STUDIES TO ELUCIDATE THE EFFECTS OF IRON(III) ON RECOGNITION OF HYALURONATE BY THE CELL SURFACE RECEPTOR CD44

Iron and the poly-anionic carbohydrate, hyaluronate (HA), have both been implicated as possible contributors to rheumatoid arthritis (RA), an autoimmune disease of unknown etiology. In this work, we hypothesized that in RA patients,iron is bound by HA in such a way as to affect HA binding by the cell surface glycoprotein CD44, a known HA receptor. To test this hypothesis, we developed cell based assays to determine the effects of iron binding to HA on HA binding byCD44. As part of this effort, we prepared biotinylated HA and biotinylated Fecomplexed HA. Mesothelioma cells expressing CD44 were treated with each of these conjugates, and the resulting materials were treated with avidin-fluoresceinand the bound fluorescein was measured using a fluorescence microtiter platereader. We found that Fe (III) decreases the apparent affinity of CD44 receptors on Mesothelioma cells for HA (Observed fluorescence intensity decreased by a factor of ~2). In competition experiments, addition of non-biotinylated Fe complexed HA did not inhibit binding of biotinylated HA. Addition of nonbiotinylated HA, however, appeared to increase the uptake of biotinylated Fe complexed HA. These results suggest that iron(lIl) may crosslink HA molecules,leading to a greater uptake of HA. This crosslinking may influence receptor clustering which is believed to be important to intra-cellular signaling

Les effets d'une viscosuppléance intra-articulaire chez des personnes âgées avec articulations tibio-fémorales arthrosées

Le but de cette étude était de vérifier d'une façon systémique les influences d'une viscosuppléance infra-articulaire au niveau des genoux chez des personnes âgées de 60 ans et plus, souffrant d'arthrose de niveau I, II et III (sauf un grade III à dans le compartiment fémoro-patellaire). Les sujets se prêtaient donc à l'exécution de multiples tests pré expérimentation afin d'établir un niveau de base pour recevoir par après le traitement (viscosuppléance inta-articulaire). Par la suite les sujets devaient revenir à intervalles de quatre semaines afin d'effectuer les mêmes tests pour la période post expérimentation. Le genou non-traité était aussi considéré comme base de comparaison. L'auteur anticipait une diminution de la douleur perçue par les sujets lors de l'exécution de divers tests fonctionnels réflétant [i.e. reflétant] des activités de la vie courante. De plus, des variables biomécaniques telles que la vitesse de marche, la force verticale maximale ainsi que le taux de chargement venaient appuyer de façon quantitative les possibles modifications encourues au niveau des membres inférieurs à l'étude. L'étude a démontré des changements significatifs au niveau des hypothèses principales ainsi que les deux premières hypothèses secondaires. Les variables biomécaniques, n'ont pour leur part, pas données de changements significatifs. L'utilisation du"Synvisc" apporte donc des résultats appréciables au niveau de la perception de la douleur chez les sujets et offre une alternative intéressante dans le traitement de l'arthrose

Picturing Texans

According to the introductory information, this text is "An index to the portraits and photographs showing recognizable individuals published in Texas pictorial historical and Genealogical references (mug books) before 1941" (p. 1). The introductory pages include an explanation of how names are listed and a list of the source materials with citations

Examining antibiotic resistance in the feedlot cattle industry using real-time, quantitative PCR (qPCR) and enterococci as an indicator bacterium

