Alien Registration- Baconnais, Marie Madeleine E. (Saint Agatha, Aroostook County)

https://digitalmaine.com/alien_docs/33182/thumbnail.jp

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

<p>Abstract</p> <p>Background</p> <p>YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs.</p> <p>Results</p> <p>We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly <it>in vitro</it>. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes.</p> <p>Conclusion</p> <p>These results suggest that YB-1 may regulate microtubule assembly <it>in vivo </it>and that its interaction with tubulin may contribute to the control of mRNA translation.</p

Macrorheology of cystic fibrosis, chronic obstructive pulmonary disease & normal sputum

<p>Abstract</p> <p>Background</p> <p>Prior microrheologic assessments of selected, microlitre plugs of cystic fibrosis (CF) sputum suggest no intrinsic rheologic abnormality. However, such analyses may not be representative of CF sputum as a whole. We therefore reassessed this question using whole sputum macrorheology. Additionally, we wished to further explore the relationships between sputum rheology, inflammation and infection.</p> <p>Methods</p> <p>Dynamic oscillatory macrorheometry was performed on whole expectorated sputum from stable adults with CF (n = 18) and COPD (n = 12) and induced sputum from normal controls (n = 7). Concomitant sputum inflammatory mediator levels were measured in CF and COPD samples. Sputum collected from CF subjects (n = 6) at commencement and completion of intravenous antibiotic therapy for an infective exacerbation was also assessed.</p> <p>Results</p> <p>CF sputum neutrophil elastase activity (NE) was significantly related to degree of sputum purulence (p = 0.049) and correlated significantly with measures of sputum viscoelasticity (r = 0.696, p = 0.008 for storage modulus G' at 9 Hz). There were significant differences in viscoelasticity between subject groups when samples were compared irrespective of appearance/degree of sputum purulence. However, the macrorheology of mucoid CF sputum did not differ from normal sputum (eg median (range) G' at 9 Hz 2.25 (0.79, 3.26) vs 2.04 (1.4,4.6) Pa, p = 1). In contrast, mucoid COPD samples demonstrated significantly greater viscoelasticity (G' at 9 Hz 4.5 (2.4, 23) Pa) than sputum from both CF (p = 0.048) & normal subjects (p = 0.009). Antibiotic therapy during exacerbations was associated with significant reductions in CF sputum viscoelasticity, with mean (SD) G' at 9 Hz decreasing from 28.5 (11.5) Pa at commencement to 6.4 (4.6) Pa on day 7 (p = 0.01).</p> <p>Conclusion</p> <p>The macrorheologic properties of whole, mucoid CF sputum are not different from normal, confirming the results of prior microrheologic studies. Instead, CF sputum viscoelasticity is related to secondary infection, decreases with intravenous antibiotic therapy and correlates with inflammation. In contrast, COPD sputum demonstrates inherently greater viscoelasticity, providing a novel target for potential therapeutic interventions.</p

Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

<p>Abstract</p> <p>Background</p> <p>In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG) and induces Ca²⁺release from endoplasmic reticulum (ER) stores.</p> <p>Methods</p> <p>In the present study, we monitored the cytosolic Ca²⁺transients using the UV light photolysis technique to uncage caged Ca²⁺or caged IP₃into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca²⁺receptors present in the ER, and measured their Ca²⁺dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin).</p> <p>Results</p> <p>We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP₃R) dependent-Ca²⁺response following both correcting treatments compared to uncorrected cells in such a way that Ca²⁺responses (CF+treatment <it>vs </it>wild-type cells) were normalized. This normalization of the Ca²⁺rate does not affect the activity of Ca²⁺-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP₃R1, we observed a decrease of the implication of IP₃R1 in the Ca²⁺response in CF corrected cells. We observed a similar Ca²⁺mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP₃R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell.</p> <p>Conclusion</p> <p>These results suggest reversal of the IP₃R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP₃R Ca²⁺release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.</p

Interferon-Inducible CXC Chemokines Directly Contribute to Host Defense against Inhalational Anthrax in a Murine Model of Infection

