The use of CRISPR/Cas associated technologies for cell transplant applications.

Science.gov (United States)

Cowan, Peter J

2016-10-01

In this review, I will summarize recent developments in the use of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) genome editing system for cell transplant applications, ranging from transplantation of corrected autologous patient stem cells to treat inherited diseases, to the tailoring of donor pigs for cell xenotransplantation. Rational engineering of the Cas9 nuclease to improve its specificity will also be discussed. Over the past year, CRISPR/Cas9 has been used in preclinical studies to correct mutations in a rapidly increasing spectrum of diseases including hematological, neuromuscular, and respiratory disorders. The growing popularity of CRISPR/Cas9 over earlier genome editing platforms is partly due to its ease of use and flexibility, which is evident from the success of complex manipulations such as specific deletion of up to 725 kb in patient-derived stem cells, and simultaneous disruption of up to 62 endogenous retrovirus loci in pig cells. In addition, high-fidelity variants of Cas9 with greatly increased specificity are now available. CRISPR/Cas9 is a fast-evolving technology that is likely to have a significant impact on autologous, allogeneic, and xenogeneic cell transplantation.

Simple Meets Single: The Application of CRISPR/Cas9 in Haploid Embryonic Stem Cells

Directory of Open Access Journals (Sweden)

Zixi Yin

2017-01-01

Full Text Available The CRISPR/Cas9 system provides a powerful method for the genetic manipulation of the mammalian genome, allowing knockout of individual genes as well as the generation of genome-wide knockout cell libraries for genetic screening. However, the diploid status of most mammalian cells restricts the application of CRISPR/Cas9 in genetic screening. Mammalian haploid embryonic stem cells (haESCs have only one set of chromosomes per cell, avoiding the issue of heterozygous recessive mutations in diploid cells. Thus, the combination of haESCs and CRISPR/Cas9 facilitates the generation of genome-wide knockout cell libraries for genetic screening. Here, we review recent progress in CRISPR/Cas9 and haPSCs and discuss their applications in genetic screening.

High performance CaS solar-blind ultraviolet photodiodes fabricated by seed-layer-assisted growth

International Nuclear Information System (INIS)

He, Qing Lin; Lai, Ying Hoi; Sou, Iam Keong; Liu, Yi; Beltjens, Emeline; Qi, Jie

2015-01-01

CaS, with a direct bandgap of 5.38 eV, is expected to be a strong candidate as the active-layer of high performance solar-blind UV photodiodes that have important applications in both civilian and military sectors. Here, we report that a seed-layer-assisted growth approach via molecular beam epitaxy can result in high crystalline quality rocksalt CaS thin films on zincblende GaAs substrates. The Au/CaS/GaAs solar-blind photodiodes demonstrated , more than five orders in its visible rejection power, a photoresponse of 36.8 mA/w at zero bias and a corresponding quantum efficiency as high as 19% at 235 nm

New applications of CRISPR/Cas9 system on mutant DNA detection.

Science.gov (United States)

Jia, Chenqiang; Huai, Cong; Ding, Jiaqi; Hu, Lingna; Su, Bo; Chen, Hongyan; Lu, Daru

2018-01-30

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management. Copyright © 2017 Elsevier B.V. All rights reserved.

La tuberculose extra-ganglionnaire primitive de la sphère ORL: à propos de 15 cas

Science.gov (United States)

Touati, Mohamed Mliha; Darouassi, Youssef; Chihani, Mehdi; Lakouichmi, Mohammed; Tourabi, Khalid; Ammar, Haddou; Bouaity, Brahim

2014-01-01

Les localisations ORL extra ganglionnaires de la tuberculose sont rares. La symptomatologie clinique ainsi que les examens paracliniques sont souvent trompeurs,posant ainsi le problème de diagnostic différentiel avec la pathologie tumorale. Nous rapportons 15 cas de localisations extra ganglionnaires de tuberculose, colligés au service ORL et CCF de l'Hopital Militaire Avicenne de Marrakech colligés entre 2009 et 2013. L’âge moyen de nos patients est de 33 ans. L’étude topographique a montré 6 cas au niveau du cavum, un cas de miliaire tuberculeuse pharyngée, 4 cas laryngés; 2 localisations auriculaires; 1 parotidienne et 1 localisation sous maxillaire. Le diagnostic était anatomopathologiquedans tous les cas. Tous nos patients ont reçu un traitement antituberculeux avec une bonne évolution. Mots-clés: Tuberculose, amygdale, rhinopharynx, larynx, glandes salivaires,Oreille moyenne. PMID:25815100

CRISPR/Cas system for yeast genome engineering: advances and applications

DEFF Research Database (Denmark)

Stovicek, Vratislav; Holkenbrink, Carina; Borodina, Irina

2017-01-01

The methods based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system have quickly gained popularity for genome editing and transcriptional regulation in many organisms, including yeast. This review aims to provide a comprehensive overview...... of CRISPR application for different yeast species: from basic principles and genetic design to applications....

CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives

DEFF Research Database (Denmark)

Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E.

2015-01-01

repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid,easy and efficient engineering of mammalian genomes. It has a wide range of applications frommodification of individual genes to genome-wide screening or regulation of genes. Facile genomeediting using CRISPR/Cas9 empowers...... researchers in the CHO community to elucidate the mechanisticbasis behind high level production of proteins and product quality attributes of interest. Inthis review, we describe the basis of CRISPR/Cas9-mediated genome editing and its applicationfor development of next generation CHO cell factories while...... highlighting both future perspectivesand challenges. As one of the main drivers for the CHO systems biology era, genome engineeringwith CRISPR/Cas9 will pave the way for rational design of CHO cell factories....

Application of the nanobiotechnology with the system CRISP-Cas

OpenAIRE

Liceth Xiomara Sáenz-Castiblanco; Maritza Angarita-Merchán; Diana Paola Lopez-Velandia

2017-01-01

Introduction: Nanobiotechnology and synthetic biology are sciences that impact today with the launching of innovative and beneficial applications for the human being. These sciences have been amalgamated to manufacture new components for the construction of totally artificial cells and the creation of synthetic biomolecules. Objective: To know the applications of nanobiotechnology related to the use of the system CRISPR/Cas in the storage of bacterial DNA and therapeutic alternatives. Materia...

Cas9 versus Cas12a/Cpf1: Structure-function comparisons and implications for genome editing.

Science.gov (United States)

Swarts, Daan C; Jinek, Martin

2018-05-22

Cas9 and Cas12a are multidomain CRISPR-associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best-characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms. Here we compare the structural and mechanistic features that distinguish Cas9 and Cas12a, and describe how these features modulate their activity. We discuss implications for genome editing, and how they may influence the choice of Cas9 or Cas12a for specific applications. Finally, we review recent studies in which Cas12a has been utilized as a genome editing tool. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes. © 2018 Wiley Periodicals, Inc.

Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma.

Science.gov (United States)

Liu, Tang; Shen, Jacson K; Li, Zhihong; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

2016-04-01

Sarcomas include some of the most aggressive tumors and typically respond poorly to chemotherapy. In recent years, specific gene fusion/mutations and gene over-expression/activation have been shown to drive sarcoma pathogenesis and development. These emerging genomic alterations may provide targets for novel therapeutic strategies and have the potential to transform sarcoma patient care. The RNA-guided nuclease CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein-9 nuclease) is a convenient and versatile platform for site-specific genome editing and epigenome targeted modulation. Given that sarcoma is believed to develop as a result of genetic alterations in mesenchymal progenitor/stem cells, CRISPR-Cas9 genome editing technologies hold extensive application potentials in sarcoma models and therapies. We review the development and mechanisms of the CRISPR-Cas9 system in genome editing and introduce its application in sarcoma research and potential therapy in clinic. Additionally, we propose future directions and discuss the challenges faced with these applications, providing concise and enlightening information for readers interested in this area. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

CRISPR/Cas9 system and its applications in human hematopoietic cells.

Science.gov (United States)

Hu, Xiaotang

2016-11-01

Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

Les leishmanioses cutanées à Leishmania major et à Leishmania tropica au Maroc: aspects épidémio-cliniques comparatifs de 268 cas

Science.gov (United States)

Chiheb, Soumia; Slaoui, Widad; Mouttaqui, Tarik; Riyad, Meriem; Benchikhi, Hakima

2014-01-01

Introduction Depuis 1995, le Maroc a connu une réactivation des foyers de leishmanioses cutanées (LC) à L. major et une nouvelle répartition géographique des foyers à L. tropica. Le but de cette étude est de comparer les aspects épidémio-cliniques associés aux LC potentiellement dûes à L. major et à L. tropica. Méthodes Une étude rétrospective a colligé 268 cas de LC au service de dermatologie du CHU Ibn Rochd de Casablanca entre Janvier 1995 et Septembre 2010. Les données étaient analysées par Epi info version 3.5.1. Le test X2 était appliqué (Différence significative = p< 0,05). Résultats Deux cent soixante-huit cas de LC ont été colligés, dont 160 femmes et 108 hommes. Ils ont été répartis en 123 patients originaires des foyers à L.major et 145 patients originaires des foyers à L. tropica. L'aspect ulcéronodulaire, ulcérovégétant ou végétant était retrouvé dans 58 cas (47,2%) des cas de LC à L. major versus 24 cas (16,7%) dans la L.C à L. tropica. L'aspect papulonodulaire était retrouvé dans 84 cas (58%) de LC à L. tropica contre 41 cas (33,3%) de LC à L. major. Conclusion Dans la LC à L. major, l'atteinte des membres et les aspects cliniques végétant ou ulcéro-végétant restent toujours prédominants. Dans la L.C à L. tropica, l'atteinte papulonodulaire unique du visage reste prédominante mais des formes ulcéronodulaires, végétantes ou ulcérovégétantes existent également dans les foyers récents à L. tropica, prêtant à confusion cliniquement avec des LC à L. major. PMID:25810796

Application of CRISPR/Cas9 Technology to HBV

Directory of Open Access Journals (Sweden)

Guigao Lin

2015-11-01

Full Text Available More than 240 million people around the world are chronically infected with hepatitis B virus (HBV. Nucleos(tide analogs and interferon are the only two families of drugs to treat HBV currently. However, none of these anti-virals directly target the stable nuclear covalently closed circular DNA (cccDNA, which acts as a transcription template for viral mRNA and pre-genomic RNA synthesis and secures virus persistence. Thus, the fact that only a small number of patients treated achieve sustained viral response (SVR or cure, highlights the need for new therapies against HBV. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 gene editing system can specifically target the conserved regions of the HBV genome. This results in robust viral suppression and provides a promising tool for eradicating the virus. In this review, we discuss the function and application of the CRISPR/Cas9 system as a novel therapy for HBV.

76 FR 79545 - Cost Accounting Standards: Change to the CAS Applicability Threshold for the Inflation Adjustment...

Science.gov (United States)

2011-12-22

... Cost Accounting Standards: Change to the CAS Applicability Threshold for the Inflation Adjustment to... Federal Procurement Policy, Cost Accounting Standards Board. ACTION: Final rule. SUMMARY: The Office of Federal Procurement Policy (OFPP), Cost Accounting Standards (CAS) Board (Board), has adopted, without...

Relance de l'aquaculture au Sri Lanka

International Development Research Centre (IDRC) Digital Library (Canada)

cas de risque et de mettre au point des systèmes de dépuration (c'est-à-dire de purification au moyen d'eau de mer stérilisée). Un élément primordial du projet de recherche consiste à protéger la population contre les contaminants (aussi bien naturels que d'origine anthropique). Pour que les collectivités puissent exploiter ...

A Broad-Spectrum Inhibitor of CRISPR-Cas9.

Science.gov (United States)

Harrington, Lucas B; Doxzen, Kevin W; Ma, Enbo; Liu, Jun-Jie; Knott, Gavin J; Edraki, Alireza; Garcia, Bianca; Amrani, Nadia; Chen, Janice S; Cofsky, Joshua C; Kranzusch, Philip J; Sontheimer, Erik J; Davidson, Alan R; Maxwell, Karen L; Doudna, Jennifer A

2017-09-07

CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome engineering in ophthalmology: Application of CRISPR/Cas to the treatment of eye disease.

Science.gov (United States)

Hung, Sandy S C; McCaughey, Tristan; Swann, Olivia; Pébay, Alice; Hewitt, Alex W

2016-07-01

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (Cas) system has enabled an accurate and efficient means to edit the human genome. Rapid advances in this technology could results in imminent clinical application, and with favourable anatomical and immunological profiles, ophthalmic disease will be at the forefront of such work. There have been a number of breakthroughs improving the specificity and efficacy of CRISPR/Cas-mediated genome editing. Similarly, better methods to identify off-target cleavage sites have also been developed. With the impending clinical utility of CRISPR/Cas technology, complex ethical issues related to the regulation and management of the precise applications of human gene editing must be considered. This review discusses the current progress and recent breakthroughs in CRISPR/Cas-based gene engineering, and outlines some of the technical issues that must be addressed before gene correction, be it in vivo or in vitro, is integrated into ophthalmic care. We outline a clinical pipeline for CRISPR-based treatments of inherited eye diseases and provide an overview of the important ethical implications of gene editing and how these may influence the future of this technology. Copyright © 2016 Elsevier Ltd. All rights reserved.

The CRISPR/Cas genome-editing tool: application in improvement of crops

Directory of Open Access Journals (Sweden)

SURENDER eKHATODIA

2016-04-01

Full Text Available The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR associated Cas9/sgRNA system is a novel fledgling targeted genome-editing technique from bacterial immune system, which is a cheap, easy and most rapidly adopted genome editing tool transforming to revolutionary paradigm. Cas9 protein is an RNA guided endonuclease utilized for creating targeted double stranded breaks with only a short RNA sequence to confer recognition of the target in animals and plants. Development of genetically edited (GE crops similar to those developed by conventional or mutation breeding using this potential technique makes it a promising and extremely versatile tool for providing sustainable productive agriculture for better feeding of rapidly growing population in changing climate. The emerging areas of research for the genome editing in plants are like, interrogating gene function, rewiring the regulatory signaling networks, sgRNA library for high-throughput loss-of-function screening. In this review, we will discuss the broad applicability of the Cas9 nuclease mediated targeted plant genome editing for development of designer crops. The regulatory uncertainty and social acceptance of plant breeding by Cas9 genome editing have also been discussed. The non-GM designer genetically edited plants could prospect climate resilient and sustainable energy agriculture in coming future for maximizing the yield by combating abiotic and biotic stresses with this new innovative plant breeding technique.

L’apprentissage au cern

CERN Multimedia

2007-01-01

pour les professions d’électronicien(ne) et de laborantin(e) en physique L’apprentissage au CERN est régi par les lois, règlements et contrats en vigueur dans le canton de Genève. En cas de réussite à l’examen de fin d’apprentissage, les apprentis obtiennent le Certificat fédéral de capacité suisse (CFC). 6 places au total sont ouvertes au recrutement pour les deux professions. L’apprentissage dure 4 ans. Minima requis pour faire acte de candidature : avoir au moins 15 ans et moins de 21 ans à la date de début de l’apprentissage ; avoir terminé la scolarité obligatoire, au minimum 9e du Cycle d’orientation genevois (3e en France) ; être ressortissant d’un pays membre du CERN (Allemagne, Autriche, Belgique, Bulgarie, Danemark, Espagne, Finlande, France, Grèce, Hongrie, Italie, Norvège, Pays-Bas, Pologne, Portugal, Royaume-Uni, République tchèque, République slovaque , Suède, Suisse) ; pour les résidents en Suisse : être ressortissant su...

Apprentissages techniques : L'apprentissage au CERN

CERN Multimedia

2004-01-01

APPRENTISSAGES TECHNIQUES GESTION ET DEVELOPPEMENT DU PERSONNEL HR/PMD L'APPRENTISSAGE AU CERN pour les professions d'électronicien(ne) et de laborantin(e) en physique L'apprentissage au CERN est régi par les lois, règlements et contrats en vigueur dans le Canton de Genève. En cas de réussite à l'examen de fin d'apprentissage, les apprentis obtiennent le Certificat Fédéral de Capacité Suisse (CFC). 7 places au total sont ouvertes au recrutement pour les deux professions. L'apprentissage dure 4 ans. Minima requis pour faire acte de candidature : • avoir au moins 15 ans et moins de 21 ans à la date de début de l'apprentissage • avoir terminé la scolarité obligatoire, au minimum 9ème du Cycle d'orientation genevois (3ème en France) • être ressortissant d'un pays membre du CERN (Allemagne, Autriche, Belgiqu...

Corps étrangers rectaux introduits par l'anus: à propos de deux cas ...

African Journals Online (AJOL)

Le but de ce travail était de rapporter notre expérience à partir de deux cas observés au cours de l'année 2014 afin de relever certains aspects de leur prise en charge. Les patients étaient de sexe masculin, âgés de 50 et 60 ans. Dans un cas, il s'agissait d'une agression à caractère punitif et dans l'autre d'un acte sexuel.

Mise au point

African Journals Online (AJOL)

et traité à temps, le risque de complications et de morbidité peut être écarté. Les auteurs rapportent 2 cas de kystes de la vallécule. La tomodensitométrie a confirmé la présence d'une formation kystique prenant origine au niveau de la val- lécule. Une laryngoscopie directe a été faite pour les 2 patients sous anesthésie ...

Mettre l'emploi au coeur des priorités au Mexique : étude de cas ...

International Development Research Centre (IDRC) Digital Library (Canada)

Depuis quelques années, on se préoccupe de plus en plus, dans de nombreux pays et sur la scène internationale, du chômage et du sous-emploi ainsi que des répercussions de l'emploi sur le bien-être, l'inclusion sociale et la stabilité politique. Ce projet produira une étude de cas pour le Rapport sur le développement ...

Modification of CAS-protocol for improvement of security web-applications from unauthorized access

Directory of Open Access Journals (Sweden)

Alexey I Igorevich Alexandrov

2017-07-01

Full Text Available Dissemination of information technologies and the expansion of their application demand constantly increasing security level for users, operating with confidential information and personal data. The problem of setting up secure user identification is probably one of the most common tasks, which occur in the process of software development. Today, despite the availability of a large amount of authentication tools, new solutions, mechanisms and technologies are being introduced regularly. Primarily, it is done to increase the security level of data protection against unauthorized access. This article describes the experience of using central user authentication service based on CAS-protocol (CAS – Central Authentication Service and free open source software, analyzing its main advantages and disadvantages and describing the possibility of its modification, which would increase security of web-based information systems from being accessed illegally. The article contains recommendations for setting a maximum time limit for users working on services, integrated with central authentication; and, analyses the research of implementing modern web-technologies while using user authentication system based on CAS-protocol. In addition, it describes the ways of CAS-server modernization for developing additional modules: a module for collecting and analyzing the use of information systems, and another one, for a user management system. Furthermore, CAS-protocol can be used at universities and other organizations for creating a unified information environment in education.

Profil épidémiologique des tumeurs malignes primitives des glandes salivaires : à propos de 154 cas

Science.gov (United States)

Setti, Khadija; Mouanis, Mohamed; Moumni, Abdelmounim; Maher, Mostafa; Harmouch, Amal

2014-01-01

Introduction Les tumeurs des glandes salivaires sont des tumeurs rares représentant 3à 5% des tumeurs de la tête et du cou. La classification de l'OMS 2005 distingue les tumeurs épithéliales, les tumeurs mésenchymateuses, les tumeurs hématologiques et les tumeurs secondaires. Méthodes Notre travail consiste en une étude rétrospective réalisée sur une période de 10 ans allant de janvier 2002 à janvier 2012. Les critères d'inclusion étaient: l'âge, le sexe, le siège de la tumeur et le type histologique. Résultats L'incidence annuelle des tumeurs malignes primitives des glandes salivaires dans notre série était de 15 cas par an. Cent cinquante quatre cas de tumeurs malignes primitives des glandes salivaires ont été colligés sans prédominance de sexe (78 femmes (50,6%) et 76 hommes (49,4%)). La moyenne d'âge était de 60 ans avec des extrêmes de 4 et 83 ans et un pic de fréquence entre 51et 70 ans. Deux tiers des cas (65%) avaient une localisation au niveau des glandes principales avec 66 cas au niveau de la parotide (43%) et 34 cas au niveau de la glande sous maxillaire (22%). Cinquante quatre patients avaient une tumeur maligne des glandes salivaires accessoires (35%) dont 61% au niveau du palais. Aucun cas de tumeur maligne de la glande sublinguale n'a été recensé dans notre étude. Le type histologique prédominant dans notre série était le carcinome adénoïde kystique et retrouvé chez 43 patients (27,9%), suivi de l'adénocarcinome sans autre indication chez 37 patients (24%) puis du carcinome mucoépidermoïde chez 16 patients (10,4%) et de l'adénocarcinome polymorphe de bas grade également chez 16 patients (10. 4%). Conclusion Les tumeurs malignes des glandes salivaires représentent un ensemble hétérogène de maladies de caractérisation complexe et de fréquence variable. PMID:25120861

CRISPR/Cas9 in Genome Editing and Beyond.

Science.gov (United States)

Wang, Haifeng; La Russa, Marie; Qi, Lei S

2016-06-02

The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

Le syndrome de Pepper: à propos de deux cas observés au Centre ...

African Journals Online (AJOL)

Le syndrome de Pepper est une forme métastatique hépatique du neuroblastome. C'est une entité spécifique du nourrisson de moins de six mois qui a la particularité de pouvoir régresser de façon spontanée avec un pronostic favorable dans 80% des cas. A cause de sa rareté, nous rapportons deux cas du syndrome de ...

CRISPR-Cas: biology, mechanisms and relevance

Science.gov (United States)

Hille, Frank

2016-01-01

Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes—termed spacers—into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent. This article is part of the themed issue ‘The new bacteriology’. PMID:27672148

Le mal de Pott: à propos de 82 cas

Directory of Open Access Journals (Sweden)

Badr Fedoul

2011-03-01

Full Text Available Nous rapportant dans cette étude, les résultats de l’expérience du service de neurochirurgie du CHU Hassan II de Fès dans la prise en charge du mal de pott dans la région de Fès. Il s’agit d’une étude rétrospective de quatre-vingt-deux cas; étalée sur une période de cinq ans (janvier 2002 au décembre 2006. L’objectif de ce travail était d’illustrer les différents aspects épidémiologiques, diagnostiques et thérapeutiques de la localisation vertébrale de la tuberculose dans notre pratique. L'âge moyen de nos patients était de 43,1 ans, avec une légère prédominance féminine (53,82%. La durée d'évolution de la maladie était longue (dix mois en moyenne; ceci est expliquée par la symptomatologie initiale insidieuse faite de rachialgies (98,78% et une admission des patients au stade de complications neurologiques (41,46%. La radiographie standard était réalisée chez tous nos patients, et complétée par la TDM dans 86.58% des cas ce qui a permis de déceler la prédominance de l'atteinte dorsale et lombaire. L'IRM est l'examen de choix, elle était demandée chez tous les malades déficitaires (37,8%.Tous nos patients ont bénéficié d'un traitement antibacillaire associé à une immobilisation du foyer pottique. Une décompression par voie antérieure était réalisée chez 29 patients (35,36 %; alors que la laminectomie n'était pratiquée que chez 5 patients (6.09 %, tandis que l'évacuation de l'abcès de psoas était réalisée chez 25 patients (30,48 %. Le diagnostic de certitude histologique était posé dans 51 cas (62,19%. Les meilleurs résultats étaient obtenus chez les malades opérés par voie antérieure, 26 cas (89,65% de récupération totale et 3 cas (10,34% partielle. L'évolution vers la consolidation et la fusion vertébrale était la règle chez tous nos malades et ceci au bout de 4 à 18 mois après le traitement.

L’addiction au pluriel : le cas des pratiquants de bodybuilding

Directory of Open Access Journals (Sweden)

Guillaume Vallet

2016-04-01

Full Text Available Cet article s’interroge sur les addictions dans le bodybuilding. A partir de données qualitatives recueillies auprès de 30 bodybuilders masculins et d’une analyse de 27 magazines spécialisés, nous mettons en évidence que les pratiquants les plus engagés ont de fortes probabilités d’expérimenter trois types d’addictions : addictions liées au surentraînement, addictions liées au mode de vie consommatoire, addictions liées au mode de vie identitaire. Ces addictions se construisant dans le temps dans le cadre d’un processus, nous interprétons celles-ci à partir du concept sociologique de « carrière », ce qui nous permet de construire une pyramide de l’engagement et des addictions dans le bodybuilding, reliée notamment à la problématique du genre.

ÉTUDE DE CAS – Kenya : Paludisme et agriculture au Kenya ...

International Development Research Centre (IDRC) Digital Library (Canada)

11 janv. 2011 ... ... au Kenya, des chercheurs du Centre international de recherche sur la .... Les membres de l'équipe ont amorcé leur collaboration par la définition des ... Mutero est, à bon droit, chargé de la coordination de SIMA, qui loge au ...

TiAu based shape memory alloys for high temperature applications

International Nuclear Information System (INIS)

Wadood, Abdul; Yamabe-Mitarai, Yoko; Hosoda, Hideki

2014-01-01

TiAu (equiatomic) exhibits phase transformaion from B2 (ordered bcc) to thermo-elastic orthorhombic B19 martensite at about 875K and thus TiAu is categorized as high temperature shape memory alloy. In this study, recent research and developments related to TiAu based high temperature shape memory alloys will be discussed in the Introduction part. Then some results of our research group related to strengthening of TiAu based high temperature shape memory alloys will be presented. Potential of TiAu based shape memory alloys for high temperature shape memory materials applications will also be discussed

Améliorer la responsabilité des gouvernements locaux au profit des ...

International Development Research Centre (IDRC) Digital Library (Canada)

Au moyen d'études de cas en Ouganda, ce projet mettra à l'épreuve les approches internationales et gouvernementales dominantes visant l'amélioration de la participation et de la responsabilité des gouvernementaux locaux. Lutter contre la pauvreté au moyen de la gouvernance. L'Ouganda fait face à plusieurs défis en ...

The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models

Science.gov (United States)

SHAO, Ming; XU, Tian-Rui; CHEN, Ce-Shi

2016-01-01

Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and biomedicine. PMID:27469250

The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models.

Science.gov (United States)

Shao, Ming; Xu, Tian-Rui; Chen, Ce-Shi

2016-07-18

Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio-medicine.

Les Brulures Electriques par Haut Voltage - A Propos de 10 Cas

Science.gov (United States)

Belmir, R.; Fejjal, N.; El Omari, M.; El Mazouz, S.; Gharib, N.; Abassi, A.; Belmahi, A.

2008-01-01

Summary Les accidents électriques par haute tension (AEHT) provoquent des brûlures profondes par effet Joule le long des axes vasculo-nerveux entre les points d'entrée et de sortie, qui sont le siège de lésions délabrantes. Les Auteurs rapportent une série de dix cas d'AEHT admis au service de chirurgie réparatrice et de brûlés de l'Hôpital Ibn Sina de Rabat à travers laquelle ils étudient les caractéristiques épidémiologiques, cliniques et thérapeutiques. Tous les patients étaient des adultes de sexe masculin dont l'âge moyen était de 31 ans. Dans 70% des cas, ces brûlures étaient secondaires à un contact avec les distributeurs d'électricité avec une surface brûlée inférieure à 20%. Le traitement des lésions électrothermiques a nécessité des interventions itératives avec amputation des segments de membres nécrosés dans 70% des cas, dont les suites étaient marquées par des séquelles fonctionnelles invalidantes. La prévention des AEHT, en particulier pour les accidents du travail au sein des professions exposées, reste fondamentale. PMID:21991124

Coinfection pulmonaire par pneumocystis jirovecii et pseudomonas aeruginosa au cours du SIDA: à propos de deux cas

Science.gov (United States)

Mamoudou, Savadogo; Bellaud, Guillaume; Ana, Canestri; Gilles, Pialoux

2015-01-01

Rapporter deux cas cliniques de coinfections pulmonaires par Pneumocystis jirovecii et par Pseudomonas aeruginosa chez des patients vivant avec le VIH. Les deux patients étaient âgés respectivement de 32 ans et 46 ans. Un patient a été pris en charge à l'hôpital Yalgado Ouédraogo de Ouagadougou au Burkina Faso et l'autre a été pris en charge à l'hôpital Ténon de Paris, en France. Les deux souffraient de pneumopathie confirmée à la radiographie et à la tomodensitométrie. L'un des patients était sévèrement immuno déprimé, contrairement à l'autre. L'examen bactériologique dans les crachats avait permis d'isoler Pseudomonas aeruginosa et Pneumocystis jirovecii chez les deux patients. Sous traitement, l’évolution a été favorable. Les coinfections morbides sont relativement fréquentes chez les patients vivant avec le VIH. Devant une symptomatologie respiratoire du sujet vivant avec le VIH, il faut savoir rechercher en plus du Bacille de Koch, Pneumocystis jirovecii et Pseudomonas aeruginosa par un lavage broncho alvéolaire. PMID:26516396

Ordered Au Nanodisk and Nanohole Arrays: Fabrication and Applications

KAUST Repository

Zheng, Yue Bing; Juluri, Bala Krishna; Kiraly, Brian; Huang, Tony Jun

2010-01-01

We have utilized nanosphere lithography (NSL) to fabricate ordered Au nanodisk and nanohole arrays on substrates and have studied the localized surface plasmon resonance (LSPR) of the arrays. Through these investigations, we demonstrate that the angle- dependent behavior of the LSPR in the Au nanodisk arrays enables real-time observation of exciton-plasmon couplings. In addition, we show that the NSL-fabricated Au nanohole arrays can be applied as templates for patterning micro-/nanoparticles under capillary force. The unique structural and plasmonic characteristics of the Au nanodisk and nano- hole arrays, as well as the low-cost and high-throughput NSL-based nanofabrication technique, render these arrays excellent platforms for numerous engineering applications. © 2010 by ASME.

