Dissecting the Interaction between p53 and TRIM24

Dissecting the Interaction of p53 and TRIM24 Aundrietta DeVan Duncan Supervisory Professor, Michelle Barton, Ph.D. p53, the “guardian of the genome”, plays an important role in multiple biological processes including cell cycle, angiogenesis, DNA repair and apoptosis. Because it is mutated in over 50% of cancers, p53 has been widely studied in established cancer cell lines. However, little is known about the function of p53 in a normal cell. We focused on characterizing p53 in normal cells and during differentiation. Our lab recently identified a novel binding partner of p53, Tripartite Motif 24 protein (TRIM24). TRIM24 is a member of the TRIM family of proteins, defined by their conserved RING, B-box, and coiled coil domains. Specifically, TRIM24 is a member of the TIF1 subfamily, which is characterized by PHD and Bromo domains in the C-terminus. Between the Coiled-coil and PHD domain is a linker region, 437 amino acids in length. This linker region houses important functions of TRIM24 including it’s site of interaction with nuclear receptors. TRIM24 is an E3-ubiquitin ligase, recently discovered to negatively regulate p53 by targeting it for degradation. Though it is known that Trim24 and p53 interact, it is not known if the interaction is direct and what effect this interaction has on the function of TRIM24 and p53. My study aims to elucidate the specific interaction domains of p53 and TRIM24. To determine the specific domains of p53 required for interaction with TRIM24, we performed co-immuoprecipitation (Co-IP) with recombinant full-length Flag-tagged TRIM24 protein and various deletion constructs of in vitro translated GST-p53, as well as the reverse. I found that TRIM24 binds both the carboxy terminus and DNA binding domain of p53. Furthermore, my results show that binding is altered when post-translational modifications of p53 are present, suggesting that the interaction between p53 and TRIM24 may be affected by these post-translational modifications. To determine the specific domains of TRIM24 required for p53 interaction, we performed GST pull-downs with in vitro translated, Flag-TRIM24 protein constructs and recombinant GST-p53 protein purified from E. coli. We found that the Linker region is sufficient for interaction of p53 and TRIM24. Taken together, these data indicate that the interaction between p53 and TRIM24 does occur in vitro and that interaction may be influenced by post-translational modifications of the proteins.

On the Pulling Power of Ice Streams

Gravity wants to pull an ice sheet to the center of the Earth, but cannot because the Earth's crust is in the way, so ice is pushed out sideways instead. Or is it? The ice sheet "sees" nothing preventing it from spreading out except air, which is much less massive than ice. Therefore, does not ice rush forward to fill this relative vacuum; does not the relative vacuum suck ice into it, because Nature abhors a vacuum? If so, the ice sheet is not only pulled downward by gravity, it is also pulled outward by the relative vacuum. This pulling outward will be most rapid where the ice sheet encounters least resistance. The least resistance exists along the bed of ice streams, where ice-bed coupling is reduced by a basal water layer, especially if the ice stream becomes afloat and the floating part is relatively unconfined around its perimeter and unpinned to the sea floor. Ice streams are therefore fast currents of ice that develop near the margins of an ice sheet where these conditions exist. Because of these conditions, ice streams pull ice out of ice sheets and have pulling power equal to the longitudinal gravitational pulling force multiplied by the ice-stream velocity. These boundary conditions beneath and beyond ice streams can be quantified by a basal buoyancy factor that provides a life-cycle classification of ice streams into inception, growth, mature, declining and terminal stages, during which ice streams disintegrate the ice sheet. Surface profiles of ice streams are diagnostic of the stage in a life cycle and, hence, of the vitality of the ice sheet.

The Jakobshanvs Effect

The Jakobshavns Effect may have been a significant factor in hastening the collapse of palaeo ice sheets with the advent of climatic warming after 18,000 years ago and may precipitate partial collapse of the present‐day Greenland and Antarctic Ice Sheets following CO2‐induced climatic warming in the decades ahead. The Jakobshavns Effect is observed today on Jakobshavns Glacier, which is located at 69°10′N on the west coast of Greenland. The Jakobshavns Effect is a group of positive feedback mechanisms which allow Jakobshavns Glacier to literally pull ice out of the Greenland Ice Sheet at a rate exceeding 7 km/a across a floating terminus 800 m thick and 6 km wide. The pulling power results from an imbalance of horizontal hydrostatic forces in ice and water columns at the grounding line of the floating terminus. Positive feedback mechanisms that sustain the rapid ice discharge rate are ubiquitous surface crevassing, high summer rates of surface melting, extending creep flow, progressive basal uncoupling, progressive lateral uncoupling, and rapid iceberg calving.

