulated myoblasts. METHODS: Murine myoblasts C2C12 cells were cultured with recombinant IL-6 protein from 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, and 100 ng/ml. Cells were labeled with MitoTracker Green dye and live cell images were captured with Confocal microscopy. Next, C2C12 cells were exposed to medium containing 1) 10% serum from control rat, 2) 10% serum from burn rat, and 3) 10% serum from burn rat and 0.5 ug/ml of IL-6 antibody. All cells were labeled as the first experiment and live cell images were recorded. The caspase 3 activity was further examined from cell protein lysate. RESULTS: The 1 ng/ml dose of r-IL6 showed a fourfold increase in mitochondrial volume (μm3) at 24 hours post challenge. The intensity signal of Mitotracker was significantly increased in the 1 ng/ml IL-6 dose group at 48 hours. The 1 ng/ml dose of r-IL6 showed an increase in mitochondrial fragmentation. In cells cultured with 10% serum from control rats, mitochondrial morphology maintained the elongated linear shape during the 48 hours. In cells cultured with 10% serum from burned rats, a reduction in mitochondrial sizes was significant. In cells cultured with 10% serum and IL-6 antibody treatment this effect was reversed. Further, the intensity signals increased in response to the burn serum challenge. However, with addition of IL-6 antibody treatment the intensity signals decreased. In addition, burn serum increased the total volume of mitochondria about 1.4 fold, while with IL-6 antibody treatment the total volume was decreased. Consistently, a significant decrease in the expression of caspase 3 in the IL-6 antibody treatment group was observed. CONCLUSION: IL-6 stimulates an increase in mitochondria fragmentation in myoblasts, while IL-6 antibody treatment decreases mitochondrial fragmentation and cell death in burn serum stimulated myoblasts. This project has shown that targeting cytokine levels may be an effective treatment strategy in the management of burn patients.Item Calcitriol Treatment Suppresses Contraction-Associated Gene Expression in Pregnant Mice Near Term(2018-01-23) Morgan, Kelsi; Yang, Ailling; Montalbano, Alina P.; Mendelson, Carole R.Preterm birth (PTB) is the leading cause of infant mortality during the first four weeks of life world-wide. This is due, in part, to our incomplete understanding of the mechanisms that mediate uterine quiescence during most of pregnancy and promote the transition to labor at term. Term and preterm labor are associated with increased levels of proinflammatory cytokines within maternal reproductive tissues where they activate inflammatory transcription factors (e.g. NF-κB) that enhance expression of genes encoding contraction-associated proteins (CAP) (i.e. connexin-43 (CX-43)), oxytocin receptor (OXTR)). By contrast, uterine quiescence is maintained throughout most of pregnancy by increased progesterone (P4) levels and enhanced progesterone receptor (PR) activity, which silence expression of proinflammatory mediators and CAP genes. Treatment of pregnant women at risk for preterm labor with progestins has negligible effects - underscoring the need for novel therapeutic targets. To identify such targets, our lab surveyed the myometrial transcriptome of timed-pregnant mice at 15.5-18.5 days post-coitum (dpc) and during labor at term (19.0 dpc) using RNA-sequencing. Interestingly, Cyp27b1 was one of the most highly downregulated transcripts at 18.5 dpc and in-labor, compared to 15.5 dpc. Cyp27b1 encodes 1α-hydroxylase, the key enzyme responsible for synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (calcitriol) which binds to the vitamin D receptor (VDR). Calcitriol/VDR have anti-inflammatory actions and are reported to mediate maternal tolerance to the hemi-allogeneic fetus. Interestingly, we previously observed that P4 treatment of timed-pregnant mice caused a significant increase in myometrial CYP27B1 mRNA levels, compared to controls. In the present study, we sought to assess effects of calcitriol treatment on CAP gene expression in timed-pregnant mice. To this end, pregnant mice were injected s.c. daily from 13.5-17.5 dpc with vehicle (n=3) of with 3 μg/kg of calcitriol (n=4). Mice were sacrificed and myometrial tissues were collected at 18.5 dpc. RT-qPCR revealed significantly reduced levels of CX43 (p<0.0001) and OXTR (p<0.05) in myometrium of calcitriol treated mice, compared to controls. Collectively, these data suggest that the decrease in Cyp27b1 expression, coupled with the decline in PR function near term may contribute to increased CAP gene expression leading to myometrial contractility and labor. Cyp27b1 may serve as a key