Antibiotics are administered to livestock at subtherapeutic levels to maintain animal health. Many of the antibiotics used are analogues or the same as those used in human medicine, raising the possibility that genes conferring resistance arise within agricultural production systems. This thesis examined antibiotic resistance in the Canadian beef feedlot industry. Real-time, quantitative PCR was used to examine differences in the relative abundance of eighteen resistance genes across five antibiotic families from feedlot cattle faeces and urban environments. The effect of infeed administration and withdrawal of tylosin phosphate on macrolide resistance was examined using enterococci as an indicator bacterium. Resistant enterococci (n=21) were selected for whole-genome sequencing and comparative genomics. Results presented here show that the relative abundance of resistance genes differs between cattle feedlots and urban environments, likely a reflection of differences in antibiotic use. Sulfonamide, fluoroquinolone and β-lactam resistance genes predominated in urban wastewater, whilst tetracycline resistance genes were more prevalent in cattle faeces. Macrolide use in cattle production increased the proportion of erythromycinand tylosin-resistant enterococci. However, withdrawal of tylosin from the diet appeared to contribute to reduced macrolide resistance in enterococci. Comparative genomics revealed resistance to macrolides was present on mobile genetic elements, specifically the Tn917 transposon harbouring erm(B). This transposon was identified in both Enterococcus hirae and Enterococcus faecium suggesting inter-species transfer of resistance genes may occur in the bovine gastrointestinal tract. Furthermore, the integrative conjugative elements (ICEs) Tn916 and Tn5801, both conferring tetracycline resistance, were identified in E. faecium. As the cost of genomic sequencing continues to decrease, further investigation of ICEs using whole genome sequencing will help determine if there are linkages between enterococci isolates from bovine environmental and human clinical sources and whether bovine enterococci represent a source to the dissemination and spread of antibiotic resistance

Caractérisation du rôle de l’amyline (IAPP) dans le diabète de type 2 : études de dérivés peptidiques et de composés inhibiteurs de la formation d’amyloïde