The Taxation of Bonuses and its Effect on Executive Compensation - Evidence from the UK Experience

This paper explores the reaction of compensation components awarded to executive directors of UK financial institutions following the adoption of the bonus tax in December 2009. Excessive bonuses are blamed for encouraging risk taking and are regarded as one of the pull factors of the financial crisis. The British government attempted to reduce bonuses and accordingly corporate risk-taking by means of a special tax on cashbased bonuses. Using a comprehensive dataset on executive compensation we show that the introduction of the bonus tax decreased the net cash bonuses awarded to directors by about 43%, accompanied however by a simultaneous increase in other compensation components leaving both variable as well as total compensation unaffected. Hence, the incidence of the bonus tax was borne by the firms which compensated their managers for the decrease in cash-based compensation by awarding them different forms of pay. Consistent with this finding our data also suggests that firms reduced dividend pay-outs as a consequence of the bonus tax.

Cell force measurements in 3D microfabricated environments based on compliant cantilevers.

We report the fabrication, functionalization and testing of microdevices for cell culture and cell traction force measurements in three-dimensions (3D). The devices are composed of bent cantilevers patterned with cell-adhesive spots not lying on the same plane, and thus suspending cells in 3D. The cantilevers are soft enough to undergo micrometric deflections when cells pull on them, allowing cell forces to be measured by means of optical microscopy. Since individual cantilevers are mechanically independent of each other, cell traction forces are determined directly from cantilever deflections. This proves the potential of these new devices as a tool for the quantification of cell mechanics in a system with well-defined 3D geometry and mechanical properties.

Influence of screw augmentation in posterior dynamic and rigid stabilization systems in osteoporotic lumbar vertebrae: a biomechanical cadaveric study.

STUDY DESIGN Biomechanical cadaveric study. OBJECTIVE To determine whether augmentation positively influence screw stability or not. SUMMARY OF BACKGROUND DATA Implantation of pedicle screws is a common procedure in spine surgery to provide an anchorage of posterior internal fixation into vertebrae. Screw performance is highly correlated to bone quality. Therefore, polymeric cement is often injected through specifically designed perforated pedicle screws into osteoporotic bone to potentially enhance screw stability. METHODS Caudocephalic dynamic loading was applied as quasi-physiological alternative to classical pull-out tests on 16 screws implanted in osteoporotic lumbar vertebrae and 20 screws in nonosteoporotic specimen. Load was applied using 2 different configurations simulating standard and dynamic posterior stabilization devices. Screw performance was quantified by measurement of screwhead displacement during the loading cycles. To reduce the impact of bone quality and morphology, screw performance was compared for each vertebra and averaged afterward. RESULTS All screws (with or without cement) implanted in osteoporotic vertebrae showed lower performances than the ones implanted into nonosteoporotic specimen. Augmentation was negligible for screws implanted into nonosteoporotic specimen, whereas in osteoporotic vertebrae pedicle screw stability was significantly increased. For dynamic posterior stabilization system an increase of screwhead displacement was observed in comparison with standard fixation devices in both setups. CONCLUSION Augmentation enhances screw performance in patients with poor bone stock, whereas no difference is observed for patients without osteoporosis. Furthermore, dynamic stabilization systems have the possibility to fail when implanted in osteoporotic bone.

Retention of Root Canal Posts: Effect of Cement Film Thickness, Luting Cement, and Post Pretreatment.

The aim of this study was to investigate the effect of the cement film thickness of a zinc phosphate or a resin cement on retention of untreated and pretreated root canal posts. Prefabricated zirconia posts (CosmoPost: 1.4 mm) and two types of luting cements (a zinc phosphate cement [DeTrey Zinc] and a self-etch adhesive resin cement [Panavia F2.0]) were used. After removal of the crowns of 360 extracted premolars, canines, or incisors, the root canals were prepared with a parallel-sided drill system to three different final diameters. Half the posts did not receive any pretreatment. The other half received tribochemical silicate coating according to the manufacturer's instructions. Posts were then luted in the prepared root canals (n=30 per group). Following water storage at 37°C for seven days, retention of the posts was determined by the pull-out method. Irrespective of the luting cement, pretreatment with tribochemical silicate coating significantly increased retention of the posts. Increased cement film thickness resulted in decreased retention of untreated posts and of pretreated posts luted with zinc phosphate cement. Increased cement film thickness had no influence on retention of pretreated posts luted with resin cement. Thus, retention of the posts was influenced by the type of luting cement, by the cement film thickness, and by the post pretreatment.