How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.

Science.gov (United States)

Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun; Ke, Ailong

2017-10-05

CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer. The prespacer is bound by Cas1-Cas2 as a dual-forked DNA, and the terminal 3'-OH of each 3' overhang serves as an attacking nucleophile during integration. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat. Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor. In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers. Here we present four structural snapshots from the type II-A system of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1-Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3' overhangs. Three molecular events take place upon encountering a target: first, the Cas1-Cas2-prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3' overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer

Intoxication par la paraphényléne-diamine (takaout au Maroc: à propos de 24 cas

Directory of Open Access Journals (Sweden)

Ali Derkaoui

2011-03-01

Full Text Available La paraphénylène-diamine (PPD est une amine aromatique dérivée de l’aniline, utilisée depuis 1863 par les femmes dans un but cosmétique comme teinture capillaire noire ou adjuvant de henné dans plusieurs pays d’Afrique et de Moyen Orient. Le but de notre travail était de décrire les caractéristiques cliniques, paracliniques et évolutives de nos patients et de les comparer avec les données de la littérature. Il s’agissait d’une étude rétrospective portant sur les cas admis en réanimation (2003-2010. Les critères d’inclusion étaient d’ordre clinique, paraclinique, thérapeutique et évolutif. Durant la période de l’étude 24 patients ont été inclus provenant de la région de Fés-Boulmane. L’intoxication à la (PPD représentait 26% de l’ensemble des intoxications admises au cours de la même période. L âge moyen était de 23,6 plus or minus 11 ,6 ans. Il existait une prédominance féminine avec un sex-ratio de 4,7. L’intoxication était volontaire dans 82,6 %, accidentelle dans 8,6 %, et criminelle dans 4,3%. Le syndrome de rhabdomyolyse caractéristique de cette intoxication était retrouvé chez 60% de nos patients, l’atteinte respiratoire chez 56,5%, l’atteinte cardiaque était présente dans 30% des cas et 17,4% des patients avaient présentés une insuffisance rénale. La prise en charge thérapeutique se basait sur l’apport volémique massif, alcalinisation des urines ainsi que l’administration de corticoïdes et de diurétiques. Le recours à une trachéotomie de sauvetage était nécessaire chez 7 patients. Trois de nos patients avaient bénéficié d’une épuration extra rénale. L’évolution était fatale chez 47,8% des cas. La PPD représente ainsi la principale cause de mortalité toxique dans notre contexte. L’intoxication à la PPD, représente la première cause de rhabdomyolyse toxique dans notre contexte. Elle est responsable d’une mortalité très élevée. Ce qui

Réduction du risque d'infection par le VIH au Botswana - essais ...

International Development Research Centre (IDRC) Digital Library (Canada)

Au Botswana, ce sont les jeunes femmes qui sont le plus touchées par les nouvelles infections par le VIH. Le présent projet a pour but de réduire le nombre de nouveaux cas d'infection par le VIH, en particulier chez les femmes de 15 à 29 ans. Bien que l'on recense des cas de VIH/sida dans la plupart des régions du ...

Prise en charge de l'hematurie au chu Sylvanus Olympio de Lome ...

African Journals Online (AJOL)

Une transfusion sanguine avait été réalisée dans 64,8% des cas et nous avions enregistré 4 cas de décès. Conclusion : Les hématuries sont relativement fréquentes au Togo et souvent de causes tumorales. Elles nécessitent une prise en charge précoce pour un meilleur pronostic. Mots clés : hématurie, épidémiologie, ...

CRISPR-Cas9 technology: applications in genome engineering, development of sequence-specific antimicrobials, and future prospects.

Science.gov (United States)

de la Fuente-Núñez, César; Lu, Timothy K

2017-02-20

The development of CRISPR-Cas9 technology has revolutionized our ability to edit DNA and to modulate expression levels of genes of interest, thus providing powerful tools to accelerate the precise engineering of a wide range of organisms. In addition, the CRISPR-Cas system can be harnessed to design "precision" antimicrobials that target bacterial pathogens in a DNA sequence-specific manner. This capability will enable killing of drug-resistant microbes by selectively targeting genes involved in antibiotic resistance, biofilm formation and virulence. Here, we review the origins and mechanistic basis of CRISPR-Cas systems, discuss how this technology can be leveraged to provide a range of applications in both eukaryotic and prokaryotic systems, and finish by outlining limitations and future prospects.

L'égalité, un effort collectif au Sénégal | CRDI - Centre de ...

International Development Research Centre (IDRC) Digital Library (Canada)

8 nov. 2011 ... Les lois et les traditions au Sénégal sont en désaccord quant aux ... C'est le cas dans l'ensemble du pays, alors que plus des trois quarts des femmes économiquement actives au Sénégal travaillent dans le secteur agricole.

Maladie de Biermer : étude de quatre cas au Centre Hospitalier ...

African Journals Online (AJOL)

Les troubles neurologiques constituaient un tableau de sclérose combinée de la moelle (SCM) dans tous les trois cas, avec un déficit moteur observé chez deux patients, une atteinte des nerfs crâniens chez un patient, et des troubles de la mémoire chez deux patients. Tous les patients avaient reçu une supplémentation en ...

Au Based Nanocomposites Towards Plasmonic Applications

Science.gov (United States)

Panniello, A.; Curri, M. L.; Placido, T.; Reboud, V.; Kehagias, N.; Sotomayor Torres, C. M.; Mecerreyes, D.; Agostiano, A.; Striccoli, M.

2010-06-01

Incorporation of nano-sized metals in polymers can transfer their unique features to the host matrix, providing nanocomposite materials with improved optical, electric, magnetic and mechanical properties. In this work, colloidal Au nanorods have been incorporated into PMMA based random co-polymer, properly functionalized with amino groups and the optical and morphological properties of the resulting nanocomposite have been investigated by spectroscopic and AFM measurements. Au nanorods have demonstrated to preserve the plasmon absorption and to retain morphological features upon the incorporation, thus making the final metal modified polymer composite exploitable for the fabrication of plasmonic devices. The prepared nanocomposites have been then patterned by Nano Imprint Lithography technique in order to demonstrate the viability of the materials towards optical applications.

L'APPRENTISSAGE AU CERN - French version only

CERN Multimedia

Roland GAY

2002-01-01

APPRENTISSAGES TECHNIQUES FORMATION ET DEVELOPPEMENT HR/TD/AP L'APPRENTISSAGE AU CERN pour les professions d'électronicien(ne) et de laborant(ine) en physique L'apprentissage au CERN est régi par les lois, règlements et contrats en vigueur dans le Canton de Genève. En cas de réussite à l'examen de fin d'apprentissage, les apprentis obtiennent le Certificat Fédéral de Capacité Suisse (CFC). 7 places au total sont ouvertes au recrutement pour les deux professions. L'apprentissage dure 4 ans. Minima requis pour faire acte de candidature :   avoir terminé la scolarité obligatoire être ressortissant d'un pays membre du CERN (Allemagne, Autriche, Belgique, Bulgarie, Danemark, Espagne, Finlande, France, Grèce, Hongrie, Italie, Norvège, Pays-Bas, Pologne, Portugal, République slovaque, République tchèque, Royaume-Uni, Su&a...

Sequelles de Brulures au Centre Hospitalier Universitaire Ibn Rochd de Casablanca: Aspects Epidemio-Cliniques

Science.gov (United States)

Chafiki, N.; Fassi Fihri, J.; Boukind, E.H.

2007-01-01

Summary Il s'agit d'une étude épidémiologique des séquelles de brûlures à propos de 100 cas colligés au service de chirurgie réparatrice et de brûlés du centre hospitalier universitaire Ibn Rochd (Casablanca). Les adultes représentent 55% de la population étudiée, l'âge moyen global est de 20 ans. Le sexe féminin est le plus touché avec 61% des cas. Les brûlures survenues à domicile sont les plus fréquentes avec 80%. L'agent causal le plus incriminé est la petite bouteille de butane avec 44,4%. Plus de la moitié de la population brûlée (55%) sont accueillis initialement au niveau d'hôpitaux régionaux. Le délai de cicatrisation moyen de 7 mois et 11 jours et par conséquent les séquelles mineures (dyschromie dans 90% des cas et prurit dans 49% des cas) et majeures (rétractions dans 86% et l'hypertrophie dans 51%) sont fréquentes. La répartition globale des séquelles montre une prédominance du segment cervicocéphalique avec 89% des cas et des membres supérieurs dans 82% des cas. Les différents aspects anatomocliniques essentiels ont été décrits au niveau de chaque segment corporel. Nos résultats ont été comparés aux données de la littérature, ce qui nous amène à considérer qu'une large campagne de prévention couplée à une meilleure prise en charge initiale, précoce, bien conduite et multidisciplinaire permet non seulement de réduire le nombre de séquelles mais aussi de diminuer leur sévérité. PMID:21991060

L'endométriose de la paroi abdominale: à propos d'un cas rare

Science.gov (United States)

Jayi, Sofia; Laadioui, Meriem; Bouguern, Hakima; Chaara, Hikmat; Melhouf, Aabdelilah

2013-01-01

L'endométriose de la paroi est une entité clinique rare, dont la physiopathologie demeure imprécise. Elle survient le plus souvent après une intervention chirurgicale gynécologique ou obstétricale. Nous rapportons le cas d'une patiente présentant une douleur cyclique, au niveau de la cicatrice de césarienne. Avec à l'examen clinique une masse de 3 cm localisée au niveau de la fosse iliaque droite. L'échographie-doppler a objectivé une image d'allure tissulaire, polylobée, à vascularisation centrale, mesurant 32/16 mm, évoquent une masse endométriosique ou une tumeur des parties molles. D'où la décision d'excision de la lésion, au cours de laquelle on a découvert une masse dure de 3/2 cm, en sous aponévrotique accolée au muscle grand droit de l'abdomen. L'étude anatomopathologique a confirmé le diagnostic d'endométriose pariétale. Les suites postopératoires étaient simples avec un recul de 2 ans et demie sans récidive de la masse ni de la douleur. A travers notre cas, nous insisterons sur les caractéristiques de cette pathologie notamment pronostic, ce qui permettra au praticien de comprendre l'intérêt du diagnostic et prise en charge précoce de cette entité - pour laquelle on ne pense jamais assez devant une masse pariétale - et de sa prévention au cours de chaque chirurgie gynécologique ou obstétricale. PMID:24171068

Harnessing CRISPR/Cas systems for programmable transcriptional and post-transcriptional regulation

KAUST Repository

Mahas, Ahmed

2017-11-29

Genome editing has enabled broad advances and novel approaches in studies of gene function and structure; now, emerging methods aim to precisely engineer post-transcriptional processes. Developing precise, efficient molecular tools to alter the transcriptome holds great promise for biotechnology and synthetic biology applications. Different approaches have been employed for targeted degradation of RNA species in eukaryotes, but they lack programmability and versatility, thereby limiting their utility for diverse applications. The CRISPR/Cas9 system has been harnessed for genome editing in many eukaryotic species and, using a catalytically inactive Cas9 variant, the CRISPR/dCas9 system has been repurposed for transcriptional regulation. Recent studies have used other CRISPR/Cas systems for targeted RNA degradation and RNA-based manipulations. For example, Cas13a, a Type VI-A endonuclease, has been identified as an RNA-guided RNA ribonuclease and used for manipulation of RNA. Here, we discuss different modalities for targeted RNA interference with an emphasis on the potential applications of CRISPR/Cas systems as programmable transcriptional regulators for broad uses, including functional biology, biotechnology, and synthetic biology applications.

Harnessing CRISPR/Cas systems for programmable transcriptional and post-transcriptional regulation

KAUST Repository

Mahas, Ahmed; Neal Stewart, C.; Mahfouz, Magdy M.

2017-01-01

Genome editing has enabled broad advances and novel approaches in studies of gene function and structure; now, emerging methods aim to precisely engineer post-transcriptional processes. Developing precise, efficient molecular tools to alter the transcriptome holds great promise for biotechnology and synthetic biology applications. Different approaches have been employed for targeted degradation of RNA species in eukaryotes, but they lack programmability and versatility, thereby limiting their utility for diverse applications. The CRISPR/Cas9 system has been harnessed for genome editing in many eukaryotic species and, using a catalytically inactive Cas9 variant, the CRISPR/dCas9 system has been repurposed for transcriptional regulation. Recent studies have used other CRISPR/Cas systems for targeted RNA degradation and RNA-based manipulations. For example, Cas13a, a Type VI-A endonuclease, has been identified as an RNA-guided RNA ribonuclease and used for manipulation of RNA. Here, we discuss different modalities for targeted RNA interference with an emphasis on the potential applications of CRISPR/Cas systems as programmable transcriptional regulators for broad uses, including functional biology, biotechnology, and synthetic biology applications.

Recent Advances in Genome Editing Using CRISPR/Cas9

Science.gov (United States)

Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

2016-01-01

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding. PMID:27252719

Apport de l'imagerie dans le diagnostic des sacroiliites infectieuses : à propos de 19 cas

Science.gov (United States)

Abid, Hanen; Chaabouni, Salim; Frikha, Faten; Toumi, Nozha; Souissi, Basma; Lahiani, Dorra; Bahloul, Zouhir; Ben Mahfoudh, Khaireddine

2014-01-01

Les sacro-iliites infectieuses méritent d’être mieux connues. Leur diagnostic est souvent retardé en raison d'une symptomatologie trompeuse et des diffcultés d'exploration de l'articulation sacro-iliaque. Notre travail est basé sur une étude rétrospective portant sur les cas de SII, recueillis sur une période comprise entre 1997 et 2008 dans notre centre universitaire Sfax-Tunisie. Le diagnostic de sacro-iliite était retenu en présence d'arguments cliniques et radiologiques d'atteinte sacroiliaque. Nous rapportons dix neuf cas de sacroiliites infectieuses (10 hommes et 9 femmes), avec un âge moyen de 32 ans. L'atteinte était unilatérale dans tous les cas. Les radiographies standard faites dans tous les cas ont été suggestives dans 14 cas et normales dans les autres cas. La TDM faite dans 13 cas a montré, un abcès des parties molles dans 8 cas et un séquestre osseux dans 2 cas. L'IRM réalisée dans 8 cas, a objectivé une infiltration des parties molles dans tous les cas et un abcès dans 3 cas. Le germe a été identifié dans 9 cas (3 cas de tuberculose, 3 cas de brucellose, 2 sacro-iliites à pyogène et un cas de candidose). Cette identification était faite par biopsie dans 3 cas, hémocultures dans 2 cas, prélèvement au niveau de la porte d'entrée dans 1 cas et sérodiagnostic dans 3 cas. Pour les autres cas, l'origine pyogène a été retenue sur des arguments cliniques et biologiques. L'imagerie joue un rôle primordial dans le diagnostic précoce et l'orientation étiologique d'une sacroiliite infectieuse. PMID:25120884

La tuberculose sternale: à propos de 2 cas | Ouarssani | Pan African ...

African Journals Online (AJOL)

On rapporte deux cas de tuberculose sternale âgés respectivement de 45 ans et de 10ans, colligés au service de pneumologie, l'examen histologique et bactériologique ont permis d'affirmer le diagnostic de tuberculose, et le traitement antibacillaire a permis une évolution favorable .A la lumière de ces observations, les ...

A thermostable Cas9 with increased lifetime in human plasma

OpenAIRE

Harrington, LB; Paez-Espino, D; Staahl, BT; Chen, JS; Ma, E; Kyrpides, NC; Doudna, JA

2017-01-01

© 2017 The Author(s). CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas...

L'APPRENTISSAGE AU CERN - French version only

CERN Multimedia

Roland Gay

2002-01-01

APPRENTISSAGES TECHNIQUES FORMATION ET DEVELOPPEMENT HR/TD/AP L'apprentissage au CERN est régi par les lois, règlements et contrats en vigueur dans le Canton de Genève. En cas de réussite à l'examen de fin d'apprentissage, les apprentis obtiennent le Certificat Fédéral de Capacité Suisse (CFC). 7 places au total sont ouvertes au recrutement pour les deux professions. L'apprentissage dure 4 ans. Minima requis pour faire acte de candidature :   avoir terminé la scolarité obligatoire être ressortissant d'un pays membre du CERN (Allemagne, Autriche, Belgique, Bulgarie, Danemark, Espagne, Finlande, France, Grèce, Hongrie, Italie, Norvège, Pays-Bas, Pologne, Portugal, République slovaque, République tchèque, Royaume-Uni, Suède, Suisse) avoir au moins 15 ans et moins de 21 ans à la date de début de ...

SYNDROME DE GELINEAU. A PROPOS D\\'UN CAS NIGERIAN ET ...

African Journals Online (AJOL)

Au Nigéria, le syndrome de Gélineau est un trouble rare avec une prévalence hospitalière de 0.026%. Nous étudions ici le cas d'un patient nigérien âgé de 14 ans atteint de narcolepsie, de cataplexie, d'hallucinations hypnagogiques visuelles et de paralysie du sommeil hypnopompique. Il a été effectué un test de latence ...

Versatile Cas9-driven subpopulation selection toolbox for Lactococcus lactis

NARCIS (Netherlands)

Els, van der Simon; James, Jennelle K.; Kleerebezem, Michiel; Bron, Peter A.

2018-01-01

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis. The Cas9 expression vector

Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

KAUST Repository

Aouida, Mustapha

2015-07-30

The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

KAUST Repository

Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; Abu Samra, Dina Bashir Kamil; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy M.

2015-01-01

The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

DEFF Research Database (Denmark)

Ferreira, Raphael; David, Florian; Nielsen, Jens

2018-01-01

for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas......9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology....

Rational Design of Mini-Cas9 for Transcriptional Activation.

Science.gov (United States)

Ma, Dacheng; Peng, Shuguang; Huang, Weiren; Cai, Zhiming; Xie, Zhen

2018-04-20

Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

Les synovites villonodulaires du genou: à propos de 20 cas

Science.gov (United States)

Margad, Omar; Boukhris, Jalal; Azriouil, Ouahb; Daoudi, Mohamed; Mortaji, Aziz; Koulali, Khalid

2017-01-01

La synovite villonodulaire pigmentée (SVNP) est une prolifération bénigne rare de la synoviale des articulations, des bourses séreuses et des gaines tendineuses, d'étiopathogénie inconnue. Notre travail porte sur 20 cas de SVN du genou colligés à l'hôpital militaire Avicenne de Marrakech sur une période de 9 ans allant de janvier 2000 au décembre 2009 Il vise à identifier les spécificités de cette lésion, et à étudier ses principaux aspects anatomocliniques et pronostiques. L'incidence annuelle était de 2,2 cas par an. Ils étaient 15 hommes et 5 femmes, d'âge moyen de 32,5 ans, atteints du coté droit dans 55%des cas sous un mode mono articulaire chez 18 patients et bi articulaire chez un seul. La douleur et la tuméfaction étaient présentes dans 80% des cas, une masse palpable dans un cas, un syndrome méniscal a été retenu dans un cas, une mono arthrite septique dans 3 circonstances de même qu'un kyste poplité dans 2 autres. L'atteinte était diffuse dans 14 cas (70%), localisée dans 6 cas. L'imagerie par résonnance magnétique(IRM) pratiquée chez 5 patients était évocatrice chez 3, l'arthroscopie diagnostique a été utilisée chez 2 malades. La confirmation s'est faite à chaque fois à l'examen anatomopathlogique. Le traitement a consisté en une synovectomie subtotale dans 15 cas et en l'exérèse de la tumeur dans les autres formes localisées, 2 cas présentant une destruction ostéocartilagineuse ont nécessité une arthroplastie. L'évolution a été marquée par la survenue de 2 récidives sous la forme diffuse avec un recul de 3, 7 ans. On a noté une raideur avec atrophie quadricipitale chez 3 patients et une arthrolyse a été réalisée. Un cas de SVN confirmé par l'histologie s'est révélé être 5 mois après la synovectomie totale un Synovialosarcome monophasique envahissant l'os d'où l'indication de l'amputation. PMID:29255556

CRISPR-Cas9 Based Engineering of Actinomycetal Genomes

DEFF Research Database (Denmark)

Tong, Yaojun; Charusanti, Pep; Zhang, Lixin

2015-01-01

. To facilitate the genetic manipulation of actinomycetes, we developed a highly efficient CRISPR-Cas9 system to delete gene(s) or gene cluster(s), implement precise gene replacements, and reversibly control gene expression in actinomycetes. We demonstrate our system by targeting two genes, actIORF1 (SCO5087......) and actVB (SCO5092), from the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2). Our CRISPR-Cas9 system successfully inactivated the targeted genes. When no templates for homology-directed repair (HDR) were present, the site-specific DNA double-strand breaks (DSBs) introduced by Cas9....... Moreover, we developed a system to efficiently and reversibly control expression of target genes, deemed CRISPRi, based on a catalytically dead variant of Cas9 (dCas9). The CRISPR-Cas9 based system described here comprises a powerful and broadly applicable set of tools to manipulate actinomycetal genomes....

Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

Science.gov (United States)

Heler, Robert; Samai, Poulami; Modell, Joshua W; Weiner, Catherine; Goldberg, Gregory W; Bikard, David; Marraffini, Luciano A

2015-03-12

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

L'atteinte vésicale au cours de la neurofibromatose de Von Recklinghausen

Science.gov (United States)

Benazzouz, Mohamed Hicham; Hajjad, Tilila; Essatara, Younes; El Sayegh, Hachem; Iken, Ali; Benslimane, Lounis; Nouini, Yassine

2014-01-01

La neurofibromatose de type 1 ou maladie de Von Recklinghausen est une maladie génétique autosomique dominante en rapport avec des mutations dans le gène suppresseur de tumeur NF1. L'atteinte uro-génitale au cours de cette maladie est rare et moins de 80 cas ont été rapportés à ce jour dans la littérature mondiale. Les auteurs rapportent un nouveau cas d'atteinte vésicale découverte fortuitement au cours du suivi d'une patiente atteinte de la maladie de Von Recklinghausen. A travers cette observation et une revue de la littérature les auteurs discutent également les difficultés diagnostiques, thérapeutiques ainsi que les modalités de suivi dans cette maladie. PMID:25328590

CAS CERN Accelerator School: Cyclotrons, linacs and their applications. Proceedings

International Nuclear Information System (INIS)

Turner, S.

1996-01-01

These proceedings present the lectures given at the eighth specialized course organized by the CERN Accelerator School (CAS), the topic this time being 'Cyclotrons, Linacs and Their Applications'. Following an introductory lecture on linacs, the fundamental features of electron, ion and induction linacs are described together with their RF systems and particle sources. Cyclotrons are then introduced followed by details of their different types, their magnet and RF design, and their injection and extraction systems, with a glance towards exotic and possible future machines. Chapters are then presented on the use of linacs and cyclotrons for medical, fission, fusion and material applications, as well as for isotope production. Finally, descriptions of the design of a radioisotope facility, the matching of accelerators to their task and the computational tools used in their design are included. (orig.)

CAS CERN Accelerator School: Cyclotrons, linacs and their applications. Proceedings

Energy Technology Data Exchange (ETDEWEB)

Turner, S [ed.

1996-03-04

These proceedings present the lectures given at the eighth specialized course organized by the CERN Accelerator School (CAS), the topic this time being `Cyclotrons, Linacs and Their Applications`. Following an introductory lecture on linacs, the fundamental features of electron, ion and induction linacs are described together with their RF systems and particle sources. Cyclotrons are then introduced followed by details of their different types, their magnet and RF design, and their injection and extraction systems, with a glance towards exotic and possible future machines. Chapters are then presented on the use of linacs and cyclotrons for medical, fission, fusion and material applications, as well as for isotope production. Finally, descriptions of the design of a radioisotope facility, the matching of accelerators to their task and the computational tools used in their design are included. (orig.).

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape.

Science.gov (United States)

Ferreira, Raphael; David, Florian; Nielsen, Jens

2018-01-24

Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPR-based applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.

L'APPRENTISSAGE AU CERN - French version only

CERN Multimedia

2003-01-01

APPRENTISSAGES TECHNIQUES FORMATION ET DEVELOPPEMENT HR/TD/AP L'APPRENTISSAGE AU CERN pour les professions d'électronicien(ne) et de laborantin(e) en physique L'apprentissage au CERN est régi par les lois, règlements et contrats en vigueur dans le Canton de Genève. En cas de réussite à l'examen de fin d'apprentissage, les apprentis obtiennent le Certificat Fédéral de Capacité Suisse (CFC). 7 places au total sont ouvertes au recrutement pour les deux professions. L'apprentissage dure 4 ans. Minima requis pour faire acte de candidature : - avoir terminé la scolarité obligatoire - être ressortissant d'un pays membre du CERN (Allemagne, Autriche, Belgique, Bulgarie, Danemark, Espagne, Finlande, France, Grèce, Hongrie, Italie, Norvège, Pays-Bas, Pologne, Portugal, République slovaque, République tchèque, Royaume-Uni, Suède, Suisse) - avoir au moins 15 ans et moins de 21 ans à la date de début de l'apprentissage - avoir un niveau scolaire correspondant à la 9ème générale ou moderne...

CRISPR/Cas9 for cancer research and therapy.

Science.gov (United States)

Zhan, Tianzuo; Rindtorff, Niklas; Betge, Johannes; Ebert, Matthias P; Boutros, Michael

2018-04-16

CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Leiomyome agressif naso-sinusien : a propos d'un cas et revue de ...

African Journals Online (AJOL)

Introduction : Les léiomyomes sont des tumeurs bénignes issues des fibres musculaires lisses. La localisation au niveau des cavités nasosinusiennes est très rare. Cas clinique : Il s'agit d'une patiente âgée de 59 ans, qui a consulté pour obstruction nasale et épistaxis gauches. L'endoscopie nasale a montré une formation ...

Anesthésie pour prothése totale de la hanche: à propos de 50 cas

Science.gov (United States)

Serghini, Issam; Qamouss, Youssef; Zoubir, Mohamed; Lalaoui, Jaafar Salim; Koulali, Idrissi Khalid; Boughalem, Mohamed

2015-01-01

La chirurgie de la prothèse totale de la hanche (PTH) est une chirurgie fonctionnelle qui consiste en un remplacement d'une articulation endommagée afin d'améliorer la qualité de vie du patient. L'anesthésie pour PTH exige une préparation rigoureuse à l'intervention, la consultation d'anesthésie sera donc la clef de cette réussite. Nous avons réalisé une étude rétrospective concernant 60 arthroplasties totales de la hanche implantées chez 50 patients adultes, colligée au sevice de Traumatologie et de chirurgie orthopédique à l'Hôpital Militaire Avicenne de Marrakech sur une période étalée de Janvier 2010 au Décembre 2012. Nous avons évalué la prise en charge anesthésique: pré, per et postopératoire des patients opérés pour une PTH. La moyenne d’âge était de 56,5 ans, le sex-ratio était de 1,63 en faveur des hommes. L'indication prédominante était la coxarthrose primitive. L'anesthésie générale était la technique anesthésique la plus utilisée (66% des cas), l'intubation difficile était rencontrée chez 6% de nos patients. La durée moyenne de l'acte chirurgical était de 114 +/- 25,33 mn. 12% de nos patients ont présenté une hypotension artérielle peropératoire. L'incidence de la transfusion homologue perop était de 14%. Nous avons noté 08 cas de complications postop: 03 cas d'infection de la PTH 15 jours après l'intervention, 03 cas de descellement aseptique, 01 cas de luxation de PTH et 01 cas de descellement septique avec un recul moyen de 54 mois. PMID:27047619

CRISPR/Cas9-Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development.