P4/PR target gene that acts cooperatively to maintain myometrial quiescence via its anti-inflammatory actions. Thus, calcitriol may be a safe and effective treatment for the prevention of PTB.Item Cdk5-Dependent Regulation of Neuronal MEK1(2013-01-22) Krishnan, Govind; Benavides, David; Tassin, Tara; Bibb, James A.INTRODUCTION: Cyclin-dependent protein kinase 5 (Cdk5) is a member of the Cdk family that is implicated in many regulatory pathways in post-mitotic neurons. The kinase plays an important role in neuronal development and synaptic transmission; its disregulation contributes to neurodegenerative diseases such as Alzheimer's disease. Cdk5 is involved in the regulation of the Ras-Raf-MEK-ERK signaling pathway. It is hypothesized that Cdk5 serves a neuroprotective role by preventing the prolonged stimulation of ERK from inducing cell cycle reentry and, hence, neuronal apoptosis. In order to address whether Cdk5 phosphorylation of MEK1 affects its in vitro kinase activity, we sought to confirm the Cdk5-phosphorylation site of MEK1. Although it has been previously shown that Cdk5 phosphorylates MEK1-T286, our previous unpublished mass spectometry analysis of in vitro kinase reactions identifies the site as T292. We corroborated this data with Western blot analysis of the in vitro Cdk5 phosphorylation of MEK with phosphate-specific antibodies to sites T286 and T292. METHODS: Glutathione S-transferase (GST)-tagged MEK1 was expressed and purified from E.coli. The tagged protein was run through column fractions, with glutathione beads that would attach to the GST tag, such that GST-MEK1 would be purified out of the bacterial lysates. This was then run through gel electrophoresis to confirm that MEK1 had indeed been purified, by comparing the bands seen in the gels to the known molecular weights of GST-MEK1. The protein concentration was determined using a Bradford assay. Non-radioactive and radioactive Cdk5 kinase assays of MEK1 were then performed. MEK1 was incubated in the absence or presence of Cdk5 with Mg2+/ATP or Mg2+/ATP plus trace amounts of [gamma-32P]-ATP, respectively. The non-radiolabeled kinase reactions were subjected to polyacrylamide gel electrophoresis (PAGE) on a 10-20% acrylamide gradient gel, followed by electrophoretic transfer and Western blot analysis with phospho-T286 and phospho-T292 MEK1 antibodies. Phosphorimages of radiolabeled kinase reactions were generated by PAGE, dried gels and standards were exposed to a phosphorimager screen and analyzed on a phosphorimager. RESULTS: Our results confirm that T292 is indeed the Cdk5 site of MEK1. The identity of this site is important because it is the location at which control can be exercised over the ERK pathway.Item Characterization of Receptor Protein Tyrosine Phosphatase Epsilon (PTPRE) Gene Promoter(2015-01-26) Isaacs, Thomas; Shukla, Abhay A.; Amatruda, JamesBACKGROUND: Receptor protein tyrosine phosphatase epsilon (PTPRE) is a receptor bound phosphatase that has been shown to be downregulated in Wilms' tumors compared to normal tissue, and could potentially be a target for future therapy. Our objective is to identify and characterize the promoter of the PTPRE gene and define the critical role of Wt1 transcription factor (commonly downregulated in Wilm's tumor) in PTPRE gene expression and in Wilms' tumor progression. METHODS: Our first step involved cloning and sequence analysis of the upstream region of the human PTPRE gene followed by PCR primer design and PCR amplification. The amplified fragment was then cloned into a promoterless reporter vector (pGl3 Basic) and transfected in Hek293 cells. Promoter DNA was used for deletion analysis where multiple PCRs were performed using a single forward primer and multiple reverse primers with nucleotides sequentially deleted from the 3' end. The different size PCR products were then cloned into pGL3 Basic vector DNA, transfected into HEK cells and had reporter assay (luciferase assay) performed to calculate fold change in PTPRE expression over promoterless vector. After the critical transcription factor binding motif was identified, PCR was performed to amplify full length promoter lacking 76 bases. The role of the deleted nucleotides was confirmed via luciferase assay. The sequence of deleted nucleotides was then analyzed for the presence of transcription factor binding motifs. Next, a predicted Wt1 transcription factor binding motif was mutated using site directed mutagenesis. Mutated fragment was cloned into pGl3Basic vector. Both wild type and mutated vector were transfected and luciferase assay was performed to confirm role of Wt1 binding motif for PTPRE promoter