Mass transport around comets and its impact on the seasonal differences in water production rates

Comets are surrounded by a thin expanding atmosphere, and although the nucleus' gravity is small, some molecules and grains, possibly with the inclusion of ices, can get transported around the nucleus through scattering (atoms/molecules) and gravitational pull (grains). Based on the obliquity of the comet, it is also possible that volatile material and icy grains get trapped in regions, which are in shadow until the comet passes its equinox. When the Sun rises above the horizon and the surface starts to heat up, this condensed material starts to desorb and icy grains will sublimate off the surface, possibly increasing the comet's neutral gas production rate on the outbound path. In this paper we investigate the mass transport around the nucleus, and based on a simplified model, we derive the possible contribution to the asymmetry in the seasonal gas production rate that could arise from trapped material released from cold areas once they come into sunlight. We conclude that the total amount of volatiles retained by this effect can only contribute up to a few percent of the asymmetry observed in some comets.

Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds

Giardia lamblia is a protozoan parasite that causes giardiasis, a diarrhoeal disease affecting humans and various animal species. Nitro drugs such as the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are used for treatment of giardiasis. Nitroreductases such as GlNR1 and GlNR2 may play a role in activation or inactivation of these drugs. The aim of this work is to characterise these two enzymes using functional assays. For respective analyses recombinant analogues from GlNR1 and GlNR2 were produced in Escherichia coli. E. coli expressing GlNR1 and GlNR2 alone or together were grown in the presence of nitro compounds. Furthermore, pull-down assays were performed using HA-tagged GlNR1 and GlNR2 as baits. As expected, E. coli expressing GlNR1 were more susceptible to metronidazole under aerobic and semi-aerobic and to nitazoxanide under semi-aerobic growth conditions whereas E. coli expressing GlNR2 were susceptible to neither drug. Interestingly, expression of both nitroreductases gave the same results as expression of GlNR2 alone. In functional assays, both nitroreductases had their strongest activities on the quinone menadione (vitamin K3) and FAD, but reduction of nitro compounds including the nitro drugs metronidazole and nitazoxanidewas clearly detected. Full reduction of 7-nitrocoumarin to 7-aminocoumarin was preferentially achieved with GlNR2. Pull-down assays revealed that GlNR1 and GlNR2 interacted in vivo forming a multienzyme complex. These findings suggest that both nitroreductases are multifunctional. Their main biological role may reside in the reduction of vitamin K analogues and FAD. Activation by GlNR1 or inactivation by GlNR2 of nitro drugs may be the consequence of a secondary enzymatic activity either yielding (GlNR1) or eliminating (GlNR2) toxic intermediates after reduction of these compounds. © 2015 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license

Titanium and hydroxyapatite coating of polyetheretherketone and carbon fiber-reinforced polyetheretherketone: A pilot study in sheep

Purpose: The purpose of this study was to evaluate the bone formation capability of polyetheretherketone (PEEK) and carbon fiber-reinforced PEEK (CFR-PEEK) implants coated with different titanium and hydroxyapatite plasma-sprayed layers after 2 and 12 weeks. Methods: In six sheep 108 implants were placed in the pelvis. Altogether six different surface modifications were tested. After 2 and 12 weeks, n = 3 implants per group were examined histologically and n = 6 implants per group were tested by a pull-out test. Results: Biomechanically (p = 0.001) as well as histologically (p > 0.05) surface coating of PEEK/CFR-PEEK led to an increase of osseointegration from 2 to 12 weeks. After 12 weeks, coated implants demonstrated significant (p < 0.001) higher pull-out values in comparison to uncoated implants. Overall, the double coating (titanium bond layer and hydroxyapatite top layer) showed the most favorable results after 2 and 12 weeks. Conclusions: Plasma-sprayed titanium and hydroxyapatite coatings on PEEK or CFR-PEEK demonstrated a significant improvement of osseointegration.

The Impact of Mobile Technology on the Viewing Experience of the Avid Sports Fan

Viewing sports on television is a very different experience than viewing a game in-person. Sports commentators on television are seemingly able to pull up random statistics as soon as something unexpected happens during a game. Because these statistics are discussed on television, any sports fan who wants to watch a game in-person misses out on the opportunity to hear them during the game. This study identifies what statistics, both common and uncommon, are considered important or interesting by avid sports fans who watch a particular sport at least two times per week. In addition, it considers the rise of mobile technology and the effects that this change of trend will have on business opportunities and experiences. The purpose of the project is to find a way to mimic the television viewing experience for fans who are watching in-person through the use of mobile technology, and in particular through the use of iPhone applications.