Science.gov (United States)

Okoli, Arinze; Okeke, Malachy I; Tryland, Morten; Moens, Ugo

2018-01-22

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them.

Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

Science.gov (United States)

Fonfara, Ines; Le Rhun, Anaïs; Chylinski, Krzysztof; Makarova, Kira S.; Lécrivain, Anne-Laure; Bzdrenga, Janek; Koonin, Eugene V.; Charpentier, Emmanuelle

2014-01-01

The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool. PMID:24270795

Optimizing CRISPR/Cas9 for the Diatom Phaeodactylum tricornutum

Directory of Open Access Journals (Sweden)

Daniel Stukenberg

2018-06-01

Full Text Available CRISPR/Cas9 is a powerful tool for genome editing. We constructed an easy-to-handle expression vector for application in the model organism Phaeodactylum tricornutum and tested its capabilities in order to apply CRISPR/Cas9 technology for our purpose. In our experiments, we targeted two different genes, screened for mutations and analyzed mutated diatoms in a three-step process. In the end, we identified cells, showing either monoallelic or homo-biallelic targeted mutations. Thus, we confirm that application of the CRISPR/Cas9 system for P. tricornutum is very promising, although, as discussed, overlooked pitfalls have to be considered.

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit.

Science.gov (United States)

Murugan, Karthik; Babu, Kesavan; Sundaresan, Ramya; Rajan, Rakhi; Sashital, Dipali G

2017-10-05

CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.

Science.gov (United States)

Hu, Johnny H; Miller, Shannon M; Geurts, Maarten H; Tang, Weixin; Chen, Liwei; Sun, Ning; Zeina, Christina M; Gao, Xue; Rees, Holly A; Lin, Zhi; Liu, David R

2018-04-05

A key limitation of the use of the CRISPR-Cas9 system for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), the required PAM sequence is NGG. No natural or engineered Cas9 variants that have been shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we use phage-assisted continuous evolution to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA and GAT. The PAM compatibility of xCas9 is the broadest reported, to our knowledge, among Cas9 proteins that are active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and cytidine and adenine base editing. Notably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility and DNA specificity.

CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

Science.gov (United States)

Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

2016-07-15

CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

Production of Purified CasRNPs for Efficacious Genome Editing.

Science.gov (United States)

Lingeman, Emily; Jeans, Chris; Corn, Jacob E

2017-10-02

CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Science.gov (United States)

Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

2015-07-23

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

CRISPR/Cas9 Genome Editing: A Promising Tool for Therapeutic Applications of Induced Pluripotent Stem Cells.

Science.gov (United States)

Zhang, Yanli; Sastre, Danuta; Wang, Feng

2018-01-01

Induced pluripotent stem cells hold tremendous potential for biological and therapeutic applications. The development of efficient technologies for targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. The revolutionary technology for genome editing known as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system is recently recognized as a powerful tool for editing DNA at specific loci. The ease of use of the CRISPR-Cas9 technology will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. More recently, this system was modified to repress (CRISPR interference, CRISPRi) or activate (CRISPR activation, CRISPRa) gene expression without alterations in the DNA, which amplified the scope of applications of CRISPR systems for stem cell biology. Here, we highlight latest advances of CRISPR-associated applications in human pluripotent stem cells. The challenges and future prospects of CRISPR-based systems for human research are also discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Une façon plus sécuritaire de combattre le paludisme au Mexique ...

International Development Research Centre (IDRC) Digital Library (Canada)

18 oct. 2010 ... Le nombre de cas de paludisme a dégringolé au Mexique — de 25 023 en 1998 (un sommet) à 4 895 en 2001, puis à 1 985 en 2008. Le pays y est arrivé sans recourir au DDT. Aux termes de l'Accord de libre-échange nord‑américain, le Mexique s'était engagé à éliminer graduellement l'utilisation du DDT ...

[CRISPR/Cas system for genome editing in pluripotent stem cells].

Science.gov (United States)

Vasil'eva, E A; Melino, D; Barlev, N A

2015-01-01

Genome editing systems based on site-specific nucleases became very popular for genome editing in modern bioengineering. Human pluripotent stem cells provide a unique platform for genes function study, disease modeling, and drugs testing. Consequently, technology for fast, accurate and well controlled genome manipulation is required. CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated) system could be employed for these purposes. This system is based on site-specific programmable nuclease Cas9. Numerous advantages of the CRISPR/Cas system and its successful application to human stem cells provide wide opportunities for genome therapy and regeneration medicine. In this publication, we describe and compare the main genome editing systems based on site-specific programmable nucleases and discuss opportunities and perspectives of the CRISPR/Cas system for application to pluripotent stem cells.

Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9.

Science.gov (United States)

Ren, Jiangtao; Zhao, Yangbing

2017-09-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR) T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.

Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9

Directory of Open Access Journals (Sweden)

Jiangtao Ren

2017-04-01

Full Text Available ABSTRACT The clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated 9 (CRISPR/Cas9 system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.

Disabling Cas9 by an anti-CRISPR DNA mimic.

Science.gov (United States)

Shin, Jiyung; Jiang, Fuguo; Liu, Jun-Jie; Bray, Nicolas L; Rauch, Benjamin J; Baik, Seung Hyun; Nogales, Eva; Bondy-Denomy, Joseph; Corn, Jacob E; Doudna, Jennifer A

2017-07-01

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

Oncogenic Human Papillomavirus: Application of CRISPR/Cas9 Therapeutic Strategies for Cervical Cancer

Directory of Open Access Journals (Sweden)

Shuai Zhen

2017-12-01

Full Text Available Oncogenic human papillomaviruses (HPVs cause different types of cancer especially cervical cancer. HPV-associated carcinogenesis provides a classical model system for clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 based cancer therapies since the viral oncogenes E6 and E7 are exclusively expressed in cancerous cells. Sequence-specific gene knockdown/knockout using CRISPR/Cas9 shows promise as a novel therapeutic approach for the treatment of a variety of diseases that currently lack effective treatments. However, CRISPR/Cas9-based targeting therapy requires further validation of its efficacy in vitro and in vivo to eliminate the potential off-target effects, necessitates verification of the delivery vehicles and the combinatory use of conventional therapies with CRISPR/Cas9 to ensure the feasibility and safety. In this review we discuss the potential of combining CRISPR/Cas9 with other treatment options as therapies for oncogenic HPVs-associated carcinogenesis. and present our assessment of the promising path to the development of CRISPR/Cas9 therapeutic strategies for clinical settings.

Genre et dynamique organisationnelle au Moyen Atlas : cas du douar Maâmar dans la région de Khénifra

Directory of Open Access Journals (Sweden)

Fatima Zahid

2014-03-01

Full Text Available Le droit au développement semble animer tous les efforts visant à promouvoir la montagne marocaine, dans toutes ses dimensions politiques, sociales, organisationnelles à travers des projets de développement dont le plus réputé au Moyen Atlas est le projet MEDA-Khénifra. Ses actions diverses et ambitieuses ont façonné un douar de la région par de nouvelles organisations qui ont impacté les relations sociales et en particulier les relations du genre. Nous analysons à travers ce cas l’impact des projets de développement sur les relations du genre et l’empowerment des femmes dans cet espace. Les enquêtes ont été menées sous forme de questionnaires et d’entretiens collectifs. Le projet a créé deux organisations formelles : une association de développement et une coopérative pour la valorisation des plantes aromatiques et médicinales. Les femmes n’ont pas été impliquées dans ces organisations et certaines d’entre elles ont été invitées à participer à deux groupements informels. Le premier, pour la production de plantes aromatiques et médicinales, est resté sous la tutelle des organisations formelles gérées par les hommes. L’autre, dédié à l’apiculture, est plus autonome et a mieux réussi. Par ailleurs, la montée en puissance des organisations formelles a conduit à une moindre capacité d’action des femmes au niveau du village, car elles n’ont plus sur ces organisations les droits qu’elles ont traditionnellement dans le cadre de la jmâa. Cette expérience montre qu’un projet de développement peut influencer négativement les relations de genre malgré les bonnes volontés. La réflexion sur la question de genre doit alors impérativement animer l’ensemble des actions des projets de développement.

Sulfonamide inhibition studies of two β-carbonic anhydrases from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2.

Science.gov (United States)

Vullo, Daniela; Lehneck, Ronny; Pöggeler, Stefanie; Supuran, Claudiu T

2018-12-01

The two β-carbonic anhydrases (CAs, EC 4.2.1.1) recently cloned and purified from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2, were investigated for their inhibition with a panel of 39 aromatic, heterocyclic, and aliphatic sulfonamides and one sulfamate, many of which are clinically used agents. CAS1 was efficiently inhibited by tosylamide, 3-fluorosulfanilamide, and 3-chlorosulfanilamide (K I s in the range of 43.2-79.6 nM), whereas acetazolamide, methazolamide, topiramate, ethoxzolamide, dorzolamide, and brinzolamide were medium potency inhibitors (K I s in the range of 360-445 nM). CAS2 was less sensitive to sulfonamide inhibitors. The best CAS2 inhibitors were 5-amino-1,3,4-thiadiazole-2-sulfonamide (the deacetylated acetazolamide precursor) and 4-hydroxymethyl-benzenesulfonamide, with K I s in the range of 48.1-92.5 nM. Acetazolamide, dorzolamide, ethoxzolamide, topiramate, sulpiride, indisulam, celecoxib, and sulthiame were medium potency CAS2 inhibitors (K I s of 143-857 nM). Many other sulfonamides showed affinities in the high micromolar range or were ineffective as CAS1/2 inhibitors. Small changes in the structure of the inhibitor led to important differences of the activity. As these enzymes may show applications for the removal of anthropically generated polluting gases, finding modulators of their activity may be crucial for designing environmental-friendly CO 2 capture processes.

CRISPR-cas System as a Genome Engineering Platform: Applications in Biomedicine and Biotechnology.

Science.gov (United States)

Hashemi, Atieh

2018-01-01

Genome editing mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated proteins (Cas) has recently been considered to be used as efficient, rapid and site-specific tool in the modification of endogenous genes in biomedically important cell types and whole organisms. It has become a predictable and precise method of choice for genome engineering by specifying a 20-nt targeting sequence within its guide RNA. Firstly, this review aims to describe the biology of CRISPR system. Next, the applications of CRISPR-Cas9 in various ways, such as efficient generation of a wide variety of biomedically important cellular models as well as those of animals, modifying epigenomes, conducting genome-wide screens, gene therapy, labelling specific genomic loci in living cells, metabolic engineering of yeast and bacteria and endogenous gene expression regulation by an altered version of this system were reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Les séquelles de brûlures cervicales: aspects épidémiologique, clinique et thérapeutique au Maroc

Science.gov (United States)

Rafik, Amine; Chabak, Hakim; Diouri, Mounia; Bahechar, Naïma; Chlihi, Abdessamad

2015-01-01

Les séquelles de brûlures cervicales représentent une entité fréquente des séquelles de brûlure, elles affectent la fonction, l'esthétique et l’état psychologique des patients et peuvent être de traitement difficile. Il s'agit d'une étude rétrospective étalée sur 5 ans de Mars 2009 au Octobre2014, réalisée au centre national des brûlés et de chirurgie plastique au CHU Ibn Rochd Casablanca. Nous avons analysé les caractéristiques épidémiologiqueset cliniques ainsi que les indications et les résultats thérapeutiques chez 300 patients présentant des rétractions cervicales post-brûlure, suivis dans notre formation. Les jeunes femmes étaient le plus souvent touchées (56%). la brûlure thermique par flamme de butane dans le cadre d'accident domestique était l’étiologie la plus fréquente (91%).75% des patients ont été pris en charge dans un délai de 18 mois après avoir présenté une incapacité fonctionnelle. Les brides cervicales modérées et sévères sont les plus fréquentes et représentent respectivement 60% et 16% des cas. Le traitement chirurgical a fait appel aux greffes cutanées dans 67%des cas, aux plasties locales dans 24%des cas et aux lambeaux dans 24% des cas, les résultats sont jugés bons dans 75%des cas et moyens dans 18% des cas, tandis que les cas restants (7%) ont nécessité une reprise chirurgicale. Le traitement des brides cervicales doit être associé à un programme de rééducation adapté, afin d'assurer la pérennité des résultats fonctionnels et esthétiques. PMID:26301017

The CAS Classroom

Science.gov (United States)

Garner, Sue

2004-01-01

The Victorian Curriculum and Assessment Authority (VCAA) Computer Algebra System (CAS)Pilot study (2001-2005) is monitoring the use of CAS in senior secondary mathematics. This article explores the author's experiences in the CAS classroom and delineates changes in teaching style, as a result of the introduction of CAS into the senior mathematics…

CRISPR/Cas9—Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development

Science.gov (United States)

Okoli, Arinze; Okeke, Malachy I.; Tryland, Morten; Moens, Ugo

2018-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them. PMID:29361752

Prévenir les maladies liées au virus Zika avec de nouvelles ...

International Development Research Centre (IDRC) Digital Library (Canada)

Ces dernières années, c'est en Amérique latine qu'on a observé le nombre le plus élevé de cas de dengue, soit au Brésil, en Colombie et au Mexique. Ces pays ont aussi été confrontés à des épidémies de chikungunya (2014-2015) et du virus Zika (2015-2016). Les trois infections sont transmises par le même vecteur de ...

Points névralgiques au regard des changements climatiques ...

International Development Research Centre (IDRC) Digital Library (Canada)

28 oct. 2016 ... Ce rapport se base sur une recension des écrits et des études de cas pour combler le vide au sujet des travaux de recherche réalisés par des consortiums. Photo collage des jeunes chercheurs de l'IRCAAA. Cette série d'articles porte sur les expériences de jeunes chercheurs travaillant dans le cadre de l' ...

CRISPR/Cas9: Transcending the Reality of Genome Editing.

Science.gov (United States)

Chira, Sergiu; Gulei, Diana; Hajitou, Amin; Zimta, Alina-Andreea; Cordelier, Pierre; Berindan-Neagoe, Ioana

2017-06-16

With the expansion of the microbiology field of research, a new genome editing tool arises from the biology of bacteria that holds the promise of achieving precise modifications in the genome with a simplicity and versatility that surpasses previous genome editing methods. This new technique, commonly named CRISPR/Cas9, led to a rapid expansion of the biomedical field; more specifically, cancer characterization and modeling have benefitted greatly from the genome editing capabilities of CRISPR/Cas9. In this paper, we briefly summarize recent improvements in CRISPR/Cas9 design meant to overcome the limitations that have arisen from the nuclease activity of Cas9 and the influence of this technology in cancer research. In addition, we present challenges that might impede the clinical applicability of CRISPR/Cas9 for cancer therapy and highlight future directions for designing CRISPR/Cas9 delivery systems that might prove useful for cancer therapeutics. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice

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Oscar Meca-Cortés

2017-09-01

Full Text Available The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs, using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene, under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells. Keywords: CRISPR/Cas9, cell therapy, mesenchymal stem cells, bystander suicide therapy, glioblastoma, non-invasive bioluminescence imaging, CRISPR/Cas9, CRISPR/Cas9 knockin

Applications of CRISPR/Cas9 in the Mammalian Central Nervous System.

Science.gov (United States)

Savell, Katherine E; Day, Jeremy J

2017-12-01

Within the central nervous system, gene regulatory mechanisms are crucial regulators of cellular development and function, and dysregulation of these systems is commonly observed in major neuropsychiatric and neurological disorders. However, due to a lack of tools to specifically modulate the genome and epigenome in the central nervous system, many molecular and genetic mechanisms underlying cognitive function and behavior are still unknown. Although genome editing tools have been around for decades, the recent emergence of inexpensive, straightforward, and widely accessible CRISPR/Cas9 systems has led to a revolution in gene editing. The development of the catalytically dead Cas9 (dCas9) expanded this flexibility even further by acting as an anchoring system for fused effector proteins, structural scaffolds, and RNAs. Together, these advances have enabled robust, modular approaches for specific targeting and modification of the local chromatin environment at a single gene. This review highlights these advancements and how the combination of powerful modulatory tools paired with the versatility of CRISPR-Cas9-based systems offer great potential for understanding the underlying genetic and epigenetic contributions of neuronal function, behavior, and neurobiological diseases.

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

Science.gov (United States)

Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

2014-05-06

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Formation of multifunctional Fe3O4/Au composite nanoparticles for dual-mode MR/CT imaging applications

International Nuclear Information System (INIS)

Hu Yong; Li Jing-Chao; Shen Ming-Wu; Shi Xiang-Yang

2014-01-01

Recent advances with iron oxide/gold (Fe 3 O 4 /Au) composite nanoparticles (CNPs) in dual-modality magnetic resonance (MR) and computed tomography (CT) imaging applications are reviewed. The synthesis and assembly of “dumbbelllike” and “core/shell” Fe 3 O 4 /Au CNPs is introduced. Potential applications of some developed Fe 3 O 4 /Au CNPs as contrast agents for dual-mode MR/CT imaging applications are described in detail. (topical review - magnetism, magnetic materials, and interdisciplinary research)

Droits des femmes et accès à la terre au Sénégal : une citoyenneté ...

International Development Research Centre (IDRC) Digital Library (Canada)

Incidence de l'accès des femmes au foncier dans le développement local : le cas des femmes du GIE Seydy Ababacar Sy de Ross Bethio dans la vallée du fleuve Sénégal. Download PDF. Papers. Plaquette de présentation du projet Droits des femmes et accès au foncier : une citoyenneté à conquérir. Download PDF.

Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

Science.gov (United States)

Lee, Ciaran M; Davis, Timothy H; Bao, Gang

2018-04-01

What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

Science.gov (United States)

Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

2016-03-18

The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

OpenAIRE

Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

2016-01-01

The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutation...

Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response.

Science.gov (United States)

Heler, Robert; Wright, Addison V; Vucelja, Marija; Bikard, David; Doudna, Jennifer A; Marraffini, Luciano A

2017-01-05

CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application. Copyright © 2017 Elsevier Inc. All rights reserved.

[Advances in CRISPR-Cas-mediated genome editing system in plants].

Science.gov (United States)

Wang, Chun; Wang, Kejian

2017-10-25

Targeted genome editing technology is an important tool to study the function of genes and to modify organisms at the genetic level. Recently, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system has emerged as an efficient tool for specific genome editing in animals and plants. CRISPR-Cas system uses CRISPR-associated endonuclease and a guide RNA to generate double-strand breaks at the target DNA site, subsequently leading to genetic modifications. CRISPR-Cas system has received widespread attention for manipulating the genomes with simple, easy and high specificity. This review summarizes recent advances of diverse applications of the CRISPR-Cas toolkit in plant research and crop breeding, including expanding the range of genome editing, precise editing of a target base, and efficient DNA-free genome editing technology. This review also discusses the potential challenges and application prospect in the future, and provides a useful reference for researchers who are interested in this field.

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

NARCIS (Netherlands)

Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke F.; Vrouwe, Valentijn; Finger Bou, Max; Naduthodi, Mihris I.S.; Gussak, Alex; Brinkman, Rudolf B.L.; Kranenburg, Van Richard; Oost, Van Der John

2017-01-01

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

DEFF Research Database (Denmark)

Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke Fenna

2017-01-01

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including...

L'amenagement du territoire au Benin : Les rendez-vous manques ...

African Journals Online (AJOL)

Le leadership est une variable-clé de l'aménagement du territoire. L'histoire, à travers l'expérience de certains pays, en donne la démonstration. Le cas certainement le plus illustratif est celui de la France au sortir de la Seconde guerre mondiale. La « politique de reconstruction de la France libérée » a connu son ...

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

Science.gov (United States)

Seamon, Kyle J; Light, Yooli K; Saada, Edwin A; Schoeniger, Joseph S; Harmon, Brooke

2018-06-05

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

Directory of Open Access Journals (Sweden)

Toshitsugu Fujita

2015-09-01

Full Text Available Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE proteins and the clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR-associated proteins (Cas (CRISPR/Cas system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

Apprentissage administratif : L'apprentissage au CERN

CERN Multimedia

2004-01-01

APPRENTISSAGE ADMINISTRATIF FORMATION ET DEVELOPPEMENT HR/PMD/RCC L'APPRENTISSAGE AU CERN pour les professions d'employé(e) de commerce et d'assistant(e) en information documentaire L'apprentissage au CERN est régi par les lois, règlements et contrats en vigueur dans le Canton de Genève. En cas de réussite à l'examen de fin d'apprentissage, les apprentis obtiennent le Certificat Fédéral de Capacité Suisse (CFC). 1 place est offerte pour la formation d'employé(e) de commerce 2 places sont offertes pour la formation d'assistant(e) en information documentaire L'apprentissage dure 3 ans. Minima requis pour faire acte de candidature : • avoir terminé la scolarité obligatoire • être ressortissant d'un pays membre du CERN (Allemagne, Autriche, Belgique, Bulgarie, Danemark, Espagne,Finlande, France, Grèce, Hongrie, Italie, Norvège...

CRISPR-Cas9 for in vivo Gene Therapy: Promise and Hurdles

Directory of Open Access Journals (Sweden)

Wei-Jing Dai

2016-01-01

Full Text Available Owing to its easy-to-use and multiplexing nature, the genome editing tool CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR associated nuclease 9 is revolutionizing many areas of medical research and one of the most amazing areas is its gene therapy potentials. Previous explorations into the therapeutic potentials of CRISPR-Cas9 were mainly conducted in vitro or in animal germlines, the translatability of which, however, is either limited (to tissues with adult stem cells amenable to culture and manipulation or currently impermissible (due to ethic concerns. Recently, important progresses have been made on this regard. Several studies have demonstrated the ability of CRISPR-Cas9 for in vivo gene therapy in adult rodent models of human genetic diseases delivered by methods that are potentially translatable to human use. Although these recent advances represent a significant step forward to the eventual application of CRISPR-Cas9 to the clinic, there are still many hurdles to overcome, such as the off-target effects of CRISPR-Cas9, efficacy of homology-directed repair, fitness of edited cells, immunogenicity of therapeutic CRISPR-Cas9 components, as well as efficiency, specificity, and translatability of in vivo delivery methods. In this article, we introduce the mechanisms and merits of CRISPR-Cas9 in genome editing, briefly retrospect the applications of CRISPR-Cas9 in gene therapy explorations and highlight recent advances, later we discuss in detail the challenges lying ahead in the way of its translatability, propose possible solutions, and future research directions.

Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.

Science.gov (United States)

Hirano, Seiichi; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Nureki, Osamu

2016-03-17

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9. Copyright © 2016 Elsevier Inc. All rights reserved.

Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

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Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

2017-11-01

CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Applications of Alternative Nucleases in the Age of CRISPR/Cas9

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Tuhin K. Guha

2017-11-01

Full Text Available Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, meganucleases (MNs, and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR associated proteins (including Cas9 have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks. No single genome-editing nuclease is optimized for all possible applications. Here, we focus on the diversity of nuclease domains available for genome editing, highlighting biochemical properties and the potential applications that are best suited to each domain.

Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

Science.gov (United States)

Fagerlund, Robert D; Wilkinson, Max E; Klykov, Oleg; Barendregt, Arjan; Pearce, F Grant; Kieper, Sebastian N; Maxwell, Howard W R; Capolupo, Angela; Heck, Albert J R; Krause, Kurt L; Bostina, Mihnea; Scheltema, Richard A; Staals, Raymond H J; Fineran, Peter C

2017-06-27

CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas1 4 -Cas2-3 2 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.

Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: A brief overview.

Science.gov (United States)

Karvelis, Tautvydas; Gasiunas, Giedrius; Siksnys, Virginijus

2017-05-15

Recently the Cas9, an RNA guided DNA endonuclease, emerged as a powerful tool for targeted genome manipulations. Cas9 protein can be reprogrammed to cleave, bind or nick any DNA target by simply changing crRNA sequence, however a short nucleotide sequence, termed PAM, is required to initiate crRNA hybridization to the DNA target. PAM sequence is recognized by Cas9 protein and must be determined experimentally for each Cas9 variant. Exploration of Cas9 orthologs could offer a diversity of PAM sequences and novel biochemical properties that may be beneficial for genome editing applications. Here we briefly review and compare Cas9 PAM identification assays that can be adopted for other PAM-dependent CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

Shape and size controlled synthesis of Au nanorods: H2S gas-sensing characterizations and antibacterial application

International Nuclear Information System (INIS)

Lanh, Le Thi; Hoa, Tran Thai; Cuong, Nguyen Duc; Khieu, Dinh Quang; Quang, Duong Tuan; Van Duy, Nguyen; Hoa, Nguyen Duc; Van Hieu, Nguyen

2015-01-01

Highlights: • We have demonstrated a facile method to prepare colloid Au nanorods. • The size and shape of Au nanorods can be controlled via seed-mediated growth method. • The H 2 S gas-sensing properties have been investigated. • The antibacterial application has been conducted. - Abstract: Controlling their size and shape is one of the important issues in the fundamental study and application of colloidal metal nanoparticles. In the current study, different sizes and shapes of Au nanorods were fabricated using a seed-mediated growth method. Material characterization by X-ray diffraction and transmission electron microscopy revealed that the obtained products were made of single-crystal Au nanorods with an average diameter and length of 10 nm and 40 nm, respectively. The Au nanorod-based sensor exhibited significantly high sensitivity and fast response/recovery time to low concentrations (2.5–10 ppm) of H 2 S at temperatures ranging from 300 °C to 400 °C. Additionally, they exhibited antibacterial effect at low concentration. These results suggested that the fabricated Au nanorods have excellent potential for practical application in air pollution monitoring and biomedicine

Cartographie et contrôle au Maroc sous le Protectorat espagnol (1912-1956

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José Luis Villanova

2010-06-01

Full Text Available La principale fonction des interventores (contrôleurs du Protectorat espagnol au Maroc entre 1912 et 1956 a consisté à surveiller l’action des autorités tribales marocaines pour asseoir la présence coloniale. Cependant, le Haut-Commissariat d’Espagne au Maroc leur a souvent demandé de préparer des rapports d’ordre économique, social ou à finalité militaire sur les tribus, généralement mal connues de l’autorité coloniale. Ces documents contiennent des cartes de qualité variée qui ont pu constituer un instrument précieux de contrôle et, dans certains cas, un outil de propagande au service de l’action coloniale espagnole.

Diagnostic ?tiologique du diab?te insipide central: ? propos de 41 cas

OpenAIRE

Chaker, Fatma; Chihaoui, Melika; Yazidi, Meriem; Slimane, Hedia

2016-01-01

La survenue d'un syndrome polyuro-polydipsiqueavec des urines hypotoniques n?cessite une strat?gie diagnostique rigoureuse. Le but de cette ?tude ?tait d??tudier les modalit?s de diagnostic du diab?te insipide central. A travers une ?tude r?trospective de 41 cas de diab?te insipide central(DIC) collig?s au service d'Endocrinologie ? l'h?pital de la Rabta de Tunis, allant de l'ann?e 1990 ? l'an 2013, nous avons relev? les circonstances de d?couverte du DIC, les anomalies du bilan ant?-hypophys...

Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities.

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Li, Ling; Hu, Shuo; Chen, Xiaoyuan

2018-07-01

In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed. Published by Elsevier Ltd.

CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity.

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Barrangou, Rodolphe; Marraffini, Luciano A

2014-04-24

Clustered regularly interspaced short palindromic repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing and can be repurposed for numerous DNA targeting applications including transcriptional control. Copyright © 2014 Elsevier Inc. All rights reserved.

CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity

Science.gov (United States)

Barrangou, Rodolphe; Marraffini, Luciano A.

2014-01-01

Summary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing, and can be repurposed for numerous DNA targeting applications including transcriptional control. PMID:24766887

CRISPR/Cas9-mediated noncoding RNA editing in human cancers.

Science.gov (United States)

Yang, Jie; Meng, Xiaodan; Pan, Jinchang; Jiang, Nan; Zhou, Chengwei; Wu, Zhenhua; Gong, Zhaohui

2018-01-02

Cancer is characterized by multiple genetic and epigenetic alterations, including a higher prevalence of mutations of oncogenes and/or tumor suppressors. Mounting evidences have shown that noncoding RNAs (ncRNAs) are involved in the epigenetic regulation of cancer genes and their associated pathways. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) system, a revolutionary genome-editing technology, has shed light on ncRNA-based cancer therapy. Here, we briefly introduce the classifications and mechanisms of CRISPR/Cas9 system. Importantly, we mainly focused on the applications of CRISPR/Cas9 system as a molecular tool for ncRNA (microRNA, long noncoding RNA and circular RNA, etc.) editing in human cancers, and the novel techniques that are based on CRISPR/Cas9 system. Additionally, the off-target effects and the corresponding solutions as well as the challenges toward CRISPR/Cas9 were also evaluated and discussed. Long- and short-ncRNAs have been employed as targets in precision oncology, and CRISPR/Cas9-mediated ncRNA editing may provide an excellent way to cure cancer.