Chemokines have been found to exert direct, defensin-like antimicrobial activity in vitro, suggesting that, in addition to orchestrating cellular accumulation and activation, chemokines may contribute directly to the innate host response against infection. No observations have been made, however, demonstrating direct chemokine-mediated promotion of host defense in vivo. Here, we show that the murine interferon-inducible CXC chemokines CXCL9, CXCL10, and CXCL11 each exert direct antimicrobial effects in vitro against Bacillus anthracis Sterne strain spores and bacilli including disruptions in spore germination and marked reductions in spore and bacilli viability as assessed using CFU determination and a fluorometric assay of metabolic activity. Similar chemokine-mediated antimicrobial activity was also observed against fully virulent Ames strain spores and encapsulated bacilli. Moreover, antibody-mediated neutralization of these CXC chemokines in vivo was found to significantly increase host susceptibility to pulmonary B. anthracis infection in a murine model of inhalational anthrax with disease progression characterized by systemic bacterial dissemination, toxemia, and host death. Neutralization of the shared chemokine receptor CXCR3, responsible for mediating cellular recruitment in response to CXCL9, CXCL10, and CXCL11, was not found to increase host susceptibility to inhalational anthrax. Taken together, our data demonstrate a novel, receptor-independent antimicrobial role for the interferon-inducible CXC chemokines in pulmonary innate immunity in vivo. These data also support an immunomodulatory approach for effectively treating and/or preventing pulmonary B. anthracis infection, as well as infections caused by pathogenic and potentially, multi-drug resistant bacteria including other spore-forming organisms

Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552–PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on β-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation

XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

International audienceThe Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 alpha/beta domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 alpha/beta domain. We observe independent recruitment of the APLF-interacting protein XRCC4 and of XLF to laser-irradiated sites via binding of A- and X-KBMs, respectively, to Ku80. Finally, we show that mutation of the X-KBM and A-KBM binding sites in Ku80 compromises both the efficiency and accuracy of end joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchor points to build the intricate interaction network required for NHEJ

A study of the geochemistry of chromium isotopes in the modern oceans

The abstract of this item is unavailable due to an embargo

Méthode intégrée de corrélation d’images et de corrélation d’images virtuelles

Digital Image Correlation (DIC) is now com-monly used in academic and industrial settings. In-deed, this method allows to measure the displace-ment field of a surface with high accuracy, good reso-lution and with a simple experimental setup. Howe-ver, image correlation does not allows accurate mea-surement in border areas, sample edges and cracks.The objective of this thesis is to use the VirtualImage Correlation (VIC) method to measure the po-sition of the edges and improve the accuracy of theDIC in these areas. The proposed strategy is basedon three points : the creation of a adapted measure-ment mesh to the geometry, the generation of a pixelmask to remove the edge pixels and the constrainedresolution of the DIC.Different test cases on synthetic images and ex-perimental data show the interest of the integra-ted method. First, the knowledge of the initial posi-tion of the border allows the automatic creation ofan adapted mesh. It is also shown that the simpleuse of a pixel mask reduces significantly the boun-dary error, both in synthetic and real cases. For thecase of constrained resolution, it is shown that itreduces measurement errors in synthetic cases. Ho-wever, this result could not be confirmed in the ap-plication cases, due to the quality of the boundarydoes not allows an accurate measurement and thusan improvement of the DIC results.La corrélation d’images numériques (DIC) est maintenant couramment utilisée dans les milieux académiques et industriels. En effet, cette méthode permet de mesurer le champ de déplacement d’une surface avec une grande précision, une bonne résolution et avec un système expérimental simple. Cependant, la corrélation d’images ne permet pas une mesure précise dans les zones de frontière, bord des échantillons et fissures.L’objectif de cette thèse est d’utiliser la méthode de corrélation d’images virtuelles (VIC) pour mesure la position des bords et améliorer la précision de laDIC dans ces zones. La stratégie proposée repose sur trois points : la création d’un maillage de mesure adapté à la géométrie, la génération d’un masque de pixel permettant de supprimer les pixels du bord et la résolution sous contrainte de la DIC.Différents cas tests sur des images synthétiques et d’application sur des données expérimentales permettent de montrer l’intérêt de la méthode intégrée de corrélation d’images et de corrélation d’images virtuelles. Tout d’abord, la connaissance de la position initiale du bord permet la création automatique d’un maillage adapté. Il est également montré que la simple utilisation d’un masque de pixel permet de ré-duire significativement l’erreur sur le bord, aussi biendans les cas synthétiques que réels. Pour le cas de la résolution sous contrainte, il est montré que celle-ci permet de réduire les erreurs de mesures dans les cas synthétiques. Cependant, ce résultat n’a pas pu être confirmé dans les cas d’applications, la qualité des bords ne permettant pas une mesure précise et donc une amélioration des résultats de DIC