DISEASE-RELATED INFORMATION NEEDS OF CANCER PATIENTS AGES 11-20: A COMPARISON OF PATIENT-PARENT AND PATIENT-PHYSICIAN PERCEPTIONS

Innovative, aggressive treatments and prolonged survival rates for patients with childhood cancers have placed new demands on the patient, parent and physician. As a result, counterproductive coping behaviors are often noted in adolescent cancer patients.^ One of the main ways the environment is manipulated by the individual to achieve personal comfort is through selectivity of information. An individual will usually pull the support personally needed to cope from the environment if sufficient resources are available. However, information provided young cancer patients is often filtered through the physicians and parents perspectives of the patient's needs without systematic input from the patient. In order to ensure that adequate information resources are available to help teenage patients cope with their illness, health professionals must have insights into the information needs of those patients. No previous efforts to address this subject were found in the literature.^ This study was designed to identify adolescent perspectives of their disease-related information needs and to compare their viewpoints with those of their parents and physicians. Sixty-five outpatient cancer patients (ages 11-20) receiving treatment at the University of Texas M. D. Anderson Hospital and Tumor Institute in Houston, Texas, 60 of their parents, and 53 physicians, who were involved in the treatment of pediatric patients at M. D. Anderson, were asked to complete self-administered questionnaires. The questionnaires used were developed, administered and analyzed by the investigator. Specific areas addressed in the questionnaires included: Perceptions of cancer-related tests and treatments, the importance of 30 disease-related items of information, responses evoked by receipt of information, current and preferred sources of information, delivery of information at the time of diagnosis, and disease-related information requested for patients, family, friends and teachers.^ Adolescent perceptions of their information needs and their preferences for delivery of information were determined. The relationships between patient-parent and patient-physician perceptions were then analyzed to determine areas in which agreements and disparities in viewpoint existed. Programmatic and research recommendations were then provided.^ Hopefully, through these efforts, the adolescent patient will be helped to receive relevant information support from those deemed to be most important to his/her efforts to cope with cancer. ^

Targeting TRAF6 for Cancer Therapeutical Development

Tumor necrosis factor (TNF)-Receptor Associated Factors (TRAFs) are a family of signal transducer proteins. TRAF6 is a unique member of this family in that it is involved in not only the TNF superfamily, but the toll-like receptor (TLR)/IL-1R (TIR) superfamily. The formation of the complex consisting of Receptor Activator of Nuclear Factor κ B (RANK), with its ligand (RANKL) results in the recruitment of TRAF6, which activates NF-κB, JNK and MAP kinase pathways. TRAF6 is critical in signaling with leading to release of various growth factors in bone, and promotes osteoclastogenesis. TRAF6 has also been implicated as an oncogene in lung cancer and as a target in multiple myeloma. In the hopes of developing small molecule inhibitors of the TRAF6-RANK interaction, multiple steps were carried out. Computational prediction of hot spot residues on the protein-protein interaction of TRAF6 and RANK were examined. Three methods were used: Robetta, KFC2, and HotPoint, each of which uses a different methodology to determine if a residue is a hot spot. These hot spot predictions were considered the basis for resolving the binding site for in silico high-throughput screening using GOLD and the MyriaScreen database of drug/lead-like compounds. Computationally intensive molecular dynamics simulations highlighted the binding mechanism and TRAF6 structural changes upon hit binding. Compounds identified as hits were verified using a GST-pull down assay, comparing inhibition to a RANK decoy peptide. Since many drugs fail due to lack of efficacy and toxicity, predictive models for the evaluation of the LD50 and bioavailability of our TRAF6 hits, and these models can be used towards other drugs and small molecule therapeutics as well. Datasets of compounds and their corresponding bioavailability and LD50 values were curated based, and QSAR models were built using molecular descriptors of these compounds using the k-nearest neighbor (k-NN) method, and quality of these models were cross-validated.