Observation of Au + Au → Au + Au + ρ0 and Au + Au → Au* + Au* + ρ0 with STAR

International Nuclear Information System (INIS)

Spencer, K.

2002-01-01

First observation of the reactions Au + Au → Au + Au + ρ 0 and Au + Au → Au* + Au* + ρ 0 with the STAR detector are reported. The ρ are produced at small perpendicular momentum, as expected if they couple coherently to both nuclei. Models of vector meson production and the correlation with nuclear breakup are discussed, as well as a fundamental test of quantum mechanics that is possible with the system. (author)

Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.

Science.gov (United States)

Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Nguyen, Nhu T; Topkar, Ved V; Zheng, Zongli; Joung, J Keith

2015-12-01

CRISPR-Cas9 nucleases target specific DNA sequences using a guide RNA but also require recognition of a protospacer adjacent motif (PAM) by the Cas9 protein. Although longer PAMs can potentially improve the specificity of genome editing, they limit the range of sequences that Cas9 orthologs can target. One potential strategy to relieve this restriction is to relax the PAM recognition specificity of Cas9. Here we used molecular evolution to modify the NNGRRT PAM of Staphylococcus aureus Cas9 (SaCas9). One variant we identified, referred to as KKH SaCas9, showed robust genome editing activities at endogenous human target sites with NNNRRT PAMs, thereby increasing SaCas9 targeting range by two- to fourfold. Using GUIDE-seq, we show that wild-type and KKH SaCas9 induce comparable numbers of off-target effects in human cells. Our strategy for evolving PAM specificity does not require structural information and therefore should be applicable to a wide range of Cas9 orthologs.

Cas9, Cpf1 and C2c1/2/3-What's next?

Science.gov (United States)

Nakade, Shota; Yamamoto, Takashi; Sakuma, Tetsushi

2017-05-04

Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012-2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence. Although Streptococcus pyogenes Cas9 (SpCas9), one of the Cas9 proteins that applications in genome engineering were first demonstrated, still has wide usage because of its high nuclease activity and broad targeting range, there are several limitations such as large molecular weight and potential off-target effect. In this commentary, we describe various improvements and alternatives of CRISPR-Cas systems, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9. These variations enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond.

ÉTUDE DE CAS – Kenya : Paludisme et agriculture au Kenya ...

International Development Research Centre (IDRC) Digital Library (Canada)

11 janv. 2011 ... Dans le miroitement du chaud soleil de midi, il reste plutôt à l'ombre d'un arbre à discuter du plus grave problème de santé qui frappe son village : le ... au Kenya, des chercheurs du Centre international de recherche sur la physiologie des insectes et l'écologie (ICIPE) et de l'Institut international de gestion ...

CAS Accelerators for Medical Applications in Vösendorf, Austria

CERN Multimedia

CERN Accelerator School

2015-01-01

The CERN Accelerator School (CAS) and MedAustron jointly organised a course on Accelerators for Medical Applications in Vösendorf, Austria between 26 May and 5 June 2015. The course was held at the Eventhotel Pyramide on the outskirts of Vienna, and was attended by 76 participants from 29 countries, coming from as far away as Canada, China, Lithuania, Thailand, Ukraine and Russia.       The intensive programme comprised 37 lectures. The emphasis was on using charged particle beams for cancer therapy and the programme began by covering the way in which particles interact with biological material, how this translates into the dose needed for treatment and how this dose is best delivered. The different accelerator options for providing the particles needed were then presented in some detail. The production of radioisotopes and how these are used for diagnostics and therapy was also covered, together with a look at novel acceleration techniques that may play a role i...

CRISPR/Cas9: A Practical Approach in Date Palm Genome Editing

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Muhammad N. Sattar

2017-08-01

Full Text Available The genetic modifications through breeding of crop plants have long been used to improve the yield and quality. However, precise genome editing (GE could be a very useful supplementary tool for improvement of crop plants by targeted genome modifications. Various GE techniques including ZFNs (zinc finger nucleases, TALENs (transcription activator-like effector nucleases, and most recently clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 (CRISPR-associated protein 9-based approaches have been successfully employed for various crop plants including fruit trees. CRISPR/Cas9-based approaches hold great potential in GE due to their simplicity, competency, and versatility over other GE techniques. However, to the best of our knowledge no such genetic improvement has ever been developed in date palm—an important fruit crop in Oasis agriculture. The applications of CRISPR/Cas9 can be a challenging task in date palm GE due to its large and complex genome, high rate of heterozygosity and outcrossing, in vitro regeneration and screening of mutants, high frequency of single-nucleotide polymorphism in the genome and ultimately genetic instability. In this review, we addressed the potential application of CRISPR/Cas9-based approaches in date palm GE to improve the sustainable date palm production. The availability of the date palm whole genome sequence has made it feasible to use CRISPR/Cas9 GE approach for genetic improvement in this species. Moreover, the future prospects of GE application in date palm are also addressed in this review.

Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

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Xiao-Hui Zhang

2015-01-01

Full Text Available CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%—RGEN (RNA-guided endonuclease-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology.

L’érysipèle sur cicatrice post opératoire en traumatologie: à propos d'un cas et revue de la littérature

Science.gov (United States)

Abid, Hatim; El Idrissi, Mohamed; Shimi, Mohamed; El Ibrahimi, Abdelhalim; El Mrini, Abdelmajid; Amraoui, Nissrine; Mernissi, Fatima Zohra

2015-01-01

L’érysipèle sur cicatrice post opératoire est une entité rare, décrite principalement chez des patients présentant les facteurs de risque classiques de la maladie au niveau des sites de veinectomie pour pontage coronarien. En traumatologie orthopédie, nous n'avons que les 3 cas rapportés dans le travail de Dhrif survenus au décours d'une implantation prothétique chez des malades à risque. Nous présentons à travers cet article, le cas d'un érysipèle post opératoire sur une cicatrice d'ostéosynthèse d'une fracture fermée du pilon tibial, ayant la particularité du terrain et des circonstances de survenues, pour enfin conclure aux principes de la prévention primaire à adopter. PMID:26401204

Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

Science.gov (United States)

van der Els, Simon; James, Jennelle K; Kleerebezem, Michiel; Bron, Peter A

2018-04-15

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN -targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis , while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria. IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the

Phage Genetic Engineering Using CRISPR–Cas Systems

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Asma Hatoum-Aslan

2018-06-01

Full Text Available Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR–Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural targets of CRISPR–Cas immunity, the viruses that specifically infect bacteria, or phages. Despite their status as the most abundant biological entities on the planet, the majority of phage genes have unassigned functions. This reality underscores the need for robust genetic tools to study them. Recent reports have demonstrated that CRISPR–Cas systems, specifically the three major types (I, II, and III, can be harnessed to genetically engineer phages that infect diverse hosts. Here, the mechanisms of each of these systems, specific strategies used, and phage editing efficacies will be reviewed. Due to the relatively wide distribution of CRISPR–Cas systems across bacteria and archaea, it is anticipated that these immune systems will provide generally applicable tools that will advance the mechanistic understanding of prokaryotic viruses and accelerate the development of novel technologies based on these ubiquitous organisms.

Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.

Science.gov (United States)

Shao, Yanjiao; Wang, Liren; Guo, Nana; Wang, Shengfei; Yang, Lei; Li, Yajing; Wang, Mingsong; Yin, Shuming; Han, Honghui; Zeng, Li; Zhang, Ludi; Hui, Lijian; Ding, Qiurong; Zhang, Jiqin; Geng, Hongquan; Liu, Mingyao; Li, Dali

2018-05-04

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Shape and size controlled synthesis of Au nanorods: H{sub 2}S gas-sensing characterizations and antibacterial application

Energy Technology Data Exchange (ETDEWEB)

Lanh, Le Thi [College of Sciences, Hue University, 77 Nguyen Hue, Hue City (Viet Nam); Hoa, Tran Thai, E-mail: trthaihoa@yahoo.com [College of Sciences, Hue University, 77 Nguyen Hue, Hue City (Viet Nam); Cuong, Nguyen Duc [College of Sciences, Hue University, 77 Nguyen Hue, Hue City (Viet Nam); Faculty of Hospitality and Tourism, Hue University, 22 Lam Hoang, Hue City (Viet Nam); Khieu, Dinh Quang [College of Sciences, Hue University, 77 Nguyen Hue, Hue City (Viet Nam); Quang, Duong Tuan [College of Education, Hue University, 34 Le Loi, Hue City (Viet Nam); Van Duy, Nguyen; Hoa, Nguyen Duc [International Training Institute for Materials Science, Hanoi University of Science and Technology, Hanoi (Viet Nam); Van Hieu, Nguyen, E-mail: hieu@itims.edu.vn [International Training Institute for Materials Science, Hanoi University of Science and Technology, Hanoi (Viet Nam)

2015-06-25

Highlights: • We have demonstrated a facile method to prepare colloid Au nanorods. • The size and shape of Au nanorods can be controlled via seed-mediated growth method. • The H{sub 2}S gas-sensing properties have been investigated. • The antibacterial application has been conducted. - Abstract: Controlling their size and shape is one of the important issues in the fundamental study and application of colloidal metal nanoparticles. In the current study, different sizes and shapes of Au nanorods were fabricated using a seed-mediated growth method. Material characterization by X-ray diffraction and transmission electron microscopy revealed that the obtained products were made of single-crystal Au nanorods with an average diameter and length of 10 nm and 40 nm, respectively. The Au nanorod-based sensor exhibited significantly high sensitivity and fast response/recovery time to low concentrations (2.5–10 ppm) of H{sub 2}S at temperatures ranging from 300 °C to 400 °C. Additionally, they exhibited antibacterial effect at low concentration. These results suggested that the fabricated Au nanorods have excellent potential for practical application in air pollution monitoring and biomedicine.

L’infarctus du myocarde du jeune adulte -Analyse rétrospective des cas colligés au CHU de Dakar

Directory of Open Access Journals (Sweden)

Nobila Valentin Yameogo

2010-09-01

Full Text Available Les données relatives à l’infarctus du myocarde chez le jeune adulte sont rares en Afrique noir. Nous rapportons une série rétrospective de 14 cas d’infarctus du myocarde chez l’adulte jeune noir africain. Pour analyser les caractéristiques épidémiologiques, cliniques, électriques, échographiques, biologiques, thérapeutiques et évolutives de l’infarctus aigu du myocarde chez le jeune adulte nous avons étudié de manière rétrospective les dossiers médicaux d’une série consécutive des patients admis entre Janvier 2003 et Décembre 2008 pour prise en charge d’infarctus aigu du myocarde. Quatre-vingt quatre (84 cas d’infarctus du myocarde ont été pris en charge durant la période d’étude ,14 patients (16,6% avaient un âge inférieur ou égal à 40 ans. L’âge moyen était de 34 +ou-5ans (extrêmes 27ans et 40 ans. Les facteurs de risque cardio-vasculaire étaient dominés par le sexe masculin (n=11, la dyslipidémie (n=7 et le tabagisme par cigarette (n=6. La contraception orale était absente chez le 1/3 des patients. Le délai moyen d’admission était tardif (15 plus ou moins 4 heures. L’IDM était antérieur dans la moitié des cas. L’acide acétylsalicylique, les inhibiteurs de l’enzyme de conversion Les bétabloquants et les statines étaient les médicaments les plus prescrits. La thrombolyse et l’angioplastie étaient respectivement réalisées chez 3 patients et 0 patient. 4 cas d’insuffisance cardiaque, 2 cas de bloc atrio-ventriculaire complet et 1 cas de décès étaient les principales complications. L’infarctus du myocarde concerne également le sujet jeune noir africain. Les facteurs de risque sont essentiellement représentés par le sexe, la dyslipidémie, le tabagisme et la contraception orale.

Ω and ϕ in Au + Au collisions at and 11.5 GeV from a multiphase transport model

Science.gov (United States)

Ye, Y. J.; Chen, J. H.; Ma, Y. G.; Zhang, S.; Zhong, C.

2017-08-01

Within the framework of a multiphase transport model, we study the production and properties of Ω and ϕ in Au + Au collisions with a new set of parameters for and with the original set of parameters for . The AMPT model with string melting provides a reasonable description at , while the default AMPT model describes the data well at . This indicates that the system created at top RHIC energy is dominated by partonic interactions, while hadronic interactions become important at lower beam energy, such as . The comparison of N(Ω++Ω-)/[2N(ϕ)] ratio between data and calculations further supports the argument. Our calculations can generally describe the data of nuclear modification factor as well as elliptic flow. Supported by National Natural Science Foundation of China (11421505, 11520101004, 11220101005, 11275250, 11322547), Major State Basic Research Development Program in China (2014CB845400, 2015CB856904) and Key Research Program of Frontier Sciences of CAS (QYZDJSSW-SLH002)

L’estime de soi : un cas particulier d’estime sociale ?

OpenAIRE

Santarelli, Matteo

2016-01-01

Un des traits plus originaux de la théorie intersubjective de la reconnaissance d’Axel Honneth, consiste dans la façon dont elle discute la relation entre estime sociale et estime de soi. En particulier, Honneth présente l’estime de soi comme un reflet de l’estime sociale au niveau individuel. Dans cet article, je discute cette conception, en posant la question suivante : l’estime de soi est-elle un cas particulier de l’estime sociale ? Pour ce faire, je me concentre sur deux problèmes crucia...

Genome editing: the road of CRISPR/Cas9 from bench to clinic

KAUST Repository

Eid, Ayman

2016-10-14

Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9). The CRISPR/Cas9 system\\'s simplicity, facile engineering and amenability to multiplexing make it the system of choice for many applications. CRISPR/Cas9 has been used to generate disease models to study genetic diseases. Improvements are urgently needed for various aspects of the CRISPR/Cas9 system, including the system\\'s precision, delivery and control over the outcome of the repair process. Here, we discuss the current status of genome engineering and its implications for the future of biological research and gene therapy.

Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

Science.gov (United States)

Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J. C.; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut

2017-01-01

Abstract The CRISPR–Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR–Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. PMID:28525578

RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

KAUST Repository

Baazim, Hatoon

2014-05-01

Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.

Au/ZnO nanoarchitectures with Au as both supporter and antenna of visible-light

Energy Technology Data Exchange (ETDEWEB)

Liu, Tianyu; Chen, Wei; Hua, Yuxiang; Liu, Xiaoheng, E-mail: xhliu@mail.njust.edu.cn

2017-01-15

Highlights: • An inversed Au/ZnO nanostructure was fabricated with ZnO loaded onto Au. • The Au/ZnO nanocomposites showed enhanced properties in visible-light photocatalysis. • The SPR effect of Au was considered important for visible-light photocatalysis. - Abstract: In this paper, we fabricate Au/ZnO nanostructure with smaller ZnO nanoparticles loaded onto bigger gold nanoparticles via combining seed-mediated method and sol-gel method. The obtained Au/ZnO nanocomposites exhibit excellent properties in photocatalysis process like methyl orange (MO) degradation and oxidative conversion of methanol into formaldehyde under visible light irradiation. The enhanced properties were ascribed to the surface plasmon resonance (SPR) effect of Au nanoparticles, which could contribute to the separation of photo-excited electrons and holes and facilitate the process of absorbing visible light. This paper contributes to the emergence of multi-functional nanocomposites with possible applications in visible-light driven photocatalysts and makes the Au/ZnO photocatalyst an exceptional choice for practical applications such as environmental purification of organic pollutants in aqueous solution and the synthesis of fine chemicals and intermediates.

Au/ZnO nanoarchitectures with Au as both supporter and antenna of visible-light

International Nuclear Information System (INIS)

Liu, Tianyu; Chen, Wei; Hua, Yuxiang; Liu, Xiaoheng

2017-01-01

Highlights: • An inversed Au/ZnO nanostructure was fabricated with ZnO loaded onto Au. • The Au/ZnO nanocomposites showed enhanced properties in visible-light photocatalysis. • The SPR effect of Au was considered important for visible-light photocatalysis. - Abstract: In this paper, we fabricate Au/ZnO nanostructure with smaller ZnO nanoparticles loaded onto bigger gold nanoparticles via combining seed-mediated method and sol-gel method. The obtained Au/ZnO nanocomposites exhibit excellent properties in photocatalysis process like methyl orange (MO) degradation and oxidative conversion of methanol into formaldehyde under visible light irradiation. The enhanced properties were ascribed to the surface plasmon resonance (SPR) effect of Au nanoparticles, which could contribute to the separation of photo-excited electrons and holes and facilitate the process of absorbing visible light. This paper contributes to the emergence of multi-functional nanocomposites with possible applications in visible-light driven photocatalysts and makes the Au/ZnO photocatalyst an exceptional choice for practical applications such as environmental purification of organic pollutants in aqueous solution and the synthesis of fine chemicals and intermediates.

L'APPRENTISSAGE AU CERN - French version only

CERN Multimedia

Laurence Van Cauter

2002-01-01

APPRENTISSAGE ADMINISTRATIF FORMATION ET DEVELOPPEMENT HR/TD/AP L'APPRENTISSAGE AU CERN pour les professions d'employé(e) de commerce et d'assistant(e) en information documentaire L'apprentissage au CERN est régi par les lois, règlements et contrats en vigueur dans le Canton de Genève. En cas de réussite à l'examen de fin d'apprentissage, les apprentis obtiennent le Certificat Fédéral de Capacité Suisse (CFC). 1 place est offerte pour la formation d'employé(e) de commerce 2 places sont offertes pour la formation d'assistant(e) en information documentaire L'apprentissage dure 3 ans. Minima requis pour faire acte de candidature : avoir terminé la scolarité obligatoire être ressortissant d'un pays membre du CERN (Allemagne, Autriche, Belgique, Bulgarie, Danemark, Espagne, Finlande, France, Grèce, Hongrie, Italie, Norvège, Pays-Bas, Pologne, Portug...

Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

Science.gov (United States)

Leenay, Ryan T; Maksimchuk, Kenneth R; Slotkowski, Rebecca A; Agrawal, Roma N; Gomaa, Ahmed A; Briner, Alexandra E; Barrangou, Rodolphe; Beisel, Chase L

2016-04-07

CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature. Copyright © 2016 Elsevier Inc. All rights reserved.

Profil hormonal chez l'homme en cas d'infertilité au laboratoire de ...

African Journals Online (AJOL)

... d'infertilité au laboratoire de radio immunologie de l'institut des radioisotopes de Niamey. ... Patients and methods: We included in our study, all patients presenting with infertility dating back at leasttwo years, with abnormal semen analysis.

Le témoignage de terrain d'une chercheuse candidate au doctorat ...

International Development Research Centre (IDRC) Digital Library (Canada)

15 mai 2017 ... Certaines d'entre elles ont été enlevées durant la guerre et d'autres ont ... que je comprenais quelque chose au phénomène que j'étudiais. Or, quand j'y retourne, je me rends compte que j'ignore encore tout du phénomène. ... ÉTUDE DE CAS — mondiale : Repenser les stratégies de recherche agricole.

Harnessing CRISPR-Cas systems for bacterial genome editing.

Science.gov (United States)

Selle, Kurt; Barrangou, Rodolphe

2015-04-01

Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

Application of CRISPR/Cas9 Genome Editing Technology for the Improvement of Crops Cultivated in Tropical Climates: Recent Progress, Prospects, and Challenges

Directory of Open Access Journals (Sweden)

Effi Haque

2018-05-01

Full Text Available The world population is expected to increase from 7.3 to 9.7 billion by 2050. Pest outbreak and increased abiotic stresses due to climate change pose a high risk to tropical crop production. Although conventional breeding techniques have significantly increased crop production and yield, new approaches are required to further improve crop production in order to meet the global growing demand for food. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9 (CRISPR-associated protein9 genome editing technology has shown great promise for quickly addressing emerging challenges in agriculture. It can be used to precisely modify genome sequence of any organism including plants to achieve the desired trait. Compared to other genome editing tools such as zinc finger nucleases (ZFNs and transcriptional activator-like effector nucleases (TALENs, CRISPR/Cas9 is faster, cheaper, precise and highly efficient in editing genomes even at the multiplex level. Application of CRISPR/Cas9 technology in editing the plant genome is emerging rapidly. The CRISPR/Cas9 is becoming a user-friendly tool for development of non-transgenic genome edited crop plants to counteract harmful effects from climate change and ensure future food security of increasing population in tropical countries. This review updates current knowledge and potentials of CRISPR/Cas9 for improvement of crops cultivated in tropical climates to gain resiliency against emerging pests and abiotic stresses.

Application of CRISPR/Cas9 Genome Editing Technology for the Improvement of Crops Cultivated in Tropical Climates: Recent Progress, Prospects, and Challenges.

Science.gov (United States)

Haque, Effi; Taniguchi, Hiroaki; Hassan, Md Mahmudul; Bhowmik, Pankaj; Karim, M Rezaul; Śmiech, Magdalena; Zhao, Kaijun; Rahman, Mahfuzur; Islam, Tofazzal

2018-01-01

The world population is expected to increase from 7.3 to 9.7 billion by 2050. Pest outbreak and increased abiotic stresses due to climate change pose a high risk to tropical crop production. Although conventional breeding techniques have significantly increased crop production and yield, new approaches are required to further improve crop production in order to meet the global growing demand for food. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 (CRISPR-associated protein9) genome editing technology has shown great promise for quickly addressing emerging challenges in agriculture. It can be used to precisely modify genome sequence of any organism including plants to achieve the desired trait. Compared to other genome editing tools such as zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs), CRISPR/Cas9 is faster, cheaper, precise and highly efficient in editing genomes even at the multiplex level. Application of CRISPR/Cas9 technology in editing the plant genome is emerging rapidly. The CRISPR/Cas9 is becoming a user-friendly tool for development of non-transgenic genome edited crop plants to counteract harmful effects from climate change and ensure future food security of increasing population in tropical countries. This review updates current knowledge and potentials of CRISPR/Cas9 for improvement of crops cultivated in tropical climates to gain resiliency against emerging pests and abiotic stresses.

Douleurs induites par les soins: la réalité au Centre Hospitalier Universitaire de Befelatanana Antananarivo, Madagascar

Science.gov (United States)

Mahavivola, Ernestho-Ghoud Indretsy; Olivah, Razanaparany Miarisoa Mireille; Mihary, Dodo; Hendriniaina, Rakotoharivelo; Lalao, Randriamboavonjy Rado; Henintsoa, Rakotonirainy Oliva; Fahafahantsoa, Rapelanoro Rabenja

2014-01-01

La douleur induite par les soins correspond à la douleur survenant lors des actes à visé diagnostique et/ou thérapeutique. A notre connaissance, nous n'avons pas encore des données disponibles pour les douleurs induites par les soins à l'Hôpital de Befelatanana. Nos objectifs étaient de décrire le profil épidémiologique de la douleur induite par les soins, d'identifier les principaux facteurs influençant sur l'intensité de la douleur et leurs retentissements chez les patients. Il s'agissait d'une étude rétrospective, transversale type un jour donné menée dans les douze services de Médecines au Centre Hospitalier Universitaire de Befelatanana en Novembre 2013. Cent deux patients ont été retenus dans l’étude et trois cent vingt trois actes douloureux étaient enregistrés. La fréquence de la douleur induite par les soins était de 69,86%. Le genre féminin prédominait dans 52% des cas (n = 53) avec un sex-ratio à 0,92. L’âge moyen était de 46 ans. Les ponctions vasculaires étaient l'acte prédominant dans 49,54% (n = 109) des cas. Les infirmiers réalisaient les soins dans 47,05% (n = 48) des cas. L'information verbale était la mesure préventive utilisée dans 57,84% des cas (n = 59). Le transport par marche à pied et au dos représentait 16,67% des cas (n = 17). Les patients naïfs des gestes étaient plus anxieux. Ces patients gardaient de mauvais souvenir dans 64,71% des cas (n = 66). La fréquence de douleur induite par les soins était trop élevée. Un effort important est nécessaire pour réduire la douleur induite par les soins PMID:25932071

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.

Science.gov (United States)

Huang, Mengqi; Zhou, Xiaoming; Wang, Huiying; Xing, Da

2018-02-06

A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Jakociunas, Tadas; Bonde, Ida; Herrgard, Markus

2015-01-01

CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces...... cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains...

Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

Science.gov (United States)

Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J C; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut; Saito, Hirohide

2017-07-27

The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Aspects epidemiologiques et cliniques des pemphigus au Senegal [Epidemiological and clinical aspects of pemphigus in Senegal

Directory of Open Access Journals (Sweden)

Moussa Diallo

2017-11-01

Full Text Available Background: The purpose of this study was to specify the epidemiological and clinical characteristics of pemphigus in the Department of Dermatology of the University Hospital of Dakar. Patients and Methodology: All medical records of patients seen for pemphigus in our department over a 11-year period were analyzed retrospectively. Results: We collected 105 cases of pemphigus, corresponding to a frequency of 10 cases/year and a hospital prevalence of 2.7%. Thus, pemphigus held the second rank of blistering autoimmune bullous diseases after bullous pemphigoid. The mean age of patients was 47.62 years and the age group [40-59 years] was predominantly affected (28 cases; 45 %. There were 41 femeles and 21 men (F/M ratio: 2. We did not find an endemic focus in our country. Clinical forms were dominated by pemphigus foliaceus (30 cases; 48.5 %, followed by pemphigus vulguaris (19 cases; 30.6 % and finally by pemphigus erythematosus (13 cases; 21%. In 16 cases (25 %, the pemphigus had begun after the consumption of a traditional medicinal plant (8 cases. An associated toxidermia was found in12 cases. Pruritus was present in 43 patients (69.35%. Discussion: In Senegal, pemphigus are common diseases, but without however an endemic focuc. They occur in young women, with a predominance of superficial forms, especially pemphigus foliaceus. The presence of pruritus and associated toxidermia to traditional medicinal plants are common. RÉSUMÉ Introduction: L’objectif de cette étude était de préciser les aspects épidémiologiques et cliniques des pemphigus au CHU de Dakar. Patients et Méthodes: Tous les dossiers des malades admis pour pemphigus ont été analysé de façon rétrospective sur une période de 11 ans. Résultats: Nous avons colligé 105 cas de pemphigus, correspondant à une fréquence de 10 cas/an et une prévalence hospitalière de 2,7 %. Les pemphigus occupaient le 2° rang des dermatoses bulleuses auto-immunes après la pemphigoide

Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

Directory of Open Access Journals (Sweden)

Wolf Yuri I

2011-07-01

Full Text Available Abstract Background The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III. Results A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs containing a distinct form of the RNA Recognition Motif (RRM domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Conclusions Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure

Colloidal Au-enhanced surface plasmon resonance imaging: application in a DNA hybridization process

International Nuclear Information System (INIS)

Manera, M G; Spadavecchia, J; Taurino, A; Rella, R

2010-01-01

The detection of the DNA hybridization mechanism using monodispersed gold nanoparticles as labels is an interesting alternative to increase the sensitivity of the SPR imaging technique. DNA-modified Au nanoparticles (DNA-Au NPs) containing single-stranded (ss) portions of DNA were prepared by monitoring their monolayer formation by UV–vis spectroscopy. The hybridization process between specific thio-oligonucleotides immobilized on the DNA–Au NPs and the corresponding complementary strands is reported and compared with the traditional hybridization process on properly self-assembled thin gold films deposited on glass substrates. A remarkable signal amplification is observed, following the incorporation of colloidal Au into a SPR biosensing experiment, resulting in an increased SPR response to DNA–DNA interactions. In particular Fusarium thiolated DNA (5'HS poly(T) 15 ATC CCT CAA AAA CTG CCG CT-3) and trichothecenes complementary DNA (5'-AGC GGC AGT TTT TGA GGG AT-3') sequences have been explored due to their possible application to agro-industry for the control of food quality

TiAu-based micro-calorimeters for space applications

International Nuclear Information System (INIS)

Dirks, B.P.F.; Popescu, M.; Bruijn, M.; Gottardi, L.; Hoevers, H.F.C.; Korte, P.A.J. de; Kuur, J. van der; Ridder, M.; Takei, Y.

