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Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing

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World Journal of Gastroenterology 2012年第42期18卷 6096-6105页

作者： Maria Homs Maria Buti David Tabernero Josep Quer Alex Sanchez Noelia Corral Rafael Esteban francisco rodriguez-frias Network Medical Research Center Hepatic and Digestive Diseases(CIBERehd)Institute of Health Carlos Ⅲ08036 BarcelonaSpain Department of Biochemistry Vall d'Hebron HospitalAutonomous University of Barcelona08035 BarcelonaSpain Department of Hepatology Vall d' Hebron HospitalAutonomous University of Barcelona08035 BarcelonaSpain Statistics and Bioinformatics Unit Research InstituteVall d'Hebron Hospital08035 BarcelonaSpain

AIM:To investigate the variability of the main immunodominant motifs of hepatitis B virus(HBV) core gene by ultra-deep-pyrosequencing(UDPS).METHODS:Four samples(2 genotype A and 2 genotype D) from 4 treatment-na ve patients were assessed for baseline *** additional samples from one patient(patient 4,genotype D) were selected for analysis:one sample corresponded to a 36-mo treatment-free period from baseline and the other to the time of viral breakthrough after 18 mo of lamivudine *** HBV region analyzed covered amino acids 40 to 95 of the core gene,and included the two main epitopic regions,Th50-69 and *** was carried out in the Genome Sequencer FLX system(454 Life Sciences,Roche).After computer filtering of UDPS data based on a Poisson statistical model,122 813 sequences were *** most conserved position detected by UDPS was analyzed by site-directed mutagenesis and evaluated in cell ***:Positions with highest variability rates were mainly located in the main core epitopes,confirming their role as immune-stimulating *** addition,the distribution of variability showed a relationship with HBV *** 1(genotype A) presented the lowest variability rates and patient 2(genotype A) had 3 codons with variability higher than 1%.Patient 3 and 4(both genotype D) presented 5 and 8 codons with variability higher than 1%,*** median baseline frequencies showed that genotype A samples had higher variability in epitopic positions than in the other positions analyzed,approaching significance(P = 0.07,sample 1 and P = 0.05,sample 2).In contrast,there were no significant differences in variability between the epitopic and other positions in genotype D ***,patient 1 presented a completely mutated motif from amino acid 64 to 67(E 64 LMT 67),which is commonly recognized by T helper ***,the variability observed in all 4 patients was particularly associated with the E 64 LMT 67 mo

关键词： Hepatitis B virus Ultra-deep-pyrosequencing Epitopes Quasispecies Linkage analysis

New real-time-PCR method to identify single point mutations in hepatitis C virus

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World Journal of Gastroenterology 2016年第43期22卷 9604-9612页

作者： Qian Chen Irene Belmonte Maria Buti Leonardo Nieto Damir Garcia-Cehic Josep Gregori Celia Perales Laura Ordeig Meritxell Llorens Maria Eugenia Soria Rafael Esteban Juan Ignacio Esteban francisco rodriguez-frias Josep Quer Liver Unit Internal Medicine Lab. Malalties Hepàtiques Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’ Hebron08035 Barcelona Spain Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas Instituto de Salud Carlos Ⅲ28029 madrid Spain Liver Pathology Unit Departments of Biochemistry and Microbiology Hospital Universitari Vall d’Hebron Autonomous University of Barcelona 08035 Bareelona Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas del Instituto de Salud Carlos Ⅲ 28029 Madrid Spain Universitat Autònoma de Barcelona 08193 Bareelona Spain

AIM To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus(HCV).METHODS In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80 K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. Light Cycler methods, based on real-time PCR with sequencespecific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10%(mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80 K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b in our setting. CONCLUSION A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This

关键词： Hepatitis C virus Resistance-associated amino acid substitutions Low-cost test Single-point mutations Q80K

Experimental study on the wear evolution of different PVD coated tools under milling operations of LDX2101 duplex stainless steel

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Advances in Manufacturing 2023年第1期11卷 158-179页

作者： Vitor F.C.Sousa francisco J.G.Silva Ricardo Alexandre Jose S.Fecheira Gustavo Pinto Andresa Baptista ISEP-School of Engineering Polytechnic of PortoRua Dr.António Bernardino de Almeida4314200–072PortoPortugal INEGI-Driving Science and Innovation Rua Dr.Roberto Frias4004200–465PortoPortugal TEandM-Technology Engineering and MaterialsS.A.Parque Industrial do TaveiroLotes 41-423045–504TaveiroPortugal

Duplex stainless steels are being used on applications that require,especially,high corrosion resistance and overall good mechanical properties,such as the naval and oil-gas exploration *** components employed in these industries are usually obtained by machining,however,these alloys have low machinability when compared to conventional stainless *** this work,a study of the wear developed when milling duplex stainless-steel,LDX 2101,is going to be presented and evaluated,employing four types of milling tools with different geometries and coatings,while studying the influence of feed rate and cutting length in the wear of these *** used have been provided with two and four flutes,as well as three different coatings,namely:TiAlN,TiAlSiN and *** cutting behavior of these tools was analyzed;data relative to the cutting forces developed during the process were obtained;and roughness measurements of the machined surfaces were *** tools were then submitted to scanning electron microscope(SEM)analysis,enabling the identification of the wear mechanisms that tools were subjected to when machining this material,furthermore,the early stages of these mechanisms were also *** this work was done with the goal of relating the machining parameters and cutting force values obtained,identifying,and discussing the wear patterns that were observed in the coating and tools after the milling tests,providing further information on the machining of these alloys.

关键词： Duplex stainless-steel Milling Tool coatings Surface roughness Wear mechanisms

Fiber Laser Sensor Based on a Phase-Shifted Chirped Grating for Acoustic Sensing of Partial Discharges

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Photonic Sensors 2013年第1期3卷 44-51页

作者： Sanderson E. U. LIMA Rubem G FARIAS francisco M. ARAUJO Luis A. FERREIRA José L. SANTOS Vladimiro MIRANDA1 Orlando FRAZAO INESC Porto Rua do Campo Alegre 687 Porto 4169-007 Portugal Faeuldade de Engenharia da Computacāo Universidade Federal do Pará Belém Brasil Faculdade de Ciēncias da Universidade do Porto Rua do Campo Alegre 687 Porto 4169-007 Portugal Faculdade de Engenharia da Universidade do Porto Rua Dr. Roberto Frias s/n Porto 4200-465Portugal

Acoustic emission monitoring is often used in the diagnosis of electrical and mechanical incipient faults in the high voltage apparatus. Partial discharges are a major source of insulation failure in electric power transformers, and the differentiation from other sources of acoustic emission is of the utmost importance. This paper reports the development of a new sensor concept - a fiber laser sensor based on a phase-shifted chirped fiber grating - for the acoustic emission detection of incipient faults in oil-filled power transformers. These sensors can be placed in the inner surface of the transformer tank wall, not affecting the insulation integrity of the structure and improving fault detection and location. The performance of the sensing head is characterized and compared for different surrounding media： air, water, and oil. The results obtained indicate the feasibility of this sensing approach for the industrial development of practical solutions.

关键词： Transformer insulation partial discharges acoustic detection fiber laser sensors