Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

Background: Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase. Methodology/Principal Findings: To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage. Conclusions/Significance: This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and th

Élaboration et caractérisation de revêtements Ti(C,N)/Al₂O₃ par CVD thermique pour les outils de coupe

Les outils de coupe principalement basés sur les carbures de tungstène sont améliorés avec des revêtements de nitrure, de carbure et d'oxyde développés par dépôt chimique en phase vapeur (CVD), optimisant considérablement les performances de l'outil. Les études de cette thèse ont porté sur la préparation et la caractérisation de CVD Ti(C,N)/Al₂O₃. Premièrement, des films de Ti(C,N) ont été déposés à partir d'un mélange gazeux TiCl₄-CH₃CN-H₂-N₂ à 850-950 °C et 70-700 mbar en faisant varier le rapport molaire N₂/H₂ de 0,27 à 3,77. La vitesse de croissance de film dépend étroitement de la pression totale et de la pression partielle d’N₂. La composition semble indépendante des conditions expérimentales, alors que la microstructure est fortement influencée. Ti(C,N) déposé à 850 °C et 70 mbar avec un rapport molaire N₂/H₂ de 0,27 présente une texture mixte avec des grains à facettes et de meilleures propriétés mécaniques. Il a été constaté que les contraintes résiduelles pouvaient être dépendantes de l'épaisseur et relaxées par les fissures de refroidissement. Deuxièmement, la croissance d’Al₂O₃ sur Ti(C,N) a été étudiée. Il a été montré que la nucléation d’α- et κ-Al₂O₃ pouvait être contrôlée par le dépôt de couches intermédiaires entre Al₂O₃ et Ti(C,N). Les propriétés mécaniques de CVD Al₂O₃ avec de couches de transition ont été étudiées et les performances d'usinage des outils revêtus ont été évaluées par des essais de tournage avec l'acier Z38CDV5. La durée de vie de notre revêtement le plus performant (κ-Al₂O₃) est beaucoup supérieure à celle d’un revêtement commercial d’Al₂O₃.Modern tools primarily based on cemented carbides are improved with protective coatings, combinations of nitride, carbide and oxide coatings grown by chemical vapor deposition (CVD) optimize dramatically the tool performance in severe conditions. Studies in this thesis focused on preparation and characterization of CVD Ti(C,N)/Al₂O₃ coatings. Firstly, a series of Ti(C,N) coatings were deposited from a TiCl₄-CH₃CN-H₂-N₂ gas mixture at 850-950 °C and 70-700 mbar with varying the N₂/H₂ molar ratio from 0.27 to 3.77. It was found that the growth rate highly depends on total pressure and N₂ partial pressure. The composition is almost independent from deposition conditions, whereas the microstructure could be strongly influenced. Ti(C,N) deposited at 850 °C and 70 mbar with the N₂/H₂ molar ratio of 0.27 exhibits a mixed texture with facetted grains and superior mechanical properties. It was also shown that residual stresses could be thickness-dependent and relaxed through cooling cracks. Secondly, influence of the nucleation surface on the growth of Al₂O₃ deposited on Ti(C,N)-based layers was investigated. It was shown that the nucleation of α- and κ-Al₂O₃ could be controlled with the deposition of specific bonding layers between Al₂O₃ and Ti(C,N) layers. Finally, mechanical properties of Al₂O₃ coatings with transition layers were studied and the machining performance of coated cutting tools was evaluated by turning tests of Z38CDV5 tool steel. Our best coating (κ-Al₂O₃) exhibits a tool life much longer than that of a commercial CVD Al₂O₃ coating

Autophagy induced by low concentrations of crotonaldehyde promotes apoptosis and inhibits necrosis in human bronchial epithelial cells