2009-01-01

We present the latest results of the performance of micro-calorimeters based on transition edge sensors (TESs) for space applications. Sensors based on TiAu superconductive layers with Cu/Bi absorbers are discussed and have been characterized. Different coupling schemes between absorber and TES have been tested leading to an optimal (preferred) design for a new batch of arrays. We discuss the progress on array development for the International X-ray Observatory (IXO) in terms of pixel uniformity and filling factor. Inter-pixel cross-talk is discussed as well.

Les masques trompeurs de la bipolarité: étude de 100 cas

Science.gov (United States)

Nabih, Fadoua Oueriagli; Benali, Abdesslam; Adali, Imane; Manoudi, Fatiha; Asri, Fatima

2015-01-01

Le trouble bipolaire (TB) est une pathologie dont la prévalence est estimée à 1-2%. Le diagnostic précoce du trouble constitue un enjeu thérapeutique majeur. L'objectif de ce travail est de déterminer les différents diagnostiques attribués aux patients bipolaires avant de recevoir le diagnostic adéquat et de préciser le délai moyen du retard diagnostique. C'est une étude descriptive transversale portant sur 100 patients atteints de TB, inclus selon les critères du DSM V, qui ont été vus en consultation ou bien hospitalisés dans le service de psychiatrie de l'hôpital Militaire Avicenne de Marrakech, durant une période de deux ans. L’âge moyen des patients était de 29,5 ans avec une prédominance masculine (80%). 40% de nos patients ont reçu au début un autre diagnostic que celui du TB et le premier diagnostic retenu était celui de l’épisode dépressif majeur dans 36% des cas, suivi de l'accès psychotique aigu dans 28% des cas, la schizophrénie dans 16,8% et le trouble de personnalité dans 10,2%. Le délai moyen du retard diagnostic était de 64 mois. 50% des patients ayant reçu un autre diagnostic avaient consulté au moins un psychiatre et 60% des patients avaient été hospitalisés au moins une fois avant le diagnostic du TB. Les errances diagnostiques du TB sont bien établies, conduisant forcément à un retard de prise en charge adéquate. Ces données devraient alerter les psychiatres pour favoriser un meilleur dépistage de la manie et de l'hypomanie qui restent les éléments clé du diagnostic du TB. PMID:26587161

CRISPR/Cas9:A powerful tool for crop genome editing

Institute of Scientific and Technical Information of China (English)

Gaoyuan Song; Meiling Jia; Kai Chen; Xingchen Kong; Bushra Khattak; Chuanxiao Xie; Aili Li; Long Mao

2016-01-01

The CRISPR/Cas9 technology is evolved from a type II bacterial immune system and represents a new generation of targeted genome editing technology that can be applied to nearly all organisms. Site-specific modification is achieved by a single guide RNA(usually about 20nucleotides) that is complementary to a target gene or locus and is anchored by a protospaceradjacent motif. Cas9 nuclease then cleaves the targeted DNA to generate double-strand breaks(DSBs), which are subsequently repaired by non-homologous end joining(NHEJ) or homology-directed repair(HDR) mechanisms. NHEJ may introduce indels that cause frame shift mutations and hence the disruption of gene functions. When combined with double or multiplex guide RNA design, NHEJ may also introduce targeted chromosome deletions,whereas HDR can be engineered for target gene correction, gene replacement, and gene knock-in. In this review, we briefly survey the history of the CRISPR/Cas9 system invention and its genome-editing mechanism. We also describe the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications of modified Cas9 variants, and discuss the potential of this system for targeted genome editing and modification for crop improvement.

CRISPR/Cas9:A powerful tool for crop genome editing

Institute of Scientific and Technical Information of China (English)

Gaoyuan Song; Meiling Jia; Kai Chen; Xingchen Kong; Bushra Khattak; Chuanxiao Xie; Aili Li; Long Mao

2016-01-01

The CRISPR/Cas9 technology is evolved from a type II bacterial immune system and represents a new generation of targeted genome editing technology that can be applied to nearly all organisms. Site-specific modification is achieved by a single guide RNA (usually about 20 nucleotides) that is complementary to a target gene or locus and is anchored by a protospacer-adjacent motif. Cas9 nuclease then cleaves the targeted DNA to generate double-strand breaks (DSBs), which are subsequently repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. NHEJ may introduce indels that cause frame shift mutations and hence the disruption of gene functions. When combined with double or multiplex guide RNA design, NHEJ may also introduce targeted chromosome deletions, whereas HDR can be engineered for target gene correction, gene replacement, and gene knock-in. In this review, we briefly survey the history of the CRISPR/Cas9 system invention and its genome-editing mechanism. We also describe the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications of modified Cas9 variants, and discuss the potential of this system for targeted genome editing and modification for crop improvement.

Les tuméfactions sous mandibulaires: à propos de 42 cas et revue de la literature

Science.gov (United States)

Darouassi, Youssef; Mliha Touati, Mohamed; Chihani, Mehdi; Nadour, Karim; Ammar, Haddou; Bouaity, Brahim

2014-01-01

Les tuméfactions de la région sous mandibulaire sont une entité pathologique fréquente, caractérisées par un tableau clinique et une démarche diagnostique stéréotypée, et une unicité presque constante au plan chirurgical. Notre travail est une étude rétrospective sur une période de 5 an d'une série consécutive de 42 cas de tuméfactions sous mandibulaires qui ont été colligés au service d'oto-rhino-laryngologie et de chirurgie cervico-faciale de l'hôpital militaire Avicenne de Marrakech. L’âge moyen des patients était de 41 ans. Une prédominance masculine a été retrouvée. Le tableau clinique a été dominé par la tuméfaction sous mandibulaire. Tous les patients ont bénéficié d'une échographie cervicale. La tomodensitométrie cervicale a été réalisée chez 7 patients. La première étiologie était la sous maxillite chronique lithiasique dans 39,02% des cas. Le traitement était chirurgical dans tous les cas. En comparant les résultats avec ceux rapportés dans la littérature, nous allons discuter les différents aspects diagnostiques et thérapeutiques des étiologies les plus fréquentes. Les tuméfactions de la région sous mandibulaire sont fréquentes, nécessitant une démarche diagnostique rigoureuse et une prise en charge thérapeutique adaptée. PMID:25469195

CRISPR-Cas9 System as a Versatile Tool for Genome Engineering in Human Cells

Directory of Open Access Journals (Sweden)

Xuelian Wang

2016-01-01

Full Text Available Targeted nucleases are influential instruments for intervening in genome revision with great accuracy. RNA-guided Cas9 nucleases produced from clustered regularly interspaced short palindromic repeats (CRISPR-Cas systems have noticeably altered the means to modify the genomes of distinct organisms. They can be notably used to facilitate effective genome manipulation in eukaryotic cells by clearly detailing a 20-nt targeting sequence inside its directed RNA. We discuss the most recent advancements in the molecular basis of the type II CRISPR/Cas system and encapsulate applications and elements affecting its use in human cells. We also propose possible applications covering its uses ranging from basic science to implementation in the clinic.

CRISPR/Cas9-mediated target validation of the Splicing Inhibitor Pladienolide B

KAUST Repository

Aouida, Mustapha; Eid, Ayman; Mahfouz, Magdy M.

2016-01-01

CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.

CRISPR/Cas9-mediated target validation of the Splicing Inhibitor Pladienolide B

KAUST Repository

Aouida, Mustapha

2016-02-24

CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation.

A versatile one-step CRISPR-Cas9 based approach to plasmid-curing

DEFF Research Database (Denmark)

Lauritsen, Ida; Porse, Andreas; Sommer, Morten Otto Alexander

2017-01-01

tool enabling rapid removal of plasmids from bacterial cells is lacking. Results Based on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have...... widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both...

Epidémie de choléra au Burkina Faso en 2005: aspects épidémiologiques et diagnostiques

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Carole Gilberte Kyelem

2011-01-01

Full Text Available INTRODUCTION: L’objectif de cette étude était de décrire les aspects épidémiologiques et diagnostiques de l’épidémie de choléra au Burkina Faso en 2005. METHODES: Etude rétrospective, d’août à octobre 2005. Elle a concerné dix districts sanitaires du Burkina Faso. A été inclus dans l’étude, tout patient présentant un syndrome cholériforme, admis dans les différentes formations sanitaires dont la coproculture s’est révélée positive à Vibrio cholerae. RESULTATS: Au cours cette épidémie, 1050 cas de diarrhées cholériformes ont été notifiés par l’ensemble des structures sanitaires du pays. Vibrio cholerae a été identifié à l’examen bactériologique des selles de 121 patients (17,2%, constituant notre population d’étude. Les hommes étaient majoritaires (57%. La moyenne d’âge était de 30 ans. Les femmes au foyer (24% et les sujets non scolarisés (62,8% représentaient les couches sociales les plus touchées. Les forages ont été la source de boisson de 39,7% des patients 72 heures avant le début de la maladie. Tous les patients ont présenté une diarrhée aqueuse. Vibrio cholerae, sérotype Ogawa, responsable de cette épidémie, était résistant au chloramphénicol et au cotrimoxazole dans respectivement 71,7% et 38,3% des cas. Ni le cas index, ni la source initiale de contamination n’ont pu être identifiés. La létalité de notre échantillon était de 3,5%. CONCLUSION: Cette épidémie a relancé la question de l’hygiène et mis à nu le problème de ces villes ou la croissance démographique galopante est en inadéquation avec le degré d’urbanisation

Mise en pratique du schéma 2BSvs dans le groupe industriel Sofiprotéol : étude de cas sur toute la filière (de l’agriculteur au pétrolier

Directory of Open Access Journals (Sweden)

Guizouarn Kristell

2015-01-01

Full Text Available Pour réduire sensiblement les émissions de CO liées aux transports routiers, l’Union européenne mise sur le développement des biocarburants, en respectant des conditions de durabilité. Afin de répondre à cette obligation et d’être en mesure de démontrer la conformité du biodiesel à ces critères, depuis l’agriculteur jusqu’au distributeur pétrolier, la filière des oléagineux a engagé la rédaction d’un schéma de vérification volontaire : 2BSvs, pour Biomasse Biocarburant Schéma volontaire sur la durabilité. Reconnu par la Commission Européenne le 19 juillet 2011, le schéma 2BSvs couvre l’ensemble de la chaîne de production des biocarburants, du producteur de biomasse à l’entrée dans un entrepôt sous douanes. À travers une étude de cas sur toute la filière, de l’agriculteur au pétrolier, cet article présente les implications pour les acteurs de la filière oléagineuse, les changements intervenus depuis avril 2013, et les défis futurs à relever.

AZO/Au/AZO tri-layer thin films for the very low resistivity transparent electrode applications

Energy Technology Data Exchange (ETDEWEB)

Chu, Chien-Hsun [Department of Materials Science and Engineering, National Cheng Kung University, Tainan 70101, Taiwan (China); Wu, Hung-Wei, E-mail: hwwu@mail.ksu.edu.tw [Department of Computer and Communication, Kun Shan University, Tainan 71003, Taiwan (China); Huang, Jow-Lay [Department of Materials Science and Engineering, National Cheng Kung University, Tainan 70101, Taiwan (China); Department of Chemical and Materials Engineering, National University of Kaohsiung, Kaohsiung 81148, Taiwan (China); Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 70101, Taiwan (China)

2014-08-01

Highlights: • High-quality Al-doped ZnO (AZO)/Au/AZO transparent conducting oxide films. • AZO films (30 nm) made by RF sputtering; ion sputtering for Au film (5–20 nm). • Effects of Au thickness on optical and electrical properties were analyzed. • The resistivity of 9 × 10{sup −5} Ω cm and the transmittance of 86.2% of the multilayer films were obtained in this study. - Abstract: Aluminum-doped ZnO (AZO)/gold/AZO tri-layer structures with very low resistivity and high transmittance are prepared by simultaneous RF magnetron sputtering (for AZO) and ion sputtering (for Au). The properties of the tri-layer films are investigated at different Au layer thicknesses (5–20 nm). The effects of Au layer thickness and the role of Au on the transmission properties of the tri-layer films were investigated. The very low resistivity of 1.01 × 10{sup −5} Ω cm, mobility of 27.665 cm{sup 2} V{sup −1} s{sup −1}, and carrier concentration of 4.563 × 10{sup 22} cm{sup −3} were obtained at an Au layer thickness of 20 nm. The peak transmittance of 86.18% at 650-nm wavelength was obtained at an Au layer thickness of 8 nm. These results show the films to be a good candidate for high-quality electrode scheme in various display applications.

A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

Science.gov (United States)

The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

[Efficient genome editing in human pluripotent stem cells through CRISPR/Cas9].

Science.gov (United States)

Liu, Gai-gai; Li, Shuang; Wei, Yu-da; Zhang, Yong-xian; Ding, Qiu-rong

2015-11-01

The RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has offered a new platform for genome editing with high efficiency. Here, we report the use of CRISPR/Cas9 technology to target a specific genomic region in human pluripotent stem cells. We show that CRISPR/Cas9 can be used to disrupt a gene by introducing frameshift mutations to gene coding region; to knock in specific sequences (e.g. FLAG tag DNA sequence) to targeted genomic locus via homology directed repair; to induce large genomic deletion through dual-guide multiplex. Our results demonstrate the versatile application of CRISPR/Cas9 in stem cell genome editing, which can be widely utilized for functional studies of genes or genome loci in human pluripotent stem cells.

The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9.

Science.gov (United States)

Tsui, Tsz Kin Martin; Hand, Travis H; Duboy, Emily C; Li, Hong

2017-06-16

Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.

Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum.

Science.gov (United States)

Liu, Jiao; Wang, Yu; Lu, Yujiao; Zheng, Ping; Sun, Jibin; Ma, Yanhe

2017-11-16

Corynebacterium glutamicum is an important industrial workhorse and advanced genetic engineering tools are urgently demanded. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) have revolutionized the field of genome engineering. The CRISPR/Cas9 system that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C. glutamicum. Herein, we developed a versatile CRISPR/Cas9 genome editing toolbox for C. glutamicum. Cas9 and gRNA expression cassettes were reconstituted to combat Cas9 toxicity and facilitate effective termination of gRNA transcription. Co-transformation of Cas9 and gRNA expression plasmids was exploited to overcome high-frequency mutation of cas9, allowing not only highly efficient gene deletion and insertion with plasmid-borne editing templates (efficiencies up to 60.0 and 62.5%, respectively) but also simple and time-saving operation. Furthermore, CRISPR/Cas9-mediated ssDNA recombineering was developed to precisely introduce small modifications and single-nucleotide changes into the genome of C. glutamicum with efficiencies over 80.0%. Notably, double-locus editing was also achieved in C. glutamicum. This toolbox works well in several C. glutamicum strains including the widely-used strains ATCC 13032 and ATCC 13869. In this study, we developed a CRISPR/Cas9 toolbox that could facilitate markerless gene deletion, gene insertion, precise base editing, and double-locus editing in C. glutamicum. The CRISPR/Cas9 toolbox holds promise for accelerating the engineering of C. glutamicum and advancing its application in the production of biochemicals and biofuels.

Apport des approches inspirées de la neuropsychologie au diagnostic d'autisme chez l'adulte: une étude de cas

OpenAIRE

Weiner , L.; Baratta , A.; Henry , J.; Di Santi , C.

2010-01-01

Resume Nous allons illustrer a partir d?un cas clinique les procedures diagnostiques des troubles du spectre autistique chez l?adulte, inspirees des theories neurocognitives actuelles. Nous relatons le cas d?un homme de 29 ans recu en consultation psychiatrique. Le diagnostic initialement retenu etait celui de personnalite evitante, compliquee de phobie sociale et d?un trouble depressif intercurrent. Malgre la diminution des symptomes anxieux et depressifs apres l?instauration d?un...

L’orientation au travail des générations : le cas de la génération X au Québec

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Charles Fleury

2011-04-01

Full Text Available Le présent article examine l’évolution de l’orientation au travail des membres de la génération X au Québec, génération dont les difficultés d’insertion professionnelle et les valeurs de jeunesse ont suscité de multiples interrogations au cours des années 1980 et au début des années 1990. S’appuyant sur des données d’enquête récoltées entre 1981 et 2006, il examine plus spécifiquement l’évolution de la valeur accordée au travail et de la signification du travail au sein de cette génération et en regard des autres générations. Il montre que, contrairement aux thèses défendues par plusieurs sociologues de la jeunesse au cours des années 1980 et 1990, l’orientation au travail des membres de la génération X se distingue assez peu de celle des autres générations. Il conclut que les difficultés économiques rencontrées de manière toute particulière par les membres de la génération X au moment de leur insertion professionnelle n’ont pas modifié, de manière durable et typique, leur orientation au travail.This paper examines the evolution of work orientation of members of the X generation in Quebec whose difficulties of employability and youth values sparked multiple questions in the 1980s and early 1990s. Based on survey data collected between 1981 and 2006, it specifically examines the evolution of the importance given to work and the meaning of work in this generation when compared with other generations. It shows that unlike numerous theses defended by some youth sociologists in the 1980s and 1990s, work orientation of the members of the X generation differs little from other generations. It concludes that the specific economic difficulties experienced by members of X generation have not changed their work orientation in a typical and sustainable way.Este artículo examina la evolución de la orientación laboral de los miembros de la generación X en Quebec cuyos problemas de inserción laboral y

Exploitations agricoles climate-intelligentes? Etudes de cas au Burkina Faso et en Colombie

NARCIS (Netherlands)

Andrieu, N.; Pédelahore, P.; Howland, F.; Descheemaeker, K.K.E.

2015-01-01

Comment satisfaire la sécurité alimentaire tout en s'adaptant au changement climatique et en l'atténuant ? Quelles sont les principales menaces pesant sur les agricultures du Sud ? Comment les agriculteurs du Sud répondent-ils à ces menaces ? Quelles sont les propositions de la recherche agronomique

[Detection of CRSPR-Cas system in Streptococcus thermophiles].

Science.gov (United States)

Li, Wan; Liang, Hongzhang; Zhang, Danqing; Wang, Nana; Tang, Yaru; Li, Bailiang; Huo, Guicheng

2016-04-14

We aimed to detect the CRSPR-Cas system of six Streptococcus thermophilus. Bioinformatics method was used to predict CRSPR-Cas system of nine S. thermophilus that published in National Center for Biotechnology Information. Four primers were designed according to the flanking sequences of standard strains and the CRISPR-Cas system of six S. thermophilus have been detected by PCR method. S. thermophilus S4 had a Cas9 gene, others all had Cas9 gene, Cas10 gene and Cas9* gene. In addition, 79 and KLDS3.0207 still had Cas3 gene. Signature genes amplification of CRSPR-Cas system could predict the type of CRSPR-Cas system in unsequenced strains, these findings will help establish the foundation for the study of CRSPR-Cas system in lactic acid bacteria.

Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis in Petunia.

Science.gov (United States)

Zhang, Bin; Yang, Xia; Yang, Chunping; Li, Mingyang; Guo, Yulong

2016-02-03

Recently, CRISPR/Cas9 technology has emerged as a powerful approach for targeted genome modification in eukaryotic organisms from yeast to human cell lines. Its successful application in several plant species promises enormous potential for basic and applied plant research. However, extensive studies are still needed to assess this system in other important plant species, to broaden its fields of application and to improve methods. Here we showed that the CRISPR/Cas9 system is efficient in petunia (Petunia hybrid), an important ornamental plant and a model for comparative research. When PDS was used as target gene, transgenic shoot lines with albino phenotype accounted for 55.6%-87.5% of the total regenerated T0 Basta-resistant lines. A homozygous deletion close to 1 kb in length can be readily generated and identified in the first generation. A sequential transformation strategy--introducing Cas9 and sgRNA expression cassettes sequentially into petunia--can be used to make targeted mutations with short indels or chromosomal fragment deletions. Our results present a new plant species amenable to CRIPR/Cas9 technology and provide an alternative procedure for its exploitation.

Ultra-relativistic Au+Au and d+Au collisions:

Science.gov (United States)

Back, B. B.; Baker, M. D.; Ballintijn, M.; Barton, D. S.; Betts, R. R.; Bickley, A. A.; Bindel, R.; Budzanowski, A.; Busza, W.; Carroll, A.; Chai, Z.; Decowski, M. P.; García, E.; Gburek, T.; George, N.; Gulbrandsen, K.; Gushue, S.; Halliwell, C.; Hamblen, J.; Hauer, M.; Heintzelman, G. A.; Henderson, C.; Hofman, D. J.; Hollis, R. S.; Hołyński, R.; Holzman, B.; Iordanova, A.; Johnson, E.; Kane, J. L.; Katzy, J.; Khan, N.; Kucewicz, W.; Kulinich, P.; Kuo, C. M.; Lin, W. T.; Manly, S.; McLeod, D.; Mignerey, A. C.; Nouicer, R.; Olszewski, A.; Pak, R.; Park, I. C.; Pernegger, H.; Reed, C.; Remsberg, L. P.; Reuter, M.; Roland, C.; Roland, G.; Rosenberg, L.; Sagerer, J.; Sarin, P.; Sawicki, P.; Seals, H.; Sedykh, I.; Skulski, W.; Smith, C. E.; Stankiewicz, M. A.; Steinberg, P.; Stephans, G. S. F.; Sukhanov, A.; Tang, J.-L.; Tonjes, M. B.; Trzupek, A.; Vale, C.; van Nieuwenhuizen, G. J.; Vaurynovich, S. S.; Verdier, R.; Veres, G. I.; Wenger, E.; Wolfs, F. L. H.; Wosiek, B.; Woźniak, K.; Wuosmaa, A. H.; Wysłouch, B.

In this talk I will review PHOBOS data on charged particle multiplicities, obtained in Au+Au and d+Au collisions at RHIC. The general features of the Au+Au pseudorapidity distributions results will be discussed and compared to those of /line{p}p collisions. The total charged particle multiplicity, scaled by the number of participant pairs, is observed to be about 40% higher in Au+Au collisions than in /line{p}p and d+Au systems, but, surprisingly at the same level of e+e- collisions. Limiting fragmentation scaling is seen to be obeyed in Au+Au collisions.

One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

DEFF Research Database (Denmark)

Grav, Lise Marie; Lee, Jae Seong; Thomsen, Signe Gerling

2015-01-01

The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously....... Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further....

A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease.

Science.gov (United States)

Ivanov, Yury V; Shariat, Nikki; Register, Karen B; Linz, Bodo; Rivera, Israel; Hu, Kai; Dudley, Edward G; Harvill, Eric T

2015-10-26

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Here we describe a novel Type II-C CRISPR and its associated genes-cas1, cas2, and cas9-in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.

Le paludisme grave d’importation chez l’adulte: étude rétrospective de treize cas admis en réanimation à Marrakech

Science.gov (United States)

El Mezouari, El Mostafa; Belhadj, Ayoub; Ziani, Mohamed; Boughanem, Mohamed; Moutaj, Redouane

2016-01-01

Le paludisme d’importation est une affection de plus en plus fréquente en zone non endémique. Les formes graves représentent 10 % des cas de paludisme à Plasmodium falciparum. Au Maroc, plus de 50 cas de paludisme sont enregistrés chaque année dont 83 % à P. falciparum. Ont été inclus dans l’étude tous les patients ayant développé un paludisme grave, admis au service de réanimation durant la période comprise entre le 1er Novembre 2009 et le 31 décembre 2015. Les principales données épidémiologiques, les motifs d’admission, la prise en charge et l’évolution ont été étudiés. Treize patients sont retenus. L’âge moyen est de 31 ans. Tous les patients ont séjourné en afrique subsaharienne et étaient non-immuns. La chimioprophylaxie était adéquate dans 33% des cas. Le délai moyen entre le début des symptômes et l’instauration du traitement était de six jours. La parasitémie moyenne initiale était de 12 %. Les motifs d’admission en réanimation étaient un coma (15%), une convulsion (07%), une détresse respiratoire (07%), une prostration (07%), une insuffisance rénale (07%), un choc associé à un ictère et une acidose (07%) et enfin une insuffisance rénale conjuguée à un coma (07%). Tous les patients ont reçu un traitement par la quinine intraveineuse avec une dose de charge dans 100 % des cas. Le taux de mortalité était de 23 %. Les causes du décès étaient dues à la défaillance multi viscérale et au syndrome de détresse respiratoire aigu. La mortalité des formes graves du paludisme reste élevée. L’adéquation de la chimioprophylaxie associée à la précocité du diagnostic et du traitement permettrait d’améliorer significativement le pronostic de cette parasitose. PMID:28292141

Incidence du cancer au Canada : tendances et projections (1983-2032

Directory of Open Access Journals (Sweden)

Lin Xie

2015-01-01

devrait continuer d'augmenter de 2 % entre 2003-2007 et 2008-2012, puis commencer à fléchir de 18 % au cours des 20 dernières années de projection. De tous les cancers féminins, on s'attend à ce que l'incidence du cancer du sein soit celle qui change le moins (une hausse de moins de 1 %. L'évolution prévue des taux pour le cancer colorectal est inférieure aux médianes pour l'ensemble des cancers, avec une diminution de 6 %, tant chez les hommes que chez les femmes, durant toute la période de projection. En ce qui concerne le cancer de la prostate, si la prévalence du dépistage se stabilise à l'avenir, les taux devraient demeurer stables. Augmentation prévue du nombre de nouveaux cas de cancer : Au Canada, on prévoit que le nombre annuel de nouveaux cas de cancer augmentera de 84 % chez les hommes, passant de 80 800 en 2003-2007 à 148 400 en 2028-2032, et de 74 % chez les femmes, passant de 74 200 à 128 800. Facteurs des variations dans les cas de cancer : La principale raison de la hausse du nombre de tous les cas de cancer nouvellement diagnostiqués sera le vieillissement de la population canadienne, et dans une moindre mesure la croissance démographique. Les variations du risque de cancer constitueront une composante relativement mineure de la hausse prévue du nombre de nouveaux cas. Les interventions préventives peuvent grandement influencer les taux d'incidence futurs de certains types de cancer. Cancers les plus courants : Les cancers les plus courants chez les hommes — cancers de la prostate, du poumon et de la vessie, cancer colorectal et lymphome non hodgkinien — devraient demeurer inchangés entre 2003-2007 et 2028-2032, mais on prévoit que le cancer colorectal remplacera le cancer du poumon au deuxième rang des cancers les plus fréquemment diagnostiqués d'ici 2028-2032. Chez les femmes, les cancers du sein, du poumon, de l'utérus ainsi que le cancer colorectal sont les quatre principaux cancers pour lesquels de nouveaux cas seront

All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.

Science.gov (United States)

Sakuma, Tetsushi; Sakamoto, Takuya; Yamamoto, Takashi

2017-01-01

CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.

Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.

Science.gov (United States)

Yamada, Mari; Watanabe, Yuto; Gootenberg, Jonathan S; Hirano, Hisato; Ran, F Ann; Nakane, Takanori; Ishitani, Ryuichiro; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

2017-03-16

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.

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Gandhi, Shashank; Piacentino, Michael L; Vieceli, Felipe M; Bronner, Marianne E

2017-12-01

The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific. Copyright © 2017 Elsevier Inc. All rights reserved.

Fabricating a Homogeneously Alloyed AuAg Shell on Au Nanorods to Achieve Strong, Stable, and Tunable Surface Plasmon Resonances

KAUST Repository

Huang, Jianfeng

2015-08-13

Colloidal metal nanocrystals with strong, stable, and tunable localized surface plasmon resonances (SPRs) can be useful in a corrosive environment for many applications including field-enhanced spectroscopies, plasmon-mediated catalysis, etc. Here, a new synthetic strategy is reported that enables the epitaxial growth of a homogeneously alloyed AuAg shell on Au nanorod seeds, circumventing the phase segregation of Au and Ag encountered in conventional synthesis. The resulting core–shell structured bimetallic nanorods (AuNR@AuAg) have well-mixed Au and Ag atoms in their shell without discernible domains. This degree of mixing allows AuNR@AuAg to combine the high stability of Au with the superior plasmonic activity of Ag, thus outperforming seemingly similar nanostructures with monometallic shells (e.g., Ag-coated Au NRs (AuNR@Ag) and Au-coated Au NRs (AuNR@Au)). AuNR@AuAg is comparable to AuNR@Ag in plasmonic activity, but that it is markedly more stable toward oxidative treatment. Specifically, AuNR@AuAg and AuNR@Ag exhibit similarly strong signals in surface-enhanced Raman spectroscopy that are some 30-fold higher than that of AuNR@Au. When incubated with a H2O2 solution (0.5 m), the plasmonic activity of AuNR@Ag immediately and severely decayed, whereas AuNR@AuAg retained its activity intact. Moreover, the longitudinal SPR frequency of AuNR@AuAg can be tuned throughout the red wavelengths (≈620–690 nm) by controlling the thickness of the AuAg alloy shell. The synthetic strategy is versatile to fabricate AuAg alloyed shells on different shaped Au, with prospects for new possibilities in the synthesis and application of plasmonic nanocrystals.