activity. Chromatin immunoprecipitation assay was then performed for further evidence. Promoter activity was also compared in two cells lines having differential expression of Wt1. Western blot and semi-quantitative PCR are used to confirm the expression levels of Wt1. RESULTS: Promoter deletion analysis confirmed that the Wt1 binding motif present at -16 position is critical for PTPRE expression and mutation of this site results in 95% loss in promoter activity in Hek293 cells. PTPRE promoter activity was shown to be high in Hek293 cells and low in Hela cells (high and low WT1 expression respectively), suggesting WT1 driving promoter activity. ChiP using WT1 antibody confirmed WT1 binding of the critical transcription factor binding motif. CONCLUSION: These results shed light on why PTPRE expression is lower in Wilms' tumors and reveals potential future targeted therapy.Item Characterization of β-glucuronidase for Enzyme Replacement Therapy in DYT6 Dystonia(2023-01-31) Lieu, Linh; Yim, Daron; Pappas, Samuel; Dauer, WilliamBACKGROUND: Dystonia is a debilitating disorder defined by sustained involuntary twisting movements. The current symptomatic treatments for dystonia offer only modest efficacy but numerous side effects. Dominantly inherited, loss of function mutations in the THAP1 transcription factor cause DYT6 dystonia (DYT-THAP1). THAP1 modulates the development of oligodendrocyte progenitor cells (OPC) by regulating the catabolism of glycosaminoglycans (GAGs), a crucial component of the extracellular matrix. The loss of THAP1 within OPCs directly reduces GAG-catabolic lysosomal enzyme β-glucuronidase (GusB) causing the accumulation of GAGs that inhibit their own maturation to myelinating cells. The result is severe dysmyelination during early CNS maturation and impaired neurodevelopment. Genetic overexpression of GusB rescues the maturation deficits and CNS myelination in THAP1 deficient mice raising the critical question of whether β-glucuronidase enzyme replacement could restore myelination in THAP1 null mice. OBJECTIVE: Characterization of β-glucuronidase for enzyme replacement therapy (ERT) in DYT6 dystonia models. METHOD: To establish a symptomatic model of DYT6 dystonia, we utilized a Cre/LoxP based Thap1 conditional knockout mouse model ("THAP1-NCKO") and a GusB transgenic mouse line ("GusB-TG"). We evaluated the enzymatic activity and biodistribution of GusB in the CNS using biochemical and histochemical assays. RESULTS: We determined that THAP1-NCKO mice had lower GusB activity than their control counterparts. Interestingly, the activity of GusB is higher in adult (P60) mice compared to juvenile (P30) mice. Visualization of GusB activity showed that distribution of GusB was highest in white matter tracts. We showed that mice with THAP1- related deficits experienced a significant reduction in GusB activity within white matter tracts but not in other surveyed GusB positive brain areas. CONCLUSIONS: Age differentially affects CNS GusB enzymatic activity in a murine model of DYT6 dystonia. GusB enzyme exhibits a distinct biodistribution that varies regionally. White matter tracts experience more severe defects with THAP1 loss. Our results provide insights into the specific locations where GusB activity is deficient and highlight the importance of a "critical period" in which genetic insults have long lasting neurodevelopmental implications.Item Clinical Applications of a Computational Voice Simulator(2016-01-19) Rao, Ashwin; Mau, TedBACKGROUND: Given input vocal fold tissue geometries and mechanical properties, a simulator can produce a virtual voice of a defined pitch and sound pressure level. A simulator gives us a tool to systematically investigate the effects of changes to vocal fold geometry or tissue properties on the voice. OBJECTIVE:To create a user-friendly graphical interface for the NCVS voice simulator, and to use it to determine gender differences in vocal fold tissue properties. METHODS: Graphical user interface (GUI): A GUI was programmed in MATLAB, with windows that separately allow user control of (1) vocal fold tissue geometries and mechanical properties, (2) ranges of inputs for "brute force" simulations in which a range of properties are investigated, and (3) optimization parameters that allow "smart" simulations with target pitch and sound pressure levels. Geometric and tissue properties: A literature search was performed to determine reasonable values for vocal fold superficial layer, the vocal ligament, and vocalis muscle to use in the simulator. Gender differences in tissue properties: Two groups of simulations were carried out, one using typical female