L’amyloïdose, une maladie progressive et incurable, implique une vaste panoplie de pathologies et de pathogénèses, qui est expliquée par la grande variabilité biologique et structurale des protéines responsables de la formation des dépôts d’amyloïde. L’amyline (polypeptide amyloïde des îlots pancréatiques, IAPP) est une protéine très susceptible de subir des changements de conformation impliquant les feuillets bêta et conférant aussi des propriétés physicochimiques distinctes. Cette protéine prend alors une forme fibrillaire et se dépose dans les îlots de Langerhans chez les humains atteints de diabète de type 2 ou d’insulinome. Ces dépôts d’amyloïde pancréatique (AIAPP) ont été décrits chez certaines espèces animales telles que les félins domestiques, les grands félins, le raton laveur et les primates non humains. La formation de dépôts d’amyloïde contribue à la pathogénèse du diabète de type 2, mais les mécanismes qui induisent la conversion de l’amyline (IAPP) en amyloïde (AIAPP) ne sont pas complètement compris. Les hypothèses du projet sont que certaines variations présentes dans les séquences peptidiques de l’IAPP provenant de différentes espèces animales jouent un rôle critique pour la formation de fibrilles et que plusieurs composés chimiques aromatiques/phénoliques sont capables d’abroger la formation de dépôts d’amyloïde. Le projet de recherche consiste donc à caractériser la propension des différentes isoformes animales d’IAPP à former de l’amyloïde in vitro afin d’identifier les acides aminés jouant un rôle clé dans cette transformation structurale et ultimement d’inhiber la formation d’amyloïde pancréatique. Le projet se divise en deux volets principaux. Le premier consiste à identifier les différentes séquences peptidiques de l’IAPP retrouvées chez les espèces animales. L’objectif est d’identifier les acides aminés jouant un rôle clé dans la formation d’amyloïde. Le gène de l’IAPP a été séquencé chez plus d’une quarantaine d’espèces. Le potentiel d’agrégation des séquences obtenues a été simulé à l’aide d’outils bioinformatique. Une librairie de 23 peptides a été commandée afin de procéder à des analyses physicochimiques in vitro permettant d’évaluer le potentiel amyloïdogénique (test fluorimétrique à la thioflavine T, essai de liaison au rouge Congo, dichroïsme circulaire, microscopie électronique à transmission) et cytotoxique (sur une lignée cellulaire provenant d’insulinome : INS-1). Les analyses effectuées à partir de la librairie constituée de 23 peptides ont permis d’identifier trois séquences ne formant pas d’amyloïde et qui proviennent des espèces animales suivantes : le tamarin lion doré (Leontopithecus rosalia), le grand dauphin (Tursiops truncatus) et l’alpaga (Vicugna pacos). Un site potentiellement critique est le segment 8-20 présentant le motif NFLVH qui ne forme plus d’amyloïde lorsqu’il est remplacé par le motif DFLGR ou KFLIR. Les acides aminés 29P, 14K et 18R sont également impliqués dans l’inhibition de la transformation structurale en fibrille. La dernière partie du projet consiste à inhiber la formation de l’amyloïde en utilisant des composés chimiques commercialisés (hypoglycémiants, anti-inflammatoires non stéroïdiens) ou nouvellement synthétisés dans notre laboratoire (les aryles éthyles urées). Un criblage d’une soixantaine de composés chimiques a été conduit dans cette étude. Leur efficacité a été testée sur l’IAPP humaine, qui possède un fort potentiel amyloïdogénique. Les techniques utilisées sont les mêmes que celles exploitées précédemment. L’essai de liaison croisée photo-induite ("photo-induced cross-linking of unmodified proteins", PICUP) a été réalisé afin d’étudier les formes intermédiaires (monomères, oligomères). Un total de 11 composés chimiques a démontré un potentiel à inhiber l’agrégation des fibrilles. Pour la classe des hypoglycémiants, le glyburide, le répaglinide et la troglitazone ont montré l’activité thérapeutique la plus élevée pour retarder et réduire la formation de fibrilles. Les anti-inflammatoires antiamyloïdogènes actifs incluaient le diclofenac, le méloxicam, le phénylbutazone, le sulindac et le ténoxicam. Les aryles étyles urées les plus intéressantes étaient la EU-362 et la EU-418. Tous ces composés ont conféré une protection cellulaire contre l’activité cytotoxique des fibrilles. Les molécules actives possèdent des éléments structuraux communs tels des substituants donneurs d’électrons (alcool, amine, halogène) sur un noyau benzène. En conclusion, ce projet de recherche a permis de caractériser l’IAPP chez diverses espèces animales, dont plusieurs chez lesquelles elle n’avait pas encore été décrite, de déterminer les sites jouant un rôle clé dans sa transformation en amyloïde