Annotation and Classification of CRISPR-Cas Systems.

Science.gov (United States)

Makarova, Kira S; Koonin, Eugene V

2015-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods.

Annotation and Classification of CRISPR-Cas Systems

Science.gov (United States)

Makarova, Kira S.; Koonin, Eugene V.

2018-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods. PMID:25981466

Introduction au dossier « Mutations de l’accompagnement dans les formations en ligne »

OpenAIRE

Simonian, Stephane; Savarieau, Béatrice

2016-01-01

Suite au colloque organisé par le laboratoire CIVIIC (Centre interdisciplinaire de recherches sur les valeurs, les idées, les identités et les compétences) qui s’est tenu à Rouen du 8 au 10 octobre 2014 intitulé « Mutations de l’accompagnement dans les formations en ligne », il nous est apparu intéressant – bien que risqué – de traiter d’un sujet pouvant être considéré comme un point aveugle lié à l’accompagnement de la rédaction de mémoires dans le cas des formations à distance ou hybrides. ...

CRISPR-Cas9: a promising genetic engineering approach in cancer research

Science.gov (United States)

Ratan, Zubair Ahmed; Son, Young-Jin; Uddin, Bhuiyan Mohammad Mahtab; Yusuf, Md. Abdullah; Zaman, Sojib Bin; Kim, Jong-Hoon; Banu, Laila Anjuman

2018-01-01

Bacteria and archaea possess adaptive immunity against foreign genetic materials through clustered regularly interspaced short palindromic repeat (CRISPR) systems. The discovery of this intriguing bacterial system heralded a revolutionary change in the field of medical science. The CRISPR and CRISPR-associated protein 9 (Cas9) based molecular mechanism has been applied to genome editing. This CRISPR-Cas9 technique is now able to mediate precise genetic corrections or disruptions in in vitro and in vivo environments. The accuracy and versatility of CRISPR-Cas have been capitalized upon in biological and medical research and bring new hope to cancer research. Cancer involves complex alterations and multiple mutations, translocations and chromosomal losses and gains. The ability to identify and correct such mutations is an important goal in cancer treatment. In the context of this complex cancer genomic landscape, there is a need for a simple and flexible genetic tool that can easily identify functional cancer driver genes within a comparatively short time. The CRISPR-Cas system shows promising potential for modeling, repairing and correcting genetic events in different types of cancer. This article reviews the concept of CRISPR-Cas, its application and related advantages in oncology. PMID:29434679

Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition.

Science.gov (United States)

Lostalé-Seijo, Irene; Louzao, Iria; Juanes, Marisa; Montenegro, Javier

2017-12-01

The discovery of RNA guided endonucleases has emerged as one of the most important tools for gene edition and biotechnology. The selectivity and simplicity of the CRISPR/Cas9 strategy allows the straightforward targeting and editing of particular loci in the cell genome without the requirement of protein engineering. However, the transfection of plasmids encoding the Cas9 and the guide RNA could lead to undesired permanent recombination and immunogenic responses. Therefore, the direct delivery of transient Cas9 ribonucleoprotein constitutes an advantageous strategy for gene edition and other potential therapeutic applications of the CRISPR/Cas9 system. The covalent fusion of Cas9 with penetrating peptides requires multiple incubation steps with the target cells to achieve efficient levels of gene edition. These and other recent reports suggested that covalent conjugation of the anionic Cas9 ribonucleoprotein to cationic peptides would be associated with a hindered nuclease activity due to undesired electrostatic interactions. We here report a supramolecular strategy for the direct delivery of Cas9 by an amphiphilic penetrating peptide that was prepared by a hydrazone bond formation between a cationic peptide scaffold and a hydrophobic aldehyde tail. The peptide/protein non-covalent nanoparticles performed with similar efficiency and less toxicity than one of the best methods described to date. To the best of our knowledge this report constitutes the first supramolecular strategy for the direct delivery of Cas9 using a penetrating peptide vehicle. The results reported here confirmed that peptide amphiphilic vectors can deliver Cas9 in a single incubation step, with good efficiency and low toxicity. This work will encourage the search and development of conceptually new synthetic systems for transitory endonucleases direct delivery.

Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

Science.gov (United States)

Sanozky-Dawes, Rosemary; Selle, Kurt; O'Flaherty, Sarah; Klaenhammer, Todd; Barrangou, Rodolphe

2015-09-01

Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5'-NTAA-3' flanking the 3' end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.

CRISPR-Cas: Adapting to change.

Science.gov (United States)

Jackson, Simon A; McKenzie, Rebecca E; Fagerlund, Robert D; Kieper, Sebastian N; Fineran, Peter C; Brouns, Stan J J

2017-04-07

Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective identification and neutralization of foreign DNA and/or RNA. CRISPR-Cas systems rely on stored genetic memories to facilitate target recognition. Thus, to keep pace with a changing pool of hostile invaders, the CRISPR memory banks must be regularly updated with new information through a process termed CRISPR adaptation. In this Review, we outline the recent advances in our understanding of the molecular mechanisms governing CRISPR adaptation. Specifically, the conserved protein machinery Cas1-Cas2 is the cornerstone of adaptive immunity in a range of diverse CRISPR-Cas systems. Copyright © 2017, American Association for the Advancement of Science.

Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy.

Science.gov (United States)

Shibata, Mikihiro; Nishimasu, Hiroshi; Kodera, Noriyuki; Hirano, Seiichi; Ando, Toshio; Uchihashi, Takayuki; Nureki, Osamu

2017-11-10

The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.

New CRISPR-Cas systems from uncultivated microbes

Science.gov (United States)

Burstein, David; Harrington, Lucas B.; Strutt, Steven C.; Probst, Alexander J.; Anantharaman, Karthik; Thomas, Brian C.; Doudna, Jennifer A.; Banfield, Jillian F.

2017-02-01

CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation

Science.gov (United States)

Rothschild, Lynn J.; Greenberg, Daniel T.; Takahashi, Jack R.; Thompson, Kirsten A.; Maheshwari, Akshay J.; Kent, Ryan E.; McCutcheon, Griffin; Shih, Joseph D.; Calvet, Charles; Devlin, Tyler D.;

Naturally Occurring Off-Switches for CRISPR-Cas9.

Science.gov (United States)

Pawluk, April; Amrani, Nadia; Zhang, Yan; Garcia, Bianca; Hidalgo-Reyes, Yurima; Lee, Jooyoung; Edraki, Alireza; Shah, Megha; Sontheimer, Erik J; Maxwell, Karen L; Davidson, Alan R

2016-12-15

CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

Science.gov (United States)

Yin, L; Maddison, L A; Chen, W

2016-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound-Propelled Nanomotors.

Science.gov (United States)

Hansen-Bruhn, Malthe; de Ávila, Berta Esteban-Fernández; Beltrán-Gastélum, Mara; Zhao, Jing; Ramírez-Herrera, Doris E; Angsantikul, Pavimol; Vesterager Gothelf, Kurt; Zhang, Liangfang; Wang, Joseph

2018-03-01

Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound-powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA-loaded nanomotors to directly penetrate through the plasma membrane of GFP-expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA-loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor-based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish.

Science.gov (United States)

Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

2018-03-02

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites ( tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. Copyright © 2018 Hu et al.

Le cancer différencié de la thyroïde chez l’enfant et l’adolescent: à propos de 22 cas

Science.gov (United States)

Anajar, Said; Tatari, Mohammed; Lakhbal, Adil; Abada, Reda; Rouadi, Sami; Roubal, Mohammed; Mahtar, Mohammed

2017-01-01

L’obectif était de mettre en relief les particularités du cancer de la thyroïde chez l’enfant et l’adolescent, et d’évaluer nos résultats par rapport à la littérature internationale a travers une série de cas la plus représentatif au Maroc: 22 cas. C'est une étude rétrospective descriptive des patients atteints de cancer différencié de la thyroïde, hospitalisés au service d’ORL et de Chirurgie Cervico-faciale de L’hopital 20 Août de Casablanca-Maroc, sur la période qui s’étend de Janvier 1995 à Mars 2015. Nous avons recueilli les données relatives à 22 cas, qui répondaient à nos critères d’inclusion. L’âge moyen de nos patients était de 14 ans, avec une sex-ratio 3,4, la plupart de nos patients ont consulté pour un nodule thyroïdien, associé dans 22,7% des cas à une adénopathie cervicale, et dans 9,1% à des signes de compression. L’ensemble des patients ont bénéficié d’une thyroïdectomie totale, suivie d’un curage ganglionnaire dans 31,82%. Le diagnostic de cancer thyroïdien a reposé sur l’examen anatomopathologique de la pièce opératoire, qui a objectivé un carcinome papillaire dans 95,4% des cas, et un carcinome vésiculaire dans 4,5%. Le traitement par l’iode radioactif 131 a été réalisé dans 100% des cas. Par la suite tous nos patients ont été mis sous hormonothérapie thyroïdienne. Une surveillance étroite et régulière a permis de détecter des métastases ganglionnaires chez 3 patients, et les métastases à distance chez 4 patients. Le cancer différencié de la thyroïde de l’enfant et l’adolescent est une entité rare mais agressive, son traitement se base sur la chirurgie, associée à l’irathérapie donnant un pronostic excellent. PMID:29255541

Not all predicted CRISPR-Cas systems are equal: isolated cas genes and classes of CRISPR like elements.

Science.gov (United States)

Zhang, Quan; Ye, Yuzhen

2017-02-06

The CRISPR-Cas systems in prokaryotes are RNA-guided immune systems that target and deactivate foreign nucleic acids. A typical CRISPR-Cas system consists of a CRISPR array of repeat and spacer units, and a locus of cas genes. The CRISPR and the cas locus are often located next to each other in the genomes. However, there is no quantitative estimate of the co-location. In addition, ad-hoc studies have shown that some non-CRISPR genomic elements contain repeat-spacer-like structures and are mistaken as CRISPRs. Using available genome sequences, we observed that a significant number of genomes have isolated cas loci and/or CRISPRs. We found that 11%, 22% and 28% of the type I, II and III cas loci are isolated (without CRISPRs in the same genomes at all or with CRISPRs distant in the genomes), respectively. We identified a large number of genomic elements that superficially reassemble CRISPRs but don't contain diverse spacers and have no companion cas genes. We called these elements false-CRISPRs and further classified them into groups, including tandem repeats and Staphylococcus aureus repeat (STAR)-like elements. This is the first systematic study to collect and characterize false-CRISPR elements. We demonstrated that false-CRISPRs could be used to reduce the false annotation of CRISPRs, therefore showing them to be useful for improving the annotation of CRISPR-Cas systems.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

Science.gov (United States)

Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

2016-06-01

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. © 2016. Published by The Company of Biologists Ltd.

Programmable type III-A CRISPR-Cas DNA targeting modules.

Directory of Open Access Journals (Sweden)

H Travis Ichikawa

Full Text Available The CRISPR-Cas systems provide invader defense in a wide variety of prokaryotes, as well as technologies for many powerful applications. The Type III-A or Csm CRISPR-Cas system is one of the most widely distributed across prokaryotic phyla, and cleaves targeted DNA and RNA molecules. In this work, we have constructed modules of Csm systems from 3 bacterial species and heterologously expressed the functional modules in E. coli. The modules include a Cas6 protein and a CRISPR locus for crRNA production, and Csm effector complex proteins. The expressed modules from L. lactis, S. epidermidis and S. thermophilus specifically eliminate invading plasmids recognized by the crRNAs of the systems. Characteristically, activation of plasmid targeting activity depends on transcription of the plasmid sequence recognized by the crRNA. Activity was not observed when transcription of the crRNA target sequence was blocked, or when the opposite strand or a non-target sequence was transcribed. Moreover, the Csm module can be programmed to recognize plasmids with novel target sequences by addition of appropriate crRNA coding sequences to the module. These systems provide a platform for investigation of Type III-A CRISPR-Cas systems in E. coli, and for introduction of programmable transcription-activated DNA targeting into novel organisms.

Progress in CRISPR/Cas9 system and its application in Duchenne's muscular dystrophy%成簇规律间隔的短回文重复序列/Cas9及在Duchenne型肌营养不良中应用的研究进展

Institute of Scientific and Technical Information of China (English)

李文彬

2017-01-01

The CRISPR/Cas9 is an adaptive immune system which exists in bacteria and archaea,providing bacteria and archaea to resist against infection of phage.Cas9 can target and degrade exogenous DNA by RNA's guiding,also provide gene over expression,stereotactical block,and high-throughput screening after modifying to broaden its application.The newly discovered CRISPR/C2c2 can target and degrade exogenous RNA,and has more specificity and efficiency than RNA interference.The CRISPR/Cas9 system has been applied to modify the dystrophin gene in Duchenne's muscular dystrophy mice models,maintaining the expression of dystrophin persistent and restoring the muscle power,with no adverse reactions on brain and germline,which provides a new direction for the treatment of Duchenne's muscular dystrophy.This paper reviews the research progress of CRISPR/Cas9 and its application in Duchenne's muscular dystrophy.%成簇规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/Cas9存在于细菌和古细菌中,是细菌抵抗噬菌体入侵的适应性免疫系统,可通过向导RNA靶向外源性DNA并对其进行剪切,还可经修饰后进行基因的过表达、阻断以及高通量筛选,扩大了CRISPR系统的应用范围.新近发现的CRISPR/C2c2系统可靶向降解外源性RNA,在特异性及效率上较RNA干扰(RNA interference,RNAi)技术更有优势.应用CRISPR/Cas9系统修正小鼠抗肌萎缩蛋白,可使抗肌萎缩蛋白表达持久,肌力恢复良好,且无明显脑部及生殖系统不良反应,为Duchenne型肌营养不良提供了新的治疗方向.本文就CRISPR/Cas9及其在Duchenne型肌营养不良中应用的研究进展作一综述.

L'endométriose périnéale profonde sur cicatrice d’épisiotomie: à propos d'un cas rare

OpenAIRE

Laadioui, Meriem; Alaoui, Fdili; Jayi, Sofia; Bouguern, Hakima; Chaara, Hikmat; Melhouf, Moulay Aabdelilah

2013-01-01

Parmi les localisations rares de l'endométriose sur cicatrice, celle du périnée demeure exceptionnelle, l'origine en est souvent iatrogène (épisiotomie). Nous rapportons le cas d'une patiente présentant une douleur cyclique, au niveau de la cicatrice d’épisiotomie. Avec à l'examen clinique une masse de 3,5 cm de grand diamètre au niveau de la cicatrice d’épisiotomie. L’écho périnéale a objectivé une image hypoéchgène hétérogène non vascularisée en regard de la cicatrice d’épisiotomie faisant ...

CRISPR/Cas9 Mediated Genome Engineering for Improvement of Horticultural Crops.

Science.gov (United States)

Karkute, Suhas G; Singh, Achuit K; Gupta, Om P; Singh, Prabhakar M; Singh, Bijendra

2017-01-01

Horticultural crops are an important part of agriculture for food as well as nutritional security. However, several pests and diseases along with adverse abiotic environmental factors pose a severe threat to these crops by affecting their quality and productivity. This warrants the effective and accelerated breeding programs by utilizing innovative biotechnological tools that can tackle aforementioned issues. The recent technique of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) has greatly advanced the breeding for crop improvement due to its simplicity and high efficiency over other nucleases such as Zinc Finger Nucleases and Transcription Activator Like Effector Nucleases. CRISPR/Cas9 tool contains a non-specific Cas9 nuclease and a single guide RNA that directs Cas9 to the specific genomic location creating double-strand breaks and subsequent repair process creates insertion or deletion mutations. This is currently the widely adopted tool for reverse genetics, and crop improvement in large number of agricultural crops. The use of CRISPR/Cas9 in horticultural crops is limited to few crops due to lack of availability of regeneration protocols and sufficient sequence information in many horticultural crops. In this review, the present status of applicability of CRISPR/Cas9 in horticultural crops was discussed along with the challenges and future potential for possible improvement of these crops for their yield, quality, and resistance to biotic and abiotic stress.

1-Hexadecylamine as both reducing agent and stabilizer to synthesize Au and Ag nanoparticles and their SERS application

International Nuclear Information System (INIS)

Hou Xiaomiao; Zhan, Xiaoling; Fang Yan; Chen Shutang; Li Na; Zhou Qi

2011-01-01

1-Hexadecylamine (HDA)-capped Au and Ag nanoparticles (NPs) have been successfully prepared by a one-pot solution growth method. The HDA is used as both reducing agent and stabilizer in the synthetic process is favorable for investigating the capping mechanism of Au and Ag NPs’ surface. The growth process and characterization of Au and Ag NPs are determined by Ultraviolet–visible (UV–vis) spectroscopy, transmission electron microscopy (TEM), and X-ray diffraction (XRD). Experimental results demonstrate that the HDA-capped Au and Ag NPs are highly crystalline and have good optical properties. Furthermore, surface-enhanced Raman scattering (SERS) spectra of 2-thionaphthol are obtained on the Au and Ag NPs modified glass surface, respectively, indicating that the as-synthesized noble metal NPs have potentially high sensitive optical detection application.

1-Hexadecylamine as both reducing agent and stabilizer to synthesize Au and Ag nanoparticles and their SERS application

Energy Technology Data Exchange (ETDEWEB)

Hou Xiaomiao; Zhan, Xiaoling, E-mail: zhangxl@bit.edu.cn [Beijing Institute of Technology, Department of Chemistry, School of Science (China); Fang Yan, E-mail: fangyan@mail.cnu.edu.cn [Capital Normal University, Beijing Key Lab for Nano-Photonics and Nano-Structure (NPNS), Department of Physics (China); Chen Shutang; Li Na; Zhou Qi [Beijing Institute of Technology, Department of Chemistry, School of Science (China)

2011-05-15

1-Hexadecylamine (HDA)-capped Au and Ag nanoparticles (NPs) have been successfully prepared by a one-pot solution growth method. The HDA is used as both reducing agent and stabilizer in the synthetic process is favorable for investigating the capping mechanism of Au and Ag NPs' surface. The growth process and characterization of Au and Ag NPs are determined by Ultraviolet-visible (UV-vis) spectroscopy, transmission electron microscopy (TEM), and X-ray diffraction (XRD). Experimental results demonstrate that the HDA-capped Au and Ag NPs are highly crystalline and have good optical properties. Furthermore, surface-enhanced Raman scattering (SERS) spectra of 2-thionaphthol are obtained on the Au and Ag NPs modified glass surface, respectively, indicating that the as-synthesized noble metal NPs have potentially high sensitive optical detection application.

A white-emitting ZnO-Au nanocomposite and its SERS applications

Energy Technology Data Exchange (ETDEWEB)

Sun Lanlan [State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, 3888 East Nan-Hu Road, Open Economic Zone, Changchun 130033 (China); Zhao Dongxu, E-mail: dxzhao2000@yahoo.com.cn [State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, 3888 East Nan-Hu Road, Open Economic Zone, Changchun 130033 (China); Ding Meng; Zhao Haifeng; Zhang Zhenzhong; Li Binghui; Shen Dezhen [State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, 3888 East Nan-Hu Road, Open Economic Zone, Changchun 130033 (China)

2012-08-01

We reported a simple method to synthesize ZnO-Au nanocomposites (hybrid A) by combining hydrothermal and electric beam evaporation deposition method. It was found that Au deposition time takes an important role in the generation of Au nanoparticles. Changing Au deposition time makes the thickness of Au formed on ZnO nanorods increase from 10 nm to 70 nm. On the other hand, white-emitting ZnO-Au nanocomposites (hybrid B) were obtained after treating hybrid A with HCl solution. Thanks to the covering of Au film and acid etching, it induces many defects on the surface of ZnO NRs, and largely enhances the visible emission of surviving ZnO and finally generates white emission on Au mesocrystals (hybrid B). Both of the ZnO-Au hybrids (A and B) can be applied as substrates in surface-enhanced Raman scattering (SERS) measurement. A typical probe molecule, 4-ATP was used to test the SERS activity of the ZnO-Au composites and the results indicated good Raman activity on the substrates.

Development and application of knowledge-based subject group integration platforms:A case study of Shanghai Institute of Ceramics,CAS

Institute of Scientific and Technical Information of China (English)

Yu; LIU; Jian; FU; Huijun; ZHENG; Hao; CHEN; Zhiping; YANG

2014-01-01

Purpose:According to the different requirements of research group users,we established the knowledge-based subject group integration platforms of Shanghai Institute of Ceramics,the Chinese Academy of Sciences(abbreviated as SIC CAS hereinafter),which were designed and constructed to better meet the needs of CAS research groups for their development,collaboration and communication.Design/methodology/approach:We first identified the requirements of users via preliminary investigation,and then chose CASI1 P,iLibrary and XKE technology,respectively as the building tools compatible with the major demands of users.These steps helped us complete the layout design of SIC CAS integration platforms,as well as its knowledge organization and integration.Findings:According to the need of users,we applied three types of platform construction technologies to five SIC integration platforms,and formulated standard norms for the further construction process,which could provide useful reference for a sustainable development for the extensive construction in CAS institutes.Research limitations:In order to make the SIC integration platforms more intelligent and have more functions,we need to enlarge the scale of the Platforms and upgrade the building tools for the platform construction.Practical implications:The nature of SIC sub-project integration platforms is to construct a content-sensitive environment which can embed knowledge services and knowledge applications seamlessly into scientific activities,so the Platform is expected to be a useful tool to help researchers better understand the recent development of the research field and form collaborations with their peers.Originality/value:SIC integration platforms are the only pilot construction that used 3different platform technologies in the first batch of knowledge-based subject group integration platforms of the Chinese Academy of Sciences.The construction is user-centered throughout the whole process,namely,from the technology

Asteroid named after CAS scientist

Institute of Scientific and Technical Information of China (English)

无

2007-01-01

@@ An asteroid has been named after CAS astronomy historian XI Zezong with the approval of the International Minor Planet Nomenclature Committee (IMPNC), announced China's National Astronomical Observatories at CAS (NAOC) on 17 August.

CRISPR/Cas9 for Human Genome Engineering and Disease Research.

Science.gov (United States)

Xiong, Xin; Chen, Meng; Lim, Wendell A; Zhao, Dehua; Qi, Lei S

2016-08-31

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.

Conductive Au nanowires regulated by silk fibroin nanofibers

Science.gov (United States)

Dong, Bo-Ju; Lu, Qiang

2014-03-01

Conductive Au-biopolymer composites have promising applications in tissue engineering such as nerve tissue regeneration. In this study, silk fibroin nanofibers were formed in aqueous solution by regulating silk self-assembly process and then used as template for Au nanowire fabrication. We performed the synthesis of Au seeds by repeating the seeding cycles for several times in order to increase the density of Au seeds on the nanofibers. After electroless plating, densely decorated Au seeds grew into irregularly shaped particles following silk nanofiber to fill the gaps between particles and finally form uniform continuous nanowires. The conductive property of the Au-silk fibroin nanowires was studied with current-voltage ( I-V) measurement. A typical ohmic behavior was observed, which highlighted their potential applications in nerve tissue regeneration.

Induced mutation and epigenetics modification in plants for crop improvement by targeting CRISPR/Cas9 technology.

Science.gov (United States)

Khan, Muhammad Hafeez Ullah; Khan, Shahid U; Muhammad, Ali; Hu, Limin; Yang, Yang; Fan, Chuchuan

2018-06-01

Clustered regularly interspaced palindromic repeats associated protein Cas9 (CRISPR-Cas9), originally an adaptive immunity system of prokaryotes, is revolutionizing genome editing technologies with minimal off-targets in the present era. The CRISPR/Cas9 is now highly emergent, advanced, and highly specific tool for genome engineering. The technology is widely used to animal and plant genomes to achieve desirable results. The present review will encompass how CRISPR-Cas9 is revealing its beneficial role in characterizing plant genetic functions, genomic rearrangement, how it advances the site-specific mutagenesis, and epigenetics modification in plants to improve the yield of field crops with minimal side-effects. The possible pitfalls of using and designing CRISPR-Cas9 for plant genome editing are also discussed for its more appropriate applications in plant biology. Therefore, CRISPR/Cas9 system has multiple benefits that mostly scientists select for genome editing in several biological systems. © 2017 Wiley Periodicals, Inc.

RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis.

Directory of Open Access Journals (Sweden)

Jong-Jin Park

Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPR and the CRISPR associated protein 9 (Cas9 system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9 offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF activation domain to dCas9 bound with the VP64 (tetramer of VP16 activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1 and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1. The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.

The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

Science.gov (United States)

Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

2018-01-09

Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

Science.gov (United States)

Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

2016-06-01

Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. Copyright © 2016 Elsevier B.V. All rights reserved.

Using CRISPR-Cas systems as antimicrobials.

Science.gov (United States)

Bikard, David; Barrangou, Rodolphe

2017-06-01

Although CRISPR-Cas systems naturally evolved to provide adaptive immunity in bacteria and archaea, Cas nucleases can be co-opted to target chromosomal sequences rather than invasive genetic elements. Although genome editing is the primary outcome of self-targeting using CRISPR-based technologies in eukaryotes, self-targeting by CRISPR is typically lethal in bacteria. Here, we discuss how DNA damage introduced by Cas nucleases in bacteria can efficiently and specifically lead to plasmid curing or drive cell death. Specifically, we discuss how various CRISPR-Cas systems can be engineered and delivered using phages or phagemids as vectors. These principles establish CRISPR-Cas systems as potent and programmable antimicrobials, and open new avenues for the development of CRISPR-based tools for selective removal of bacterial pathogens and precise microbiome composition alteration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

Science.gov (United States)

Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

2011-04-06

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

L’usage politique de l’islam : l’universel au service d’un État. Le cas du Maroc The Political Use of Islam. The Universal in the Service of a State. The Moroccan Case

Directory of Open Access Journals (Sweden)

Abdessamad Belhaj

2011-03-01

Full Text Available Lorsqu’il s’agit d’étudier les rapports des pays arabes à la mondialisation, il est nécessaire d’évoquer l’islam. En tant que facteur local, la symbolique islamique offre des ressources de légitimation au pouvoir. Le cas marocain illustre bien la manière dont les rapports existant entre le Makhzen et le répertoire religieux (Amīr al-Mu’minīne, la bayca , la chérifibilité…  font de l’introduction de la modernité un simple espace virtuel et fonctionnel. L’émergence de la contestation islamiste participe à la consécration du répertoire traditionnel. D’autre part, depuis les trois dernières décennies, l’essor de l’islam mondialisé offre au Maroc des opportunités politiques à l’échelle mondiale. Ceci est observable au niveau du rôle joué par le pays dans le cadre de la diplomatie islamique (OCI, ISESCO…  ou au niveau de son positionnement dans l’islam européen. La gestion de l’interaction entre l’islam local et l’islam global, adaptée à la conjoncture internationale, assure le maintien au pouvoir du régime.One cannot study the relationships between Arab countries and globalization without taking Islam into account for it offers symbolic support to legitimizing local power. Morocco is a good example : the relations between the royal court (Makhzen and Islamic features such as being the Commander of the Faithful or Sheriffhood make of modernity a mere virtual or functional sphere. Even the emergence of Islamic contestation contributes to the consecration of the traditional repertory. On the other hand, the rise of world Islam during the last thirty years has opened up political opportunities for Morocco on the international stage – for instance the agencies of Islamic diplomacy (OCI, ISESCO or the quest for a new role in European Islam. By managing the interaction between the local and the global, the regime is able to keep itself in power.