geometries with the average female speaking voice frequency of 200 Hz, the other using typical male geometries and 100 Hz as the desired target. Assessment of outcome: Each group of simulations comprised of several thousand simulations, with each simulation corresponding to a specific combination of frequency, sound pressure level, and corresponding tissue properties that generated those specific voice qualities. Clusters of solutions were identified with cluster analysis. RESULTS: Solutions visualized in 2D plots showing a strong linear dependence of voice frequency on the longitudinal shear modulus of the vocal ligament, consistent with the general understanding of vocal physiology. There was no clear dependence of frequency on the other tissue properties. Cluster analysis for the male simulation showed two groups of solutions. The first group had the superficial layer longitudinal shear moduli (μ'1) roughly equal to that of the muscle (μ'3). The second group had a ratio of μ'1/μ'3 between 10-20. This indicates that, for a male voice output of 100 Hz, tissue parameters are most optimal if the superficial layer and muscle have similar stiffness, or if the superficial layer is about 10-20 times stiffer than the muscle. The translayer ratio of μ'2/μ'3 also showed two main clusters: one close to 1 and another at a ratio of around 100. This indicated that, for an average male voice, the vocal ligament and muscle should have comparable stiffness, or the ligament is about 100 times stiffer than the muscle. On the other hand, cluster analysis for the female simulation only showed one solution group, with both μ'1/μ'3 and μ'2/μ'3 ratios of around 1. This indicated that, for a female voice output around 100 Hz, the superficial layer, muscle, and ligament layers all should have comparable stiffness levels. CONCLUSIONS: We have shown that simulations for female geometric parameters calculate different clusters than those for male parameters, given the same target pitch and SPL. By creating a GUI for the NCVS voice simulator, we are now enabling clinician users explore how various morphological defects and changes can affect the human voice, ultimately leading to improved clinical and surgical outcomes.Item Comparison of Bioluminescence and Fluorescence Imaging as Tools for Evaluating Growth of MCF7 and 4T1 Mammary Tumors(2017-01-17) Lin, Elisa B.; Winters, Alex; Gerberich, Jeni; Campbell, Trey; Liu, Li; O'Kelly, Devin; Mason, Ralph P.INTRODUCTION: Tumor growth can be assessed by a variety of small animal imaging modalities which are cheap, easy, and efficient. In particular, bioluminescence imaging (BLI) and fluorescence imaging (FLI) have received attention for their ability to measure tumor growth and response to treatment. Both imaging modalities are accurate and well established, however, each method has its own unique advantages and limitations. This study compared the use of BLI and FLI to characterize and monitor growth of mammary 4T1-luc and MCF7-luc-GFP-mCherry tumors in nude mice. Strong correlations were established between BLI, FLI, and tumor volume, providing evidence that each method could be used to validate the other and reduce overall error. METHODS: BLI and FLI image sequences were performed with the IVIS(r) Spectrum. BLI was used for 4T1 (n = 8) and MCF7 (n = 6) tumors. FLI was only used for MCF7 tumors. Tumor volume was measured with calipers. RESULTS: Evaluating the area under each BLI and FLI curve (the AUC method) proved to be more accurate than only evaluating at one time point or wavelength. For both BLI and FLI, the AUC method greatly simplified the imaging workflow and removed the need for perfect temporal accuracy, since all times and wavelengths were considered. BLI and FLI both showed strong correlation with tumor volume (R2 = 0.91 and 0.87, respectively). The BLI and FLI signals were also correlated (R2 = 0.79). Experimental difficulties like tumor scarring and a mid-experiment C. bovis infection compromised data quality. DISCUSSION: The strong correlations between each measurement are very reassuring. Each offers specific benefits, e.g., BLI and FLI allow detection of sub-palpable volumes and additional metastases in some cases. BLI offers particularly strong contrast to noise, but requires the administration of luciferin substrate. FLI signal is subject to background auto fluorescence; this became a particular problem when the C. bovis infection occurred. Caliper measurements are simple for subcutaneous tumors, but the optical imaging can also reveal deeper tumors. The investigations to date largely confirm growth characteristics and the utility of available imaging