et, ultimement, de tester le potentiel thérapeutique de nouveaux agents antiamyloïdogènes dans le diabète de type 2. Nous espérons que ce projet ouvrira ainsi la porte à de nouvelles stratégies de traitement.Amyloidosis is a progressive and, as of now, incurable disease caused by the deposition of insoluble proteins. Amyloid research over the past decades focused on the characterization of the substantive biological variability of amyloid deposits. Amyloidosis encompasses a diversity of pathological manifestations, explained by the diversity of underlying causal proteins. In the pancreas of susceptible species, islet amyloid polypeptide (IAPP) is a precursor for an amyloid protein (AIAPP), which has a characteristic fibrillar structure and resistance to physical agents. This folded protein deposits in the islets of Langerhans of patients with type 2 diabetes or islet cell tumors (insulinoma). Amyloid deposits have also been well characterized, anatomically, in feline and non-human primate species. Amyloid fibril formation contributes to the pathogenesis of diabetes mellitus but the precise pathophysiologic factors involved in the fibrillization of IAPP as well as resultant islet injury remain to be elucidated. Further understanding of the causative factors in the fibrillogenesis of IAPP will be requisite in the development of therapeutic strategies to disrupt the amyloidosis process. This project hypothesizes that the specific variations found in IAPP peptide sequences among different animal species are critical for IAPP fibrillization. Also, some aromatic/polyphenolic compounds can abrogate fibrillization. The main objective forms the basis for development of new therapeutic tactics to impede amyloid formation and associated cellular injury. Thus this project has two specific aims. The first specific aim was to identify critical variations in IAPP amino acid sequences from different animal species and to assess their amyloidogenic potential. To accomplish this, the IAPP gene was isolated and sequenced from paraffin-embedded tissues from various animals (40 species). The aggregation potency was assessed for each sequence using in silico analysis. A library of 23 peptides was prepared from sequences that were distinctly different and their amyloidogenic potential was assessed in vitro using physicochemical analysis (thioflavin-T assay, Congo red binding assay, far-UV circular dichroism, transmission electron microscopy) and cytotoxicity assays (insulinoma cell line INS-1). Among this peptide library, three were non-amyloidogenic and corresponded to the following animal species: golden lion tamarin (Leontopithecus rosalia), commun bottlenose dolphin (Tursiops truncates) and alpaca (Vicugna pacos). Segment 8-20 of the peptide was critical for amyloid formation and the substitution of the NFLVH motif found in this region by a DFLGR or KFLIR motif impeded fibrillization. The amino acids 29P, 14K and 18R were also demonstrated to abrogate fibril formation. The second objective consisted in abrogating IAPP fibrillogenesis using conceptualized aromatic/polyphenolic structures, specifically hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl urea agents. This part of the project involved molecular screening of more than 60 compounds. Their efficacy at inhibiting amyloid formation was assessed in vitro on human IAPP, which exhibits the highest amyloidogenic potential. Techniques included the above-mentioned methods, with the addition of photo-induced cross-linking of unmodified proteins (PICUP). A total of 11 compounds showed potential in abrogating IAPP aggregation. Among the hypoglycemic agents evaluated, glyburide, repaglinide and troglitazone showed the highest potency in reducing fibril formation. The NSAIDs that displayed anti-amyloidogenic activity were diclofenac, meloxicam, phenylbutazone, sulindac and tenoxicam. EU-362 and EU-418 were the hit compounds resulting from the screening of the aryl ethyl urea (EU) class. Additionally, these anti-amyloidogenic molecules conferred a protection against fibril cytotoxicity. All of the active molecules bear a commun motif composed of benzene ring with electron donor moieties, such as alcohol, amine or halide. To conclude, this project characterized IAPP in several animal species in which it has not been previously described and improves our understanding of the amyloidogenesis process. Moreover, the therapeutic potential of hypoglycemic, non-steroidal anti-inflammatory and aryl ethyl ureas agents as anti-amyloidogenic compounds was evaluated. It is conceivable that the additional information hereby gained on the regulation of amyloidogenesis may point towards new therapeutic strategies for diabetic patients