The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET

Directory of Open Access Journals (Sweden)

Mengyi Yang

2018-01-01

Full Text Available Summary: Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. : Yang et al. revealed significant conformational dynamics of Cas9 at global and local scales using single-molecule FRET. They uncovered surprising long-range allosteric communication between the HNH nuclease domain and the RNA/DNA heteroduplex at the PAM-distal end that serves as a proofreading checkpoint to govern the nuclease activity and specificity of Cas9. Keywords: CRISPR, Cas9, single-molecule, FRET, conformational dynamics, proofreading, off-target, allosteric communication, genome editing

Evolution and classification of the CRISPR-Cas systems

Science.gov (United States)

S. Makarova, Kira; H. Haft, Daniel; Barrangou, Rodolphe; J. J. Brouns, Stan; Charpentier, Emmanuelle; Horvath, Philippe; Moineau, Sylvain; J. M. Mojica, Francisco; I. Wolf, Yuri; Yakunin, Alexander F.; van der Oost, John; V. Koonin, Eugene

2012-01-01

The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a `polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci. PMID:21552286

Les tumeurs malignes naso-sinusiennes: à propos de 32 cas et revues de la littérature

Science.gov (United States)

Darouassi, Youssef; Touati, Mohamed Mliha; Chihani, Mehdi; El Alami, Jihane; Bouaity, Brahim; Ammar, Haddou

2015-01-01

Sous l’appellation tumeurs malignes naso-sinusiennes est regroupé un vaste éventail de tumeurs, aux histologies et localisations variées, mais aux tableaux cliniques souvent similaires. Le diagnostic de ces tumeurs est difficile, nécessitant une approche multidisciplinaire, à savoir oto-rhino-laryngologique, radiologique et anatomopathologique. Notre étude rétrospective concerne 32 cas de tumeurs malignes naso-sinusiennes, colligées au service d’ORL de l’hôpital militaire Avicenne de Marrakech, entre Janvier 2004 et Décembre 2014. L’analyse des données a noté que la fréquence des tumeurs épithéliales (75% des cas) était supérieure à celle des tumeurs non épithéliales (25% des cas), avec en tête de file l’adénocarcinome de l’ethmoïde (31,25%) et le carcinome épidermoïde du sinus maxillaire (18,75%). Ces tumeurs surviennent le plus souvent chez le sujet âgé avec une moyenne d’âge de 52 ans et une répartition équitable entre les deux sexes. Le délai de consultation moyen était de 12 mois avec une symptomatologie dominée par un syndrome nasosinusien (71,8%), associé dans certains cas à des signes ophtalmologiques (12,5%) ou neurologiques (15,6%). Tous nos patients ont bénéficié d’un examen clinique notamment endoscopique, d’une exploration radiologique des tumeurs et de leurs extensions, et d’une confirmation diagnostique par un examen anatomopathologique. Le traitement a consisté en une exérèse chirurgicale de la tumeur dès que cela était possible, soit dans 81,3% des cas (26 patients), généralement complété par un traitement adjuvant radio-chimiothérapique (77%). Le suivi à un an de nos patients a permis de noter une bonne évolution pour 08 d’entre eux (25%), une récidive dans 6 cas (18,75%), le décès de neuf patients (28,1%), et l’absence d’information concernant les autres cas (28,1%). PMID:26985260

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

OpenAIRE

Sebastian N. Kieper; Cristóbal Almendros; Juliane Behler; Rebecca E. McKenzie; Franklin L. Nobrega; Anna C. Haagsma; Jochem N.A. Vink; Wolfgang R. Hess; Stan J.J. Brouns

2018-01-01

Summary: CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp....

Study relating to the physico-chemical behaviour of heavy water in nuclear reactors; Etudes relatives au comportement physico-chimique de l'eau lourde dans les reacteurs nucleaires

Energy Technology Data Exchange (ETDEWEB)

Chenouard, J; Dirian, G; Roth, E; Vignet, P; Platzer, R [Commissariat a l' Energie Atomique, Grenoble (France). Centre d' Etudes Nucleaires

1959-07-01

Chemical and isotope pollution, and radiolytic decomposition are the two most important ways in which heavy water becomes degraded in nuclear reactors. Chemical pollution has led to the creation of ion exchange purification loops specially designed for reactors: the report contains a description in detail of the application of this purification method in CEA research reactors, including the analysis required, results obtained, and their interpretation. The intelligence obtained on radiolytic decomposition with the same facilities is also discussed, as well as the recombination apparatus and control equipment utilized. Finally, investigation to date in the CEA on recombination circuits for power reactors is also discussed. (author) [French] Parmi les degradations subies par l'eau lourde dans les reacteurs nucleaires, les deux plus importantes sont la pollution chimique et isotopique et la decomposition radiolytique. La pollution chimique a conduit a mettre au point pour le cas particulier des reacteurs, des circuits d'epuration par echange d'ions. On decrit ici en detail la mise en oeuvre de cette methode dans les reacteurs de recherche du CEA; les controles qu'elle necessite, les resultats obtenus et leur interpretation. En ce qui concerne la dissociation radiolytique de l'eau, les renseignements obtenus sur ces memes reacteurs sont communiques, ainsi que les details des dispositifs de recombinaison et des moyens de controle. Enfin, on fait le point des etudes poursuivies au CEA sur ces memes problemes de recombinaison dans le cas des reacteurs de puissance. (auteur)

Self-standing corrugated Ag and Au-nanorods for plasmonic applications

DEFF Research Database (Denmark)

Habouti, S.; Mátéfi-Tempfli, M.; Solterbeck, C.-H.

2011-01-01

We use home-made Si-supported anodized alumina thin film templates for the electrodeposition of large area self-standing Ag- and Au-nanorod (Au-NR) arrays. The deposition conditions chosen, i.e. electrolyte composition and deposition voltage, lead to a corrugated rod morphology, particularly for Au...

CRISPR-Cas9 Structures and Mechanisms.

Science.gov (United States)

Jiang, Fuguo; Doudna, Jennifer A

2017-05-22

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

A guild of 45 CRISPR-associated (Cas protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.

Directory of Open Access Journals (Sweden)

Daniel H Haft

2005-11-01

Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPRs are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21-37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer "immunity" against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.

Les maisons bâties au village d’origine dans les représentations des migrants roumains

Directory of Open Access Journals (Sweden)

Andra Larionescu

2013-06-01

Full Text Available Les maisons des migrants bâties dans le pays d’origine revêtent de significations différentes: certains migrants les perçoivent comme un moyen de valider leur statut social au sein de la communauté d’origine, tandis que d’autres les conçoivent comme un investissement ou comme une « sécurité » à la retraite, ou bien comme un abri en cas d’échec. Néanmoins, pour la majorité des migrants, la maison reste un « chez soi» lié soit au pays d’origine, soit au pays de destination, le sentiment d’appartenance étant déterminé par l’âge du sujet, le gendre, le niveau d’éducation, les liens familiaux, l’expérience de la migration et les caractéristiques du marché du travail. En utilisant un travail de terrain mené entre 2009 et 2011 dans le village de Marginea, au nord de la Roumanie, l’article examine les facteurs qui influent sur la sémantique de la maison bâtie au village d’origine.

Tenth anniversary of CAS ONLINE service : What CAS services should be in the new era of chemical information

Science.gov (United States)

Kostakos, Charles N.

Chemical Abstracts Service celebrated 10th anniversary of CAS online information service in 1990. A speech given on the occasion reviewed history of the CAS ONLINE, in relation to its most important benefits for scientists and engineers. The development of STN international, the network through which CAS ONLINE is accessible around the world, was also discussed in the speech. The CAS ONLINE now contains a wide variety of files relating to chemical field including CA file, Registry file. CA previews,. CASREACT, CIN. MARPAT, etc for supplying chemical information worldwide.

Progress and Prospects of CRISPR/Cas Systems in Insects and Other Arthropods

Directory of Open Access Journals (Sweden)

Dan Sun

2017-09-01

Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPR and the CRISPR-associated gene Cas9 represent an invaluable system for the precise editing of genes in diverse species. The CRISPR/Cas9 system is an adaptive mechanism that enables bacteria and archaeal species to resist invading viruses and phages or plasmids. Compared with zinc finger nucleases and transcription activator-like effector nucleases, the CRISPR/Cas9 system has the advantage of requiring less time and effort. This efficient technology has been used in many species, including diverse arthropods that are relevant to agriculture, forestry, fisheries, and public health; however, there is no review that systematically summarizes its successful application in the editing of both insect and non-insect arthropod genomes. Thus, this paper seeks to provide a comprehensive and impartial overview of the progress of the CRISPR/Cas9 system in different arthropods, reviewing not only fundamental studies related to gene function exploration and experimental optimization but also applied studies in areas such as insect modification and pest control. In addition, we also describe the latest research advances regarding two novel CRISPR/Cas systems (CRISPR/Cpf1 and CRISPR/C2c2 and discuss their future prospects for becoming crucial technologies in arthropods.

A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9

DEFF Research Database (Denmark)

Vad-Nielsen, Johan; Lin, Lin; Jensen, Kristopher Torp

2016-01-01

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve...

Lithiases vésicales géantes: A propos de 2 cas

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S. Ouedraogo

2016-12-01

Full Text Available Les lithiases vésicales sont fréquentes. Généralement, les calculs vésicaux sont de petite taille et leur traitement est relativement aisé. Les macro-lithiases vésicales sont rares. Nous présentons deux cas de macro-lithiase vésicale diagnostiqués dans un contexte social particulier. Il s’agit de deux patients de sexe masculin, l’un âgé de 47 ans et l’autre de 66 ans, reçus en consultation pour dysurie et douleur hypogastrique. Cette symptomatologie évoluait depuis plusieurs années. Les patients n’ont consulté que lorsque cette symptomatologie est devenue très gênante. L’examen physique a noté une masse hypogastrique. L’imagerie médicale a permis d’aboutir au diagnostic de macro-lithiase vésicale occupant quasiment toute l’aire vésicale. Une extraction par cystotomie sus-pubienne a été réalisée dans les 2 cas. Les suites opératoires furent simples. Ces observations mettent en lumière une consultation tardive dans une société où les questions touchant à la sphère uro-génitale restent un tabou.

Study relating to the physico-chemical behaviour of heavy water in nuclear reactors; Etudes relatives au comportement physico-chimique de l'eau lourde dans les reacteurs nucleaires

Energy Technology Data Exchange (ETDEWEB)

Chenouard, J.; Dirian, G.; Roth, E.; Vignet, P.; Platzer, R. [Commissariat a l' Energie Atomique, Grenoble (France). Centre d' Etudes Nucleaires

1959-07-01

Chemical and isotope pollution, and radiolytic decomposition are the two most important ways in which heavy water becomes degraded in nuclear reactors. Chemical pollution has led to the creation of ion exchange purification loops specially designed for reactors: the report contains a description in detail of the application of this purification method in CEA research reactors, including the analysis required, results obtained, and their interpretation. The intelligence obtained on radiolytic decomposition with the same facilities is also discussed, as well as the recombination apparatus and control equipment utilized. Finally, investigation to date in the CEA on recombination circuits for power reactors is also discussed. (author) [French] Parmi les degradations subies par l'eau lourde dans les reacteurs nucleaires, les deux plus importantes sont la pollution chimique et isotopique et la decomposition radiolytique. La pollution chimique a conduit a mettre au point pour le cas particulier des reacteurs, des circuits d'epuration par echange d'ions. On decrit ici en detail la mise en oeuvre de cette methode dans les reacteurs de recherche du CEA; les controles qu'elle necessite, les resultats obtenus et leur interpretation. En ce qui concerne la dissociation radiolytique de l'eau, les renseignements obtenus sur ces memes reacteurs sont communiques, ainsi que les details des dispositifs de recombinaison et des moyens de controle. Enfin, on fait le point des etudes poursuivies au CEA sur ces memes problemes de recombinaison dans le cas des reacteurs de puissance. (auteur)

Effets du statut d’emploi sur la santé et la sécurité au travail : le cas des auxiliaires familiales et sociales et des infirmières de soins à domicile au Québec

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Élise Ledoux

2005-05-01

Full Text Available Les données de cette recherche corroborent les principaux résultats de nombreuses études sur les liens entre la précarité d’emploi et les risques accrus à la santé et à la sécurité du travail (SST. Constituée de quatre études de cas de services de soutien à domicile (SAD de Centres locaux de services communautaires (CLSC, cette recherche montre comment l’organisation du travail peut soutenir les stratégies protectrices auxquelles ont recours les auxiliaires sociales et familiales (AFS et les infirmières de services de SAD ou leur nuire. Il s’avère que les pratiques organisationnelles positives auraient les mêmes effets sur les salariées régulières que sur celles qui ont un statut précaire, cependant ces dernières bénéficient rarement de ce soutien. Quant aux dysfonctionnements organisationnels, les personnels précaires disposent de moins de marges de manœuvre pour les pallier. Ils sont donc doublement à risque au regard de leur santé et de leur sécurité au travail.The data from this study corroborate the main results of many studies on the relationships between job precariousness and increased occupational health and safety (OHS risks. This project consists of four studies of cases of local community service centre (CLSC homecare services (HS and shows how work organization can have a positive or negative effect on the protective strategies used by HS homecare workers and nurses. Positive organizational practices would have the same effects on regular employees as on those with a precarious status; however, the latter rarely benefit from this support. As for organizational dysfunction, precarious employees have less margin of maneuver to compensate for it. They therefore face a two-fold greater occupational health and safety risk.

How Does Amino Acid Ligand Modulate Au Core Structure and Characteristics in Peptide Coated Au Nanocluster?

Science.gov (United States)

Li, Nan; Li, Xu; Zhao, Hongkang; Zhao, Lina

2018-03-01

The atomic structures and the corresponding physicochemical properties of peptide coated Au nanoclusters determine their distinctive biological targeting applications. To learn the modulation of amino acid ligand on the atomic structure and electronic characteristics of coated Au core is the fundamental knowledge for peptide coated Au nanocluster design and construction. Based on our recent coated Au nanocluster configuration study (Nanoscale, 2016, 8, 11454), we built the typically simplified Au13(Cys-Au-Cys) system to more clearly learn the basic modulation information of amino acid ligand on Au core by the density functional theory (DFT) calculations. There are two isomers as ligand adjacent bonding (Iso1) and diagonal bonding (Iso2) to Au13 cores. The geometry optimizations indicate the adjacent bonding Iso1 is more stable than Iso2. More important, the Au13 core of Iso1 distorts much more significantly than that of Iso2 by Cys-Au-Cys bonding through the root-mean-square deviation (RMSD) analysis, which modulate their electronic characteristics in different ways. In addition, the frontier molecular orbital results of Au13(Cys-Au-Cys) isomers confirm that the Au cores mainly determine the blue shifts of Au13(Cys-Au-Cys) systems versus the original Au13 core in their UV-visible absorption spectrum studies. The configuration of Au13 core performs deformation under Cys-Au-Cys ligand modulation to reach new stability with distinct atomic structure and electronic properties, which could be the theory basis for peptide coated AuNCs design and construction.

Investigating CRISPR-Cas systems in Clostridium botulinum via bioinformatics tools.

Science.gov (United States)

Negahdaripour, Manica; Nezafat, Navid; Hajighahramani, Nasim; Rahmatabadi, Seyyed Soheil; Ghasemi, Younes

2017-10-01

The Clustered regularly interspaced short palindromic repeats (CRISPR) systems are a type of innate immunity found in some prokaryotes, which protect them against alien genetic elements by targeting foreign nucleic acids. Some other functions are also attributed to these systems. Clostridium botulinum bacteria produce botulinum neurotoxins (BoNT), one of the deadliest known toxins for humans and some animals. Food poisoning due to these bacteria is still a challenge in food industries. On the other hand, BoNT has been widely investigated for therapeutic applications including different muscle disorders. Bont genes may be located on bacterial chromosomes, plasmids, or even prophages. Generally, the genomes of Cl. botulinum show a high level of plasticity. In order to investigate the presence and characteristics of CRISPRs in these anaerobe bacteria, an in silico study on 113 CRISPR arrays identified in 38 Cl. botulinum strains was performed. A high occurrence of CRISPR arrays (80%) were found, with a remarkable frequency on plasmids. Several (CRISPR-associated) Cas proteins from different types were recognized in the studied strains, which were mostly Cas6. The CRISPR-Cas systems were identified as type I or III, but no type II. The spacers showed more homology with bacterial plasmids than phages. Active CRISPR-Cas systems can prevent the transfer of foreign genes, which may also include bont genes. This study provides the first insight into the probable roles of CRISPR-Cas systems in Cl. botulinum strains such as toxigenicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Intérêt des collectivités pour l'implantation d'arbres nourriciers dans les parcs urbains : le cas de Villa El Salvador, au Pérou

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Lafontaine, M.

2017-01-01

Full Text Available Intérêt des collectivités pour l'implantation d'arbres nourriciers dans les parcs urbains : le cas de Villa El Salvador, au Pérou. A study was conducted in Villa El Salvador, Peru, in order to evaluate the interest of local community members in establishing food trees within neighbourhood public green areas. Focus groups and semi-directed interviews conducted with the community actors allowed to identify their perceptions regarding the role these trees could play, the most appropriate management methods for their establishment and maintenance, as well as the best ways of allocating their products. SWOT ( Strength - Weaknesses – Opportunities- Threats and AHP (Analytical Hierarchisation Process analyses permitted to identify the most important favourable and restrictive factors affecting the long-term success of this production strategy. Results show that the possibility of harvesting edible products for family consumption, neighbourhood food aid programs or sale would motivate the inhabitants to contribute to the activities related to the establishment and maintenance of food trees. However, in order to be successful, the implementation of food trees within public green areas has to be in line with neighbourhood policies developed by the local population and to carefully anticipate possible sources of conflicts.

Charge transport through O-deficient Au-MgO-Au junctions

KAUST Repository

Fadlallah, M. M.; Eckern, Ulrich; Rungger, Ivan; Schuster, Cosima; Schwingenschlö gl, Udo

2009-01-01

Metal-oxide heterostructures have been attracting considerable attention in recent years due to various technological applications. We present results of electronic structure and transport calculations for the Au-MgO-Au (metal-insulator-metal) heterostructure based on density-functional theory and the nonequilibrium Green’s functions method. The dependence of the conductance of the heterostructure on the thickness of the MgO interlayer and the interface spacing is studied. In addition, we address the effects of O vacancies. We observe deviations from an exponentially suppressed conductance with growing interlayer thickness caused by Au-O chemical bonds. Electronic states tracing back to O vacancies can increase the conductance. Furthermore, this effect can be enhanced by enlarging the interface spacing as the vacancy induced Mg states are shifted toward the Fermi energy.

Charge transport through O-deficient Au-MgO-Au junctions

KAUST Repository

Fadlallah, M. M.

2009-12-29

Metal-oxide heterostructures have been attracting considerable attention in recent years due to various technological applications. We present results of electronic structure and transport calculations for the Au-MgO-Au (metal-insulator-metal) heterostructure based on density-functional theory and the nonequilibrium Green’s functions method. The dependence of the conductance of the heterostructure on the thickness of the MgO interlayer and the interface spacing is studied. In addition, we address the effects of O vacancies. We observe deviations from an exponentially suppressed conductance with growing interlayer thickness caused by Au-O chemical bonds. Electronic states tracing back to O vacancies can increase the conductance. Furthermore, this effect can be enhanced by enlarging the interface spacing as the vacancy induced Mg states are shifted toward the Fermi energy.

CRISPR/Cas-Mediated Knockin in Human Pluripotent Stem Cells.

Science.gov (United States)

Verma, Nipun; Zhu, Zengrong; Huangfu, Danwei

2017-01-01

Fluorescent reporter and epitope-tagged human pluripotent stem cells (hPSCs) greatly facilitate studies on the pluripotency and differentiation characteristics of these cells. Unfortunately traditional procedures to generate such lines are hampered by a low targeting efficiency that necessitates a lengthy process of selection followed by the removal of the selection cassette. Here we describe a procedure to generate fluorescent reporter and epitope tagged hPSCs in an efficient one-step process using the CRISPR/Cas technology. Although the method described uses our recently developed iCRISPR platform, the protocols can be adapted for general use with CRISPR/Cas or other engineered nucleases. The transfection procedures described could also be used for additional applications, such as overexpression or lineage tracing studies.

Arthropathie destructrice des epaules au cours d�une acromegalie

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Nessrine Akasbi

2011-10-01

Full Text Available L�acromegalie est une maladie endocrinienne rare, en rapport avec une hypersecretion d�hormone de croissance. Elle a des consequences rhumatologiques: l�arthropathie peripherique, l�atteinte rachidienne et les syndromes canalaires. L�atteinte articulaire accompagne une acromegalie active, sa survenue apres un traitement radical et une remission complete est rare. Nous presentons le cas d�une patiente de 70 ans ayant un antecedent d�acromegalie sur adenome hypophysaire il y a 25 ans, traitee chirurgicalement et declaree en remission complete, a developpe une arthropathie destructrice des deux epaules. Le but de notre observation est de mettre le point sur la possibilite d�une atteinte articulaire au cours de l�acromegalie et de son retentissement fonctionnelle.

Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.

Science.gov (United States)

Xu, Rongfang; Wei, Pengcheng; Yang, Jianbo

2017-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation.

A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

Science.gov (United States)

Farasat, Iman; Salis, Howard M.

2016-01-01

The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9’s off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits. PMID:26824432

The new CAS-DIS digital ionosonde

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Wang Shun

2013-04-01

Full Text Available A high quality digital ionosonde called the Chinese Academy of Sciences digital ionosonde (CAS-DIS has been developed for investigations of the ionosphere. Two important features are used for the CAS-DIS; first, the technique of analog down-conversion has been replaced by the new approach of digital down-conversion technology. Secondly, to solve the problem of large instantaneous receiving bandwidth in digital receivers, an analog narrowband tracking filter is used for the CAS-DIS. The center frequency of the filter tracks the carrier frequency transmitted in real-time, to ensure that the frequency components are filtered out of the effective bandwidth. This report describes the system architecture of the CAS-DIS, its main features, and its test results for ionosphere detection. 

MacSyFinder: a program to mine genomes for molecular systems with an application to CRISPR-Cas systems.

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Sophie S Abby

Full Text Available Biologists often wish to use their knowledge on a few experimental models of a given molecular system to identify homologs in genomic data. We developed a generic tool for this purpose.Macromolecular System Finder (MacSyFinder provides a flexible framework to model the properties of molecular systems (cellular machinery or pathway including their components, evolutionary associations with other systems and genetic architecture. Modelled features also include functional analogs, and the multiple uses of a same component by different systems. Models are used to search for molecular systems in complete genomes or in unstructured data like metagenomes. The components of the systems are searched by sequence similarity using Hidden Markov model (HMM protein profiles. The assignment of hits to a given system is decided based on compliance with the content and organization of the system model. A graphical interface, MacSyView, facilitates the analysis of the results by showing overviews of component content and genomic context. To exemplify the use of MacSyFinder we built models to detect and class CRISPR-Cas systems following a previously established classification. We show that MacSyFinder allows to easily define an accurate "Cas-finder" using publicly available protein profiles.MacSyFinder is a standalone application implemented in Python. It requires Python 2.7, Hmmer and makeblastdb (version 2.2.28 or higher. It is freely available with its source code under a GPLv3 license at https://github.com/gem-pasteur/macsyfinder. It is compatible with all platforms supporting Python and Hmmer/makeblastdb. The "Cas-finder" (models and HMM profiles is distributed as a compressed tarball archive as Supporting Information.

Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System.

Science.gov (United States)

Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S; Kim, Dong H; Deng, Wenbin; Liu, Ying

2015-12-15

Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼ 33% correctly targeted clones) compared to conventional targeting protocol (∼ 3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations.

Harnessing type I and type III CRISPR-Cas systems for genome editing

DEFF Research Database (Denmark)

Li, Yingjun; Pan, Saifu; Zhang, Yan

2016-01-01

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any...... report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR...... and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided...

CRISPR-Cas9 Toolkit for Actinomycete Genome Editing

DEFF Research Database (Denmark)

Tong, Yaojun; Robertsen, Helene Lunde; Blin, Kai

2018-01-01

engineering approaches for boosting known and discovering novel natural products. In order to facilitate the genome editing for actinomycetes, we developed a CRISPR-Cas9 toolkit with high efficiency for actinomyces genome editing. This basic toolkit includes a software for spacer (sgRNA) identification......, a system for in-frame gene/gene cluster knockout, a system for gene loss-of-function study, a system for generating a random size deletion library, and a system for gene knockdown. For the latter, a uracil-specific excision reagent (USER) cloning technology was adapted to simplify the CRISPR vector...... construction process. The application of this toolkit was successfully demonstrated by perturbation of genomes of Streptomyces coelicolor A3(2) and Streptomyces collinus Tü 365. The CRISPR-Cas9 toolkit and related protocol described here can be widely used for metabolic engineering of actinomycetes....

CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production.

Science.gov (United States)

Li, Hung; Shen, Claire R; Huang, Chun-Hung; Sung, Li-Yu; Wu, Meng-Ying; Hu, Yu-Chen

2016-11-01

Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, but genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7942 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7942 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 435.0±35.0μg/L, an ≈11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Synthesis of Au Nanostars and Their Application as Surface Enhanced Raman Scattering-Activity Tags Inside Living Cells.

Science.gov (United States)

Cao, Xiaowei; Shi, Chaowen; Lu, Wenbo; Zhao, Hang; Wang, Man; Tong, Wei; Dong, Jian; Han, Xiaodong; Qian, Weiping

2015-07-01

This work presents the synthesis and characterization of Au nanostars (AuNSs) and demonstrates their application as surface enhanced Raman scattering (SERS)-activity tags for cellular imaging and sensing. Nile blue A (NBA) and bovine serum albumin (BSA) were used as Raman reporter molecules and capping materials, respectively. The SERS-activity tags were tested on human lung adenocarcinoma cell (A549) and alveolar type II cell (AT II) and found to present a low level of cytotoxicity and high chemical stability. These SERS-activity tags not only can be applied in multiplexed cellular imaging, including dark field imaging, transmission electron microscopy (TEM) and SERS imaging, but also can be used for cellular sensing. The SERS spectra clearly identified cellular important components such as proteins, nucleic acids, lipids, and carbohydrates. This study also shows that endocytosis is the main channel of tags internalized in cells. The AuNSs exhibiting strong surface enhanced Raman effects are utilized in the design of an efficient, stable SERS-activity tag for intracellular applications.

Colloidal Au and Au-alloy catalysts for direct borohydride fuel cells: Electrocatalysis and fuel cell performance

Science.gov (United States)

Atwan, Mohammed H.; Macdonald, Charles L. B.; Northwood, Derek O.; Gyenge, Elod L.

Supported colloidal Au and Au-alloys (Au-Pt and Au-Pd, 1:1 atomic ratio) on Vulcan XC-72 (with 20 wt% metal load) were prepared by the Bönneman method. The electrocatalytic activity of the colloidal metals with respect to borohydride electro-oxidation for fuel cell applications was investigated by voltammetry on static and rotating electrodes, chronoamperometry, chronopotentiometry and fuel cell experiments. The fundamental electrochemical techniques showed that alloying Au, a metal that leads to the maximum eight-electron oxidation of BH 4 -, with Pd or Pt, well-known catalysts of dehydrogenation reactions, improved the electrode kinetics of BH 4 - oxidation. Fuel cell experiments corroborated the kinetic studies. Using 5 mg cm -2 colloidal metal load on the anode, it was found that Au-Pt was the most active catalyst giving a cell voltage of 0.47 V at 100 mA cm -2 and 333 K, while under identical conditions the cell voltage using colloidal Au was 0.17 V.

Colloidal Au and Au-alloy catalysts for direct borohydride fuel cells: Electrocatalysis and fuel cell performance

Energy Technology Data Exchange (ETDEWEB)

Atwan, Mohammed H.; Northwood, Derek O. [Department of Mechanical, Auto and Materials Engineering, University of Windsor, Windsor (Canada N9B 3P4); Macdonald, Charles L.B. [Department of Chemistry and Biochemistry, University of Windsor, Windsor (Canada N9B 3P4); Gyenge, Elod L. [Department of Chemical and Biological Engineering, The University of British Columbia, Vancouver, BC (Canada V6T 1Z4)

2006-07-14

Supported colloidal Au and Au-alloys (Au-Pt and Au-Pd, 1:1 atomic ratio) on Vulcan XC-72 (with 20wt% metal load) were prepared by the Bonneman method. The electrocatalytic activity of the colloidal metals with respect to borohydride electro-oxidation for fuel cell applications was investigated by voltammetry on static and rotating electrodes, chronoamperometry, chronopotentiometry and fuel cell experiments. The fundamental electrochemical techniques showed that alloying Au, a metal that leads to the maximum eight-electron oxidation of BH{sub 4}{sup -}, with Pd or Pt, well-known catalysts of dehydrogenation reactions, improved the electrode kinetics of BH{sub 4}{sup -} oxidation. Fuel cell experiments corroborated the kinetic studies. Using 5mgcm{sup -2} colloidal metal load on the anode, it was found that Au-Pt was the most active catalyst giving a cell voltage of 0.47V at 100mAcm{sup -2} and 333K, while under identical conditions the cell voltage using colloidal Au was 0.17V. (author)

[CRISPR/CAS9, the King of Genome Editing Tools].