methods matching the extant literature. The correlations had not been examined for 4T1-luc at UTSW previously. Furthermore, these methods provide a foundation for my forthcoming medical school research activity. Notably, future plans include continued investigation of metastases and utilizing Multispectral Optoacoustic Tomography (MSOT) for integrated hypoxia studies.Item Confirmation of Hypoglycemia in Goat -/- Mice When Total Body Fat Falls below 2% of Body Weight(2013-01-22) Singh, Ashish; Goldstein, Joseph L.; Zhao, Tongjin; Brown, Michael S.Ghrelin is an octanoylated peptide hormone first identified in stomach, with the octanoyl group being essential to its biological activity. The enzyme that attaches the octanoyl group to ghrelin is called Ghrelin-Oacyltransferase (GOAT). By studying mice that have the GOAT gene knocked out (GOAT KO mice), we have shown that these mice develop severe hypoglycemia under a 60% calorie restricted diet. In order for this hypoglycemia to occur, depletion of fat deposits is required. Specifically, GOAT knockout mice will not develop severe hypoglycemia until the total fat mass drops to 2% of the total body weight. These observations were made in 8-week-old mice with an average starting fat mass between 8-10% of total body weight. In our present work, we wanted to know whether we could reproduce the results using older mice with a higher percentage of fat mass. The mice used in this study were 32-34 week old male mice (wild type and GOAT knockout mice, n=8/group), and both groups had an average starting fat mass of 17% of total body weight. We then subjected these mice to a 60% calorie restriction and monitored their fat mass and blood glucose level everyone or two days. For the first 7 days of calorie restriction, both wild type and GOAT knockout mice were able to maintain their blood glucose around 60 mg/dl. After that, the GOAT knockout mice start to develop hypoglycemia when their body fat mass dropped below 2% of the body weight. However, the wild type mice were able to maintain their blood glucose level above 40 mg/dl throughout the course even when their fat mass dropped below 2% of their body weight. The results here further confirm that in order to develop hypoglycemia in the GOAT knockout mice, the fat mass needs to be depleted from these mice during calorie restriction , even in older mice (32-34 weeks versus 8 weeks).Item Corneal Stromal Remodeling after Photorefractive Keratectomy(2017-01-17) Su, Shan; Kivanany, Pouriska; Grose, Kyle; Petroll, W. MatthewThe cornea is an optically clear tissue that contributes 2/3 of the eye's refractive power, making it a target for vision correction procedures. A small percentage of patients who receive corneal surgery experience loss of corneal transparency (haze). Haze occurs when stromal cells in the cornea (keratocytes) become activated and transform into fibroblasts or myofibroblasts, which can generate contractile forces that may disrupt the collagen architecture. We investigated the wound healing process following photorefractive keratectomy (PRK, a clinical vision correction procedure) using 3-D imaging both in vivo and in situ. We hypothesized that following PRK in rabbits, keratocytes located within the injured stroma, where collagen is intact, would align with the collagen lamellae and will not produce fibrosis. In contrast, we expected keratocytes anterior to the wound, where collagen is disrupted, to undergo myofibroblast transformation and produce significant haze. Twelve New Zealand white rabbits were scanned one week before surgery using an in vivo confocal microscope. PRK was performed on the right eyes of the rabbits (9 diopter spherical photocorrection) with subsequent scans at 7 days, 21 days, 3 months, and 6 months. Custom software was used to build 3-D reconstructions and measure stromal thickness and haze. Some rabbits were sacrificed at each time point to obtain in situ confocal images. Thickness measurements at pre-scan, 7 days, 21 days, 3 months, and 6 months were: 335, 208, 265, 293, 313um. The decrease and subsequent increase in thickness is consistent with removal of tissue for PRK, followed by tissue regeneration. Haze measurements at the same time points were: 1797, 4453, 6906, 3212, 3433 intensity units. The increase and subsequent decrease in backscatter suggests two phases of wound healing. In situ confocal imaging showed that cells within the native stroma were aligned with collagen lamellae without prominent stress fibers at 7 and 21 days, while cells anterior to the injured stroma at 21 days aligned randomly and displayed prominent stress fibers. At 3 months, these cells