Development and Characterization of Novel Ras Inhibitors

Ras genes are mutated in 15% of human cancers. Ras GTPases operate as molecular switches regulating cellular processes including proliferation, differentiation, and apoptosis. The three main isoforms of Ras – H-Ras, K-Ras, and N-Ras – inhabit distinct nanodomains of the plasma membrane and intracellular compartments including the Golgi. However, the role of single endogenous Ras isoforms on these compartments remains unclear as most studies have utilized ectopically expressed and mutant forms of Ras proteins. In an effort to develop novel tools that will allow us to abrogate individual endogenous Ras isoforms, we targeted the catalytic domain of p120RasGAP to the plasma membrane with the hypervariable region (HVR) of H-Ras (GAP-CTH) or K-Ras (GAP-CTK) and to the Golgi using the HVR of H-Ras with insertion of a point mutation (GAP-CTH181S). We performed GST-RBD pull-downs on cells expressing each GAP construct and stimulated with epidermal growth factor (EGF). We found that GAP-CTH and GAP-CTK specifically inhibited H-Ras or K-Ras, respectively. However, we did not detect any effect of GAP-CTH181S on Ras activation. Additionally, we used confocal microscopy to verify the ability of GAP constructs to abrogate Ras activation in distinct sub-cellular compartments. We found that GAP-CTH inhibits H-Ras activation on the plasma membrane, while GAP-CTK inhibits K-Ras activation on the plasma membrane. On the contrary, GAP-CTH181S inhibited H-Ras activation on the Golgi. We also analyzed the effects of these GAP constructs on the activation of ERK and Akt in response to EGF stimulation. We found that EGF stimulation of the MAPK pathway was inhibited by GAP-CTK but none of the other GAP constructs, while Akt activation was not inhibited by any GAP construct. Finally, we assayed cellular proliferation and differentiation. We found that GAP-CTK and GAP-CTH were equipotent inhibitors of cellular growth, whereas GAP-CTH181S was less potent. We also found that GAP-CTK and GAP-CTH inhibited differentiation with similar potency, while GAP-CTH181S was more potent. This approach may be adapted to investigate any Ras-dependent signaling pathway. Therefore, it has the potential to become a powerful tool for studying Ras isoform-specific signaling outputs.

Evaluation of memory B cells among perinatally HIV infected children from Houston Texas

Background: HIV associated B cell exhaustion is a notable characteristic of HIV viremic adults. However, it is not known if such alterations are present in perinatal HIV infected children, whose viral dynamics differs from those seen in adults. In the present study we perform an analysis of B cells subsets and measure antigen-specific memory B cells (MBC) in a pediatric HIV infected cohort. ^ Methods: Peripheral mononuclear cells (PBMC) of perinatal HIV infected individuals are characterized into naïve (CD21hi/CD27−), classic (CD27+), tissue like (CD21lo/CD27 −) and activated MBC (CD27+CD21− ) by FACS. A memory ELISPOT assay is used to detect antibody secreting cells. We measure total IgG and antibodies specific for influenza, HBV, mumps, measles, rubella and VZV. Memory was expressed as spot forming cells (SPC) /million of PBMC. Wilcoxon rank-sum was used to compare unpaired groups and linear regression analysis was used to determine predictors of B cell dysfunction ^ Results: 41 HIV perinatal infected children are included (51.2% females and 65.9% Black). Age at study is median (range) 8.78 years (4.39-11.57). At the time of testing they have a CD4% of 30.9 (23.2-39.4), a viral load (VL) of 1.95 log10 copies/ml (1.68-3.29) and a cumulative VL of 3.4 log10 copy × days (2.7-4.0). Ninety two percent of the children are on cARV for > 6 months. Overall, HIV+ children compared with controls have a significant lower number of IgG and antigen specific SFC. In addition, they have a lower proportion of classical MBC 12.9 (8.09-19.85) vs 29.4 (18.7-39.05); 0.01, but a significant higher proportion of tissue like memory MBC 6.01 (2.79-12.7) vs 0.99 (0.87-1.38); 0.003, compared with controls. Patients are parsed on VL (<400 and ≥ 400 copies/ml) with the objective to evaluate the effect of VL on B cell status. Patients with a VL ≥ 400 copies/ml have a significantly lower IgG, HBV, measles, rubella and VZV SPC compared with those with a VL < 400 copies/ml. There are no significant differences in B cell subpopulations between the groups. A moderate negative correlation was observed between the time of cARV initiation and the frequency of IgG memory B cells, suggesting that early initiation of cARV appears to lead to a better functionality of the IgG memory B cells (P=0.05). A statistically significant positive correlation was observed between the total number of IgG memory cells and the number of antigen-specific memory B cells/SPCs. Suggesting that the progressive recovery of the IgG memory B cell pull goes along with a progressive increase in the number of antigen-specific SPCs. ^ Conclusion: A pediatric cohort in overall good status with respect to HIV infection and on ART has defects in B cell function and numbers (reduced total and antigen specific MBC and increased tissue like and reduced classical MBC).^