Acute Leukemia

This book provides a comprehensive overview of he basic mechanisms underlying areas of acute leukemia, current advances, and future directions in management of this disease. The first section discusses the classification of acute leukemia, taking into account diagnoses dependent on techniques that are essential, and thankfully readily available, in the laboratory. The second section concerns recent advances in molecular biology, markers, receptors, and signaling molecules responsible for disease progression, diagnostics based on biochips and other molecular genetic analysis. These advances provide clinicians with important understanding and improved decision making towards the most suitable therapy for acute leukemia. Biochemical, structural, and genetic studies may bring a new era of epigenetic based drugs along with additional molecular targets that will form the basis for novel treatment strategies. Later in the book, pediatric acute leukemia is covered, emphasizing that children are not small adults when it comes to drug development. The last section is a collection of chapters about treatment, as chemotherapy-induced toxicity is still a significant clinical concern. The present challenge lies in reducing the frequency and seriousness of adverse effects while maintaining efficacy and avoiding over-treatment of patients

Investigating the impact of gold nanoparticles on cells: from transcription to behavior

Gold nanoparticles (Au NPs) are being research extensively for various biomedical applications; their applicability arises from a unique set of optical and physical properties brought on by their nanoscale dimensions.1 Furthermore, facile and scalable synthetic and surface functionalization strategies for Au NPs make these properties highly tunable.1 These areas of research are still relatively new and the number of publications per year referring to gold nanomaterials has skyrocketed from 11 in 1990, to 673 in 2000, to more than 31,400 in 2015 (Web of Science database, topic search = “gold nano*”, accessed March 2, 2016). The potential application of Au NPs for disease detection, diagnosis and therapy has motivated numerous analyses of their interactions with biomolecules, cells, animals, humans and the environment.1,2 A vast majority of studies aimed at gaining a better understanding of how cells interact with and are influenced by Au NPs have focused mainly on measuring cytotoxicity and simple cell processes like proliferation or NP uptake. While Au NPs larger than 4-5 nm in diameter (with appropriate, non-toxic surface coatings) have been shown to be largely non-cytotoxic, there can be subtle non-toxic effects induced by Au NPs.3 The adsorption of soluble proteins onto NP surfaces (the protein corona) is highly studied, but little attention has been paid to how those interactions perturb gene expression of cells or to understanding NP interactions with other types of biomolecules. This thesis aims to look deeper into how molecular level effects of NPs in cells and cellular environments can lead to down-stream changes to cell gene expression and behavior. Firstly, the impact of Au nanorods (Au NRs) on 3D cancer cell migration via interactions between Au NRs and extracellular matrix (ECM) structural proteins was examined.4 While experiments on the influence of NPs on cell behaviors exist, nearly all of these studies neglect the impact of the ECM. In vivo cells exist in complex, fibrous 3D environments and series of intricate biochemical, cell-cell and cell-ECM interactions govern behaviors such as migration. Cancer cell migration allows tumor cells to spread and metastasize to new areas of the body, but little is known about how Au NR interaction with the ECM after injection and targeting to tumors may affect this process. The inevitable contact of in vivo Au NRs with the ECM presents a possible source of unintended side effects. In order to study how gold nanoparticles can influence ECM properties and cell-ECM interactions, we have created a nested-gel type I collagen matrix for measuring whether Au NRs alter the migration of MDA-MB-231 human breast cancer cells in 3D collagen environments. In contrast to the few studies of Au NR-induced migration changes in 2D environments, our results show that Au NRs in a model ECM increase the frequency of spontaneous cellular migration. The presence of negatively-charged polyelectrolyte-coated Au NRs during the collagen self-assembly process was shown to induce mechanical and structural changes, to alter molecular diffusion, and to affect cellular adhesion, morphology, locomotion strategy and protease expression. The results demonstrate the indirect impact nanoparticles can exert on cell behaviors within three-dimensional ECMs. The shape and surface chemistry of Au NPs was also investigated in terms of the role of these factors in cellular transcriptomic (gene expression) responses after both short- and long-term exposures.5 Respectively, human dermal fibroblasts (HDF) and prostate cancer (PC3) cells were exposed to 0.1 nM (24 hours) and 1.0 nM (48 hours) concentrations of Au NPs of four different, but related surface chemistries. A combination of microarray gene expression analysis techniques and typical cellular characterization was used to learn more about how the nature of the Au NP surface coating influences cells on a molecular level. Identity, charge and lability of surface coatings (and cell type and dosing parameters) were all implicated as important factors to consider in order to predict gene expression responses. In a separate study, HDF cells were exposed to 0.1 nM (low-dose) Au NPs of different shapes and surface coatings for 20 weeks. The long-term effects of acute (24 hour) and chronic (20 weeks) exposure were measured by viability, proliferation, NP uptake, and morphology studies combined with gene expression analysis of genes related to stress and toxicity pathways. It is rare to find chronic exposure studies, especially with Au NPs, and these experiments showed that acute, sub-cytotoxic doses of NPs may induce long-term stress on cells. These cells were found to react very differently to acute versus chronic doses of the same NPs after 20 weeks. Additionally, surface coating was shown to have a much larger impact on determining NP-cell interactions than shape of Au NPs. In all, we have expanded the collective understanding of the molecular interactions Au NPs experience inside cells based on surface chemistry, shape, dosage and exposure time and parameters. References 1. Dreaden, E.C.; Alkilany, A.M.; Huang, X.; Murphy, C.J.; El-Sayed, M.A. The Golden Age: Gold Nanoparticles for Biomedicine. Chem. Soc. Rev. 2012, 41, 2740–2779. 2. Yang, X.; Yang, M.; Pang, B.; Vara, M.; Xia, Y. Gold Nanomaterials at Work in Biomedicine. Chem. Rev. 2015, 115, 10410–10488. 3. Alkilany, A.M.; Lohse, S.E.; Murphy, C.J. The Gold Standard: Gold Nanoparticle Libraries to Understand the Nano-Bio Interface. Acc. Chem. Res. 2013, 46, 650–661. 4. Grzincic, E.M.; Murphy, C.J. Gold Nanorods Indirectly Promote Migration of Metastatic Human Breast Cancer Cells in Three-Dimensional Cultures. ACS Nano 2015, 9, 6801–6816. 5. Grzincic, E.M.; Yang, J.A.; Drnevich, J.; Falagan-Lotsch, P.; Murphy, C.J. Global Transcriptomic Analysis of Model Human Cell Lines Exposed to Surface-Modified Gold Nanoparticles: The Effect of Surface Chemistry. Nanoscale 2015, 7, 1349–1362