aligned with correlation to the collagen and did not express stress fibers. At 6 months, the cell/collagen arrangement was similar to uninjured tissue. Our results suggest that the collagen lamellae direct fibroblast patterning during repopulation of the native stroma, without inducing fibrosis or significant haze. In contrast, cells accumulating on top of the stroma initially align randomly and produce hazy, fibrotic tissue. Remarkably, over time, cells remodel the fibrotic tissue to produce a lamellar structure that is similar to the native corneal stroma.Item Developing a Real-Time, Axially Resolving Optical Monitor of Spinal Cord Blood Flow(2019-01-22) Gao, Feng; Busch, David R.; Goh, Chia Chieh; Lin, Wei; Yodh, Arjun G.; Floyd, Thomas F.BACKGROUND: Spinal cord ischemia is a disease of high morbidity and mortality often caused by surgeries repairing the thoracic and abdominal aorta. Current methods to monitor spinal cord hemodynamics such as electrophysiology methods, MR arterial spin labeling, and laser Doppler either have a slow response time or are unfeasible intra-operatively. In this study, we developed an optical probe to monitor spinal cord blood flow and oxygenation in real-time at multiple sites along the spine. METHODS: Experiments were conducted on 8 adult domestic pigs. Probes were inserted into the epidural space through a laminotomy prior to asphyxia and local ischemia via catheter balloon inflation. Vital signs, anesthetic parameters, and spinal hemodynamics were measured continuously prior to intervention, throughout asphyxia, and during inflation/deflation of the balloon catheter. Optical blood flow measurements were compared against microspheres. Optical hemoglobin saturation of spinal cord was compared to mixed venous blood gases. RESULTS: The fiber optic probe detected changes in flow and oxygenation in all asphyxia and balloon inflation trials across multiple sites along the spine. We observed significant changes in spinal cord blood flow during balloon inflation in the epidural space. We also observed a significant correlation between optically measured hemoglobin saturation and mixed venous blood gases. CONCLUSION: We developed an intra-operative tool that provides continuous, real-time monitoring of spinal cord hemodynamics at multiple sites along the spine. We hope this tool can more safely guide surgeons in reducing the incidence of spinal cord ischemia.Item Development of Ex Vivo Femoral Head Deformity Model to Test Restorative Surgical Techniques(2022-02-01) Edwards, David; Niese, Brad; Ma, Chi; Kim, Harry K.W.Legg-Calve-Perthes disease (LCPD) is a childhood ischemic hip disorder which produces femoral head deformity due to weakened bone structure. The femoral head deformity causes pain, stiffness, and debilitating osteoarthritis; if left untreated, a total hip replacement is eventually required. Currently there is no reliable ex-vivo deformity model to develop and test the efficacy of new surgical devices and methods to improve the deformity and to restore the round shape. We hypothesize that a reliable model of deformity comparable to LCPD can be created using porcine cadaver bone by applying compressive force methods. Due to the availability of porcine humeral heads in the lab, we performed our preliminary studies using the humeral heads. We tested four mechanical approaches. Our first approach involved the application of static and cyclic compressions to mature humeral heads. However, the method failed to deform the heads due the high compressive strength of the mature bone. Our second approach involved drilling into the mature heads to create stress risers before applying the compression forces. This resulted in undesirable fracture of the bone. Our third approach used juvenile porcine humeral heads with cyclic compressions which resulted in deformity, but was inconsistent in the number of cycles required to achieve the appropriate deformity. Finally, we successfully achieved a reliable deformity model resembling LCPD by mounting juvenile humeral heads in a specific orientation and applying successive increases in static compression force at 50, 100, 150, 200, and 250 lbs using a Bose Electroforce 3330 test instrument. The amount of collapse after each static compression was measured using calipers. Identical measurements were taken after each compression test, up to 250lbs of force. This method created a 2 mm collapse of the humeral head which was reproducible. In summary, we developed a novel ex-vivo model of deformity which will facilitate the development of new restorative surgical devices and techniques to improve the femoral head deformity of LCPD.