Crotonaldehyde is a common environmental contaminant. Autophagy, apoptosis, and necrosis, were all respectively reported to be induced by crotonaldehyde. However, the relationships between programmed cell deaths, especially between autophagy and apoptosis, have not been elucidated. In the present study, alterations of autophagy, apoptosis and necrosis were investigated in human bronchial epithelial cells (BEAS-2B) exposed to crotonaldehyde, and effects of autophagy on apoptosis and necrosis were detected. We found that a high con- centration (160 mu mol/L mu M) of crotonaldehyde did not induce apoptosis, while a low concentration (80 mu M) of crotonaldehyde induced autophagy, apoptosis and necrosis. In 80 mu M crotonaldehyde-exposed BEAS-2B cells, autophagy and apoptosis exhibited a trend of increasing prior to decreasing with the increase of time, while the time point inducing the highest level of autophagy was 2 h, and that of apoptosis was 4 h. With the pretreatment of bafilomycin A(1), the apoptosis was inhibited and the necrosis was enhanced significantly in cells exposed to 80 mu M crotonaldehyde. Autophagy mediated the induction of apoptosis via the intrinsic apoptotic pathway. The results indicate that autophagy mediates the initiation of apoptosis and plays a role in protecting from necrosis in low concentrations of crotonaldehyde-exposed BEAS-2B cells

Crotonaldehyde induces autophagy-mediated cytotoxicity in human bronchial epithelial cells via PI3K, AMPK and MAPK pathways

Crotonaldehyde is an ubiquitous hazardous pollutant in the environment which can be produced naturally, artificially and endogenously. Acute exposure of crotonaldehyde was reported to induce severe lung injury in humans and experimental animals. However, the exact toxicity mechanisms of crotonaldehyde in organisms have not been fully explored. In the present study, we explored the role autophagy played in the cytotoxicity induced by crotonaldehyde in human bronchial epithelial cells (BEAS-2B), and the pathways that mediated autophagy, including the phosphatidylinositol 3-kinase (PI3K) pathway, the AMP-activated protein kinase (AMPK) pathway and the mitogen-activated protein kinase (MAPK) pathways, were examined and validated. We found that crotonaldehyde induced cytotoxicity and autophagy simultaneously in BEAS-2B cells, and blockage of autophagic flux significantly elevated the viability of BEAS-2B exposed to high concentrations of crotonaldehyde. Crotonaldehyde down regulated the activity of PI3K pathway, and elevated the activities of AMPK and MAPK pathways. Pretreatment of specific agonist or antagonist of these pathways could inhibit autophagy and partly improve the viability. These results suggested that acute exposure of crotonaldehyde induced cell death mediated by autophagy, which might be helpful to elucidate the toxicity mechanisms of crotonaldehyde and contribute to environmental and human health risk assessment. (C) 2017 Elsevier Ltd. All rights reserved

The significance of low PU.1 expression in patients with acute promyelocytic leukemia

Abstract Background Although the importance of the hematopoietic transcription factor PU.1 in acute myeloid leukemia (AML) has been demonstrated, the expression of PU.1 in acute promyelocytic leukemia (APL) patient samples awaits further investigation. The current study used APL patient samples to assess the expression pattern of PU.1 in the initiation and progression of APL. Findings We used real-time RT-PCR to compare PU.1 expression between de novo APL patient samples and normal blood specimens, and the results indicated that PU.1 expression was significantly lower in newly diagnosed APL patient samples as compared to normal hematopoietic cells. Further evidence showed a significant inverse correlation between the expression level of PML-RARα and that of PU.1. In addition, we analyzed the correlation between PML-RARα and PU.1 expression in a large population of AML patients retrieved from the expression profiles. The results showed that PU.1 expression was lower in patients with APL than other AML subtypes and there was also a trend towards increasing PU.1 expression from AML-M0 to AML-M5, with the exception of AML-M3 (APL). These observations suggested that PU.1 expression was reduced by PML-RARα in APL patients. Furthermore, we measured PU.1 expression in APL-initiating cells isolated from de novo APL patients by side population cell analysis and found that suppression of PU.1 expression occurred concurrently with PML-RARα expression, indicating the pivotal role of PU.1 in APL initiation. Conclusion Our findings provide evidence that low PU.1 expression in APL patients is required for disease initiation and progression.</p