Science.gov (United States)

Bannikov, A V; Lavrov, A V

2017-01-01

The discovery of CRISPR/Cas9 brought a hope for having an efficient, reliable, and readily available tool for genome editing. CRISPR/Cas9 is certainly easy to use, while its efficiency and reliability remain the focus of studies. The review describes the general principles of the organization and function of Cas nucleases and a number of important issues to be considered while planning genome editing experiments with CRISPR/Cas9. The issues include evaluation of the efficiency and specificity for Cas9, sgRNA selection, Cas9 variants designed artificially, and use of homologous recombination and nonhomologous end joining in DNA editing.

Electrical conductivity dependence of thin metallic films of Au and Pd as a top electrode in capacitor applications

International Nuclear Information System (INIS)

Nazarpour, S.; Langenberg, E.; Jambois, O.; Ferrater, C.; Garcia-Cuenca, M.V.; Polo, M.C.; Varela, M.

2009-01-01

Electrical conductivity dependence of thin metallic films of Au and Pd over the different perovskites was investigated. It is found from electrical properties that crystallographic growth orientation of Au and Pd thin layers attained from X-ray diffraction results indicate the slop of current (I)-voltage (V) plots. Besides, surface morphology and topography was considered using Field Emission Scanning Electron Microscopy and Atomic Force Microscopy, respectively. Obtained results showed the Stranski-Krastanov growth of the Pd and Au. Indeed, diminishing of the root-mean-square roughness of Pd/BiMnO 3 /SrTiO 3 following by Au deposition should be concerned due to growth of Au onto the crack-like parts of the substrate. These crack-like parts appeared due to parasitic phases of the Bi-Mn-O system mainly Mn 3 O 4 (l 0 l) and Mn 3 O 4 (0 0 4 l). The different response in the electrical properties of heterostructures suggests that electrical conductance of the Au and Pd thin metallic films have the crystallographic orientation dependence. Furthermore, polycrystallinity of the thin metallic films are desired in electrode applications due to increase the conductivity of the metallic layers.

Facile Synthesis of Quasi-One-Dimensional Au/PtAu Heterojunction Nanotubes and Their Application as Catalysts in an Oxygen-Reduction Reaction.

Science.gov (United States)

Cai, Kai; Liu, Jiawei; Zhang, Huan; Huang, Zhao; Lu, Zhicheng; Foda, Mohamed F; Li, Tingting; Han, Heyou

2015-05-11

An intermediate-template-directed method has been developed for the synthesis of quasi-one-dimensional Au/PtAu heterojunction nanotubes by the heterogeneous nucleation and growth of Au on Te/Pt core-shell nanostructures in aqueous solution. The synthesized porous Au/PtAu bimetallic nanotubes (PABNTs) consist of porous tubular framework and attached Au nanoparticles (AuNPs). The reaction intermediates played an important role in the preparation, which fabricated the framework and provided a localized reducing agent for the reduction of the Au and Pt precursors. The Pt7 Au PABNTs showed higher electrocatalytic activity and durability in the oxygen-reduction reaction (ORR) in 0.1 M HClO4 than porous Pt nanotubes (PtNTs) and commercially available Pt/C. The mass activity of PABNTs was 218 % that of commercial Pt/C after an accelerated durability test. This study demonstrates the potential of PABNTs as highly efficient electrocatalysts. In addition, this method provides a facile strategy for the synthesis of desirable hetero-nanostructures with controlled size and shape by utilizing an intermediate template. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Les conjonctivites néonatales dans le canton de Glidji au Sud du Togo: une étude transversale à propos de 159 nouveau-nés

Science.gov (United States)

Kokou, Vonor; Nidain, Maneh; Kassoula, Nononsaa Batomguela; Kwassi, Fiaty- Amenouvor; Meba, Banla; Patrice, Balo Komi

2016-01-01

Introduction Le but de l’étude était décrire les aspects épidémiologiques des conjonctivites néonatales dans le canton de Glidji au Sud du Togo. Methodes Nous avons mené une étude transversale dans les 4 Unités Sanitaires Périphériques du canton de Glidji du 19 Mars au 13 Mai 2009 soit 8 semaines. Tous les nouveau-nés ont été inclus et la conjonctivite néonatale était définie par la présence chez un nouveau-né d'au moins deux des signes suivants: hyperhémie conjonctivale, œdème palpébral, chémosis, sécrétions purulentes, larmoiement. Les paramètres étudiés étaient l’âge, le sexe, les facteurs de risque, les antécédents, la présence ou non de conjonctivite, les germes en causes et l’évolution sous traitement. Resultats Sur la période, 159 nouveau-nés ont été examinés. L’âge moyen était de 10,9 jours avec des extrêmes de 0 à 28 jours. Il y avait 80 garçons pour 79 filles soit un sex-ratio de 1,01. Sur les 159 nouveau-nés, 7 cas de conjonctivite ont été diagnostiqués soit une prévalence de 4,4%. Les facteurs de risque identifiés étaient l'accouchement par voie basse et la présence d'IST chez la mère pendant la grossesse. Sur les 7 cas de conjonctivite, l'examen cytobactériologique a permis d'isoler le staphylococcus aureus dans 2 cas. L’évolution des cas de conjonctivite sous traitement était favorable avec régression des signes dès le 3è jour. Conclusion Les conjonctivites néonatales avaient une prévalence de 4,4% dans le canton de Glidji au sud du Togo et le staphylocoque doré était le germe en cause. Leur prévention passe par un bon suivi lors de la consultation prénatale et l'instillation de collyre antibiotique à la naissance PMID:27642383

Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

Science.gov (United States)

Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

2017-06-01

Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus.

Science.gov (United States)

Park, Joo Youn; Moon, Bo Youn; Park, Juw Won; Thornton, Justin A; Park, Yong Ho; Seo, Keun Seok

2017-03-21

Discovery of clustered, regularly interspaced, short palindromic repeats and the Cas9 RNA-guided nuclease (CRISPR/Cas9) system provides a new opportunity to create programmable gene-specific antimicrobials that are far less likely to drive resistance than conventional antibiotics. However, the practical therapeutic use of CRISPR/Cas9 is still questionable due to current shortcomings in phage-based delivery systems such as inefficient delivery, narrow host range, and potential transfer of virulence genes by generalized transduction. In this study, we demonstrate genetic engineering strategies to overcome these shortcomings by integrating CRISPR/Cas9 system into a temperate phage genome, removing major virulence genes from the host chromosome, and expanding host specificity of the phage by complementing tail fiber protein. This significantly improved the efficacy and safety of CRISPR/Cas9 antimicrobials to therapeutic levels in both in vitro and in vivo assays. The genetic engineering tools and resources established in this study are expected to provide an efficacious and safe CRISPR/Cas9 antimicrobial, broadly applicable to Staphylococcus aureus.

Magnetic susceptibilities of liquid Cr-Au, Mn-Au and Fe-Au alloys

Energy Technology Data Exchange (ETDEWEB)

Ohno, S.; Shimakura, H. [Niigata University of Pharmacy and Applied Life Sciences, Higashijima, Akiha-ku, Niigata 956-8603 (Japan); Tahara, S. [Faculty of Science, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213 (Japan); Okada, T. [Niigata College of Technology, Kamishin’eicho, Nishi-ku, Niigata 950-2076 (Japan)

2015-08-17

The magnetic susceptibility of liquid Cr-Au, Mn-Au, Fe-Au and Cu-Au alloys was investigated as a function of temperature and composition. Liquid Cr{sub 1-c}Au{sub c} with 0.5 ≤ c and Mn{sub 1-c}Au{sub c} with 0.3≤c obeyed the Curie-Weiss law with regard to their dependence of χ on temperature. The magnetic susceptibilities of liquid Fe-Au alloys also exhibited Curie-Weiss behavior with a reasonable value for the effective number of Bohr magneton. On the Au-rich side, the composition dependence of χ for liquid TM-Au (TM=Cr, Mn, Fe) alloys increased rapidly with increasing TM content, respectively. Additionally, the composition dependences of χ for liquid Cr-Au, Mn-Au, and Fe-Au alloys had maxima at compositions of 50 at% Cr, 70 at% Mn, and 85 at% Fe, respectively. We compared the composition dependences of χ{sub 3d} due to 3d electrons for liquid binary TM-M (M=Au, Al, Si, Sb), and investigated the relationship between χ{sub 3d} and E{sub F} in liquid binary TM-M alloys at a composition of 50 at% TM.

Cas9-triggered chain ablation of cas9 as a gene drive brake

OpenAIRE

Wu, Bing; Luo, Liqun; Gao, Xiaojing J.

2016-01-01

With the advent of clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) technology, researchers can construct gene drives that can bias the inheritance of edited alleles to alter entire populations. As demonstrated with the mutagenic chain reaction in Drosophila4, the CRISPR-Cas9 system can propagate genomic modification together with the genome-editing machinery itself. Although gene drives might have the potential to control insect-borne di...

A lentivirus-free inducible CRISPR-Cas9 system for efficient targeting of human genes.

Science.gov (United States)

Bisht, Kamlesh; Grill, Sherilyn; Graniel, Jacqueline; Nandakumar, Jayakrishnan

2017-08-01

CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Features of CRISPR-Cas Regulation Key to Highly Efficient and Temporally-Specific crRNA Production

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Andjela Rodic

2017-11-01

Full Text Available Bacterial immune systems, such as CRISPR-Cas or restriction-modification (R-M systems, affect bacterial pathogenicity and antibiotic resistance by modulating horizontal gene flow. A model system for CRISPR-Cas regulation, the Type I-E system from Escherichia coli, is silent under standard laboratory conditions and experimentally observing the dynamics of CRISPR-Cas activation is challenging. Two characteristic features of CRISPR-Cas regulation in E. coli are cooperative transcription repression of cas gene and CRISPR array promoters, and fast non-specific degradation of full length CRISPR transcripts (pre-crRNA. In this work, we use computational modeling to understand how these features affect the system expression dynamics. Signaling which leads to CRISPR-Cas activation is currently unknown, so to bypass this step, we here propose a conceptual setup for cas expression activation, where cas genes are put under transcription control typical for a restriction-modification (R-M system and then introduced into a cell. Known transcription regulation of an R-M system is used as a proxy for currently unknown CRISPR-Cas transcription control, as both systems are characterized by high cooperativity, which is likely related to similar dynamical constraints of their function. We find that the two characteristic CRISPR-Cas control features are responsible for its temporally-specific dynamical response, so that the system makes a steep (switch-like transition from OFF to ON state with a time-delay controlled by pre-crRNA degradation rate. We furthermore find that cooperative transcription regulation qualitatively leads to a cross-over to a regime where, at higher pre-crRNA processing rates, crRNA generation approaches the limit of an infinitely abrupt system induction. We propose that these dynamical properties are associated with rapid expression of CRISPR-Cas components and efficient protection of bacterial cells against foreign DNA. In terms of synthetic

Le syndrome néphrotique idiopathique (SNI) de l’enfant à Dakar: à propos de 40 cas

Science.gov (United States)

Keita, Younoussa; Lemrabott, Ahmed Tall; Sylla, Assane; Niang, Babacar; Ka, El Hadji Fary; Dial, Chérif Mohamed; Ndongo, Aliou Abdoulaye; Sow, Amadou; Moreira, Claude; Niang, Abdou; Ndiaye, Ousmane; Diouf, Boucar; Sall, Mouhamadou Guélaye

2017-01-01

Introduction L’objectif de ce travail était d’analyser les caractéristiques diagnostiques, thérapeutiques et évolutives de l’enfant atteint de néphrose dans un service de pédiatrie de Dakar. Méthodes L’étude était réalisée au service de pédiatrie de l’hôpital Aristide Le Dantec. Il s’agissait d’une étude rétrospective sur une période de 03 ans allant du 1er janvier 2012 au 31 décembre 2014. Ont été inclus tous les patients âgés de 02 ans à 12 ans présentant un tableau de Syndrome néphrotique idiopathique. Résultats Quarante cas de néphrose étaient colligés soit une prévalence de 23% parmi les néphropathies prises en charge dans le service. L’âge moyen était de 7,11± 3,14 ans. Le syndrome néphrotique était pur chez 72,5% (n=29) des patients. Les œdèmes des membres inférieurs étaient présents chez 100% des patients, l’oligurie dans 55% (n=22) et l’HTA dans 5% (n=2) des cas. La protéinurie moyenne était de 145,05 ± 85,54 mg/kg/24heures. La protidémie moyenne était de 46,42 ±7,88 g/L et l’albuminémie moyenne de 17,90 ± 7,15 g/L. Trente-neuf patients avaient reçu une corticothérapie à base de prednisone. La corticosensibilité était retenue chez 77% (n=30) des patients et la corticorésistance chez 13% (n=5) des cas. Le facteur de mauvaise réponse à la corticothérapie était un niveau de protéinurie initiale supérieure à 150 mg/kg/jour (p = 0,024). La biopsie rénale était réalisée chez 18% (n=7) des patients et retrouvait dans 57,2% (n=4) des cas une hyalinose segmentaire et focale. Le cyclophosphamide et l’azathioprine étaient associés aux corticoïdes dans 10% (n=4) des cas chacun. Le taux de rémission globale était de 89,8%. L’évolution vers l’insuffisance rénale chronique était notée chez trois (03) des patients. Conclusion La néphrose représentait près du quart des néphropathies prises en charge dans notre service. Le taux de rémission globale était élevé. Le

Profil épidémiologique des hémoglobinopathies: étude transversale descriptive autour du cas index

Science.gov (United States)

Dahmani, Fatima; Benkirane, Souad; Kouzih, Jaafar; Woumki, Aziz; Mamad, Hassan; Masrar, Azlarab

2017-01-01

Les hémoglobinopathies sont des affections constitutionnelles conséquentes à des anomalies des hémoglobines. Elles sont souvent graves dans leurs formes majeures, leur prise en charge est lourde avec un grand impact psycho-social sur les patients et leur famille. Classées parmi les maladies rares, elles sont encore insuffisamment connues des professionnels de santé. Cette méconnaissance est à l'origine d'une errance diagnostique, d'un retard dans leur prise en charge et par conséquent une morbidité et une mortalité élevée chez ces patients. L'Organisation Mondiale de la Santé (OMS) a publié en 2008 des données concernant l'épidémiologie des hémoglobinopathies: plus de 330000 cas naissent chaque année avec une hémoglobinopathie (83% des cas de drépanocytose, 17% des cas de thalassémie). Les troubles de l'hémoglobine sont responsables d'environ 3,4% des décès chez les moins de 5 ans. A l'échelle mondiale, 7% environ des femmes enceintes seraient porteuses d'une forme de la thalassémie et 1% des couples sont à risque. Toutefois, elles sont relativement fréquentes dans certaines régions du globe où les mariages consanguins sont communs. Afin de décrire les caractéristiques épidémiologiques des familles à risque d'hémoglobinopathies (étude autour du cas) dont les cas index sont suivis au service de pédiatrie à l'Hôpital Provincial El Idrisi de Kenitra au Maroc, une étude transversale descriptive a été réalisée durant deux enquêtes la première en mai 2015 et la deuxième en juin de la même année lors des journées de vaccination des cas index contre le pneumocoque. Après avoir recueilli les données épidémiologiques de nos patients, nous avons réalisé une étude biologique comportant: l'hémogramme avec étude morphologique des globules rouges en coloration MGG et numération automatique des réticulocytes; les électrophorèses de l'hémoglobine à pH alcalin (8.8) et secondairement à pH acide (5.4) sur gel d

Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9

OpenAIRE

Hidetaka Kaya; Masafumi Mikami; Akira Endo; Masaki Endo; Seiichi Toki

2016-01-01

The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adj...

Synthesis of biocompatible AuAgS/Ag2S nanoclusters and their applications in photocatalysis and mercury detection

International Nuclear Information System (INIS)

Zhao, Qian; Chen, Shenna; Zhang, Lingyang; Huang, Haowen; Liu, Fengping; Liu, Xuanyong

2014-01-01

In this paper, a facile approach for preparation of AuAgS/Ag 2 S nanoclusters was developed. The unique AuAgS/Ag 2 S nanoclusters capped with biomolecules exhibit interesting excellent optical and catalytic properties. The fluorescent AuAgS/Ag 2 S nanoclusters show tunable luminescence depending on the nanocluster size. The apoptosis assay demonstrated that the AuAgS/Ag 2 S nanoclusters showed low cytotoxicity and good biocompatibility. Therefore, the nanoclusters can be used not only as a probe for labeling cells but also for their photocatalytic activity for photodegradation of organic dye. Moreover, a highly selective and sensitive assay for detection of mercury including Hg 2+ and undissociated mercury complexes was developed based on the quenching fluorescent AuAgS/Ag 2 S nanoclusters, which provides a promising approach for determining various forms of Hg in the mercury-based compounds in environment. These unique nanoclusters may have potential applications in biological labeling, sensing mercury, and photodegradation of various organic pollutants in waste water.Graphical Abstract

L’équité au cœur des politiques climatiques : l’exemple des négociations relatives au climat et de la recherche de solutions à la crise énergétique

Directory of Open Access Journals (Sweden)

Loïc Aubrée

2011-02-01

Full Text Available L’article débat de la question de l’équité dans la lutte contre les changements climatiques. Il établit un parallèle entre le développement pris au niveau international de la négociation entre Etats d’une part, et la pauvreté au niveau d’une collectivité d’autre part. Dans les deux cas, il s’agit à la fois d’un principe moral essentiel, mais aussi d’une condition centrale des consensus et donc de la réussite des politiques face au défi climatique. La question de l’équité renvoie aussi très vite au changement nécessaire des modes de vie vers plus de sobriété.The article debates Equity Issues in the Climate Change debate is debated. A parallel is set between the development issues at the international level of negotiations between States, and poverty alleviation at the local community level. In both cases, the discussions stem from an essential moral principle, but also as a requisite for consensus and thus a condition of success of climate change policies to combat Climate Change. The issue of equity brings also forward the necessary modification of consumption patterns, into more frugal modes.

A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.

Science.gov (United States)

Qian, Pengge; Wang, Xu; Yang, Zhenke; Li, Zhenkui; Gao, Han; Su, Xin-Zhuan; Cui, Huiting; Yuan, Jing

2018-06-01

The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii. Copyright © 2018 Elsevier B.V. All rights reserved.

AuCu@Pt Nanoalloys for Catalytic Application in Reduction of 4-Nitrophenol

Directory of Open Access Journals (Sweden)

Sadia Mehmood

2016-01-01

Full Text Available To enhance and optimize nanocatalyst ability for nitrophenol (4-NP reduction reaction we look beyond Au-metal nanoparticles and describe a new class of Au nanoalloys with controlled composition for core of AuCu-metals and Pt-metal shell. The reduction of 4-NP was investigated in aqueous media spectroscopically on 7.8 nm Au nanospheres (AuNSs, 8.3 nm AuCuNSs, and 9.1 nm AuCu@Pt core-shell NSs in diameter. The rate constants of the catalyzed reaction at room temperature, activation energies, and entropies of activation of reactions catalyzed by the AuCu@Pt core-shell NSs are found to have different values to those of the pure metal NSs. The results strongly support the proposal that catalysis by nanoparticles is taking place efficiently on the surface of NSs. These core-shell nanocatalysts exhibited stability throughout the reduction reaction and proved that heterogonous type mechanisms are most likely to be dominant in nanoalloy based catalysis if the surface of the NSs is not defected upon shell incorporation.

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

Science.gov (United States)

Zhang, Qiang; Xing, Hui-Li; Wang, Zhi-Ping; Zhang, Hai-Yan; Yang, Fang; Wang, Xue-Chen; Chen, Qi-Jun

2018-03-01

We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

Une reine entre ombres et lumières ou le pouvoir au féminin : le cas d’Isabelle Bourbon, reine d’Espagne, première femme de Philippe IV (1603-1644

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Frédérique Sicard

2009-09-01

Full Text Available Le rôle de la Reine et sa relation au pouvoir sont strictement réglementés, mais son influence politique est paradoxalement difficile à apprécier car elle s’exerce à l’ombre de ce qui est permis. Isabelle de Bourbon, née Princesse de France, épouse en 1615 le Prince héritier Philippe d’Espagne et fait partie de ces Reines de l’ombre, peu étudiées. Elevée dans une certaine liberté, le contraste avec la cour d’Espagne et la surveillance de l’omniprésent Favori, le Comte-Duc d’Olivares, lui pèsent d’autant plus. Quel éclairage le cas de la Reine Isabelle permet-il d’apporter à la relation entre genre et pouvoir au Siècle d’Or ? Trois points seront abordés : les tensions et dominations qui se jouent autour du Roi Philippe IV, le poids de la famille, au sens large, indissociable des relations entretenues entre genre et pouvoir. Enfin, nous nous intéresserons à l’activité politique de la Reine Isabelle.The role of French queens and their relation to power were strictly regulated, but their political influence is difficult to assess given the shadows within which they worked. In 1615, Isabelle of Bourbon, born Princess of France, married Philip of Spain and became one of these seldom-studied Queens of the shadows. What illumination does Queen Isabelle’s case bring to the gender-power relationship during the Golden Age? Three points are addressed in the article: the tensions and power dynamics around Philip IV; the weight of the family, in a broad sense, which necessarily affected relations between gender and power. Finally, we will focus on the political activity of Queen Isabelle; her example constitutes a concrete example of women’s power in the Golden Age.

Les coûts du diabète sur 10 ans au Canada : intégration des coûts en soins de santé imputables au diabète à un modèle de prédiction de son incidence

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Anja Bilandzic

2017-01-01

Full Text Available Introduction : Notre objectif était d’estimer les coûts de santé directs liés au traitement du diabète au Canada sur 10 ans, à l’aide de données de sondages nationaux et d’un outil de prévision du risque de diabète, ainsi que les coûts par personne. Méthodologie : Nous avons utilisé le Diabetes Population Risk Tool pour estimer le nombre de nouveaux cas de diabète chez les personnes âgées de 20 ans et plus sur 10 ans (jusqu'en 2022 à l’aide des données de 2011 et de 2012 de l’Enquête sur la santé dans les collectivités canadiennes. Nous avons évalué les coûts liés au diabète à partir d’une étude de cohorte faisant appel à l’appariement par score de propension, en utilisant la base de données sur le diabète de l’Ontario ainsi que d’autres données administratives. Nous avons calculé les coûts totaux en utilisant les coûts associés aux nouveaux cas, en tenant compte du sexe, de l’année du diagnostic et des taux de mortalité annuels dus à la maladie. Résultats : D'après nos calculs, le risque de développer le diabète sur 10 ans s’élevait pour la population canadienne en 2011-2012 à 9,98 %, soit 2,16 millions de nouveaux cas. Les coûts totaux en soins de santé imputables au diabète pendant cette période sont de 15,36 milliards (7,55 milliards pour les femmes et 7,81 milliards pour les hommes. Ce sont les hospitalisations de courte durée qui constituent la majeure partie de ces coûts (43,2 %. Des interventions menées au sein de la population entraînant une perte de poids de 5 % au sein de la population permettraient de réduire les coûts des soins de santé de 2,03 milliards de dollars. Une réduction du risque de 30 % obtenue grâce à des interventions auprès des personnes à plus haut risque de développer le diabète (c.-à-d. les 10 % au sommet du groupe à risque le plus élevé entraînerait des économies de l’ordrede 1,48 million de dollars. Conclusion : D'ici 2022

Amélioration des tribunaux du travail au Mexique : le cas de Cuautitlán

International Development Research Centre (IDRC) Digital Library (Canada)

Ce projet de recherche dirigé par l'Instituto Tecnológico de México s'intéressera au contexte juridique du pays et aux procédures administratives liées aux décisions et aux ... Monitoring and notification : evidence from a field experiment in a Mexican labor court ... Delay and corruption : a simple model with empirical tests.

Enhanced middle-infrared light transmission through Au/SiO(x)N(y)/Au aperture arrays.

Science.gov (United States)

Xiao, Gongli; Yao, Xiang; Ji, Xinming; Zhou, Jia; Bao, Zongming; Huang, Yiping

2011-12-01

The enhanced middle-infrared light transmission through Au/SiO(x)N(y)/Au aperture arrays by changing the refractive index and the thickness of a dielectric layer was studied experimentally. The results indicated that the transmission spectra was highly dependent on the refractive index and the thickness of SiO(x)N(y). We found that the transmission peaks redshifted regularly along with the refractive index from 1.6 to 1.8, owing to the role of surface plasmon polaritons (SPP) coupling in the Au/SiO(x)N(y)/Au cascaded metallic structure. Simultaneously, a higher transmission efficiency and narrower transmission peak was obtained in Au/SiO2.1N0.3/Au cascaded metallic structure with small refractive index (1.6) than in Au/SiO0.6N1/Au cascaded metallic structure with large refractive index (1.8). When the thickness of SiO(x)N(y) changes from 0.2 to 0.4 microm, the shape of transmission spectra exhibits a large change. It was found that a higher transmission efficiency and narrower transmission peak was obtained in Au/SiO(x)N(y)/Au cascaded metallic structure with a thin dielectric film (0.2 microm), with the increase of SiO(x)N(y) film's thickness, the transmission peak gradually widened and disappeared finally. This effect is useful in applications of biochemical sensing and tunable integrated plasmonic devices in the middle-infrared region.

Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.

Science.gov (United States)

Lee, Hayun; Zhou, Yi; Taylor, David W; Sashital, Dipali G

2018-04-05

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration. Copyright © 2018 Elsevier Inc. All rights reserved.

The genome editing revolution: A CRISPR-Cas TALE off-target story.

Science.gov (United States)

Stella, Stefano; Montoya, Guillermo

2016-07-01

In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains. © 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.

Nouveaux modes de coordination des acteurs dans le développement local: cas des zones rurales au Bénin

Directory of Open Access Journals (Sweden)

Bernard G. Hounmenou

2003-10-01

Full Text Available Les politiques de développement en cours dans plusieurs pays du sud, notamment dans ceux de l’Afrique sub-saharienne ont amorcé un nouveau tournant à partir de la fin des années 1980. A la faveur de l’instauration du climat de démocratie dans plusieurs de ces pays à partir de cette période, nombre d’entre eux ont procédé à de profondes réformes, donnant à la société civile et aux populations à la base, un droit de regard plus important dans la conduite des affaires les concernant. L’une des manifestations les plus importantes de ces réformes se traduit par la mise en œuvre de la décentralisation. Ce processus vise entre autres, la prise en compte des réalités locales et la responsabilisation des communautés à la base dans la gestion de leur développement. Il s’agit en d’autres termes, de favoriser le renforcement des capacités des populations locales, en vue de leur permettre de prendre en charge de façon durable, la gestion de leurs propres affaires dans le cadre de la gouvernance participative. Amorcé en janvier 1993, avec les états généraux de l’administration territoriale, le processus de la décentralisation au Bénin n’a connu sa phase de concrétisation qu’avec les élections et l’installation des élus locaux en début 2003. En prélude à l’avènement de la décentralisation, plusieurs localités béninoises font l’objet, depuis le milieu des années 1990, d’expériences d’appui au développement des communautés locales. C’est le cas notamment, des villages des départements de l’Atlantique et des Collines. A travers ces expériences, se mettent en place, divers dispositifs de gouvernance locale au sein des systèmes locaux d’action publique relatifs aux localités rurales. Il s’agit notamment, des comités villageois de suivi ou de gestion, des dispositifs de comités villageois de développement dans le département de l’Atlantique, et du dispositif des projets de

Near-infrared-responsive, superparamagnetic Au@Co nanochains

Directory of Open Access Journals (Sweden)

Varadee Vittur

2017-08-01

Full Text Available This manuscript describes a new type of nanomaterial, namely superparamagnetic Au@Co nanochains with optical extinctions in the near infrared (NIR. The Au@Co nanochains were synthesized via a one-pot galvanic replacement route involving a redox-transmetalation process in aqueous medium, where Au salt was reduced to form Au shells on Co seed templates, affording hollow Au@Co nanochains. The Au shells serve not only as a protective coating for the Co nanochain cores, but also to give rise to the optical properties of these unique nanostructures. Importantly, these bifunctional, magneto-optical Au@Co nanochains combine the advantages of nanophotonics (extinction at ca. 900 nm and nanomagnetism (superparamagnetism and provide a potentially useful new nanoarchitecture for biomedical or catalytic applications that can benefit from both activation by light and manipulation using an external magnetic field.

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

Science.gov (United States)

Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

2016-01-01

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

Fabrication of Au-Pd Core-shell Nanoparticles using Au Thin-Film Dewetting at High Temperature and Chemical Synthesis Methods

Energy Technology Data Exchange (ETDEWEB)

Kim, Min-Gyu; Lee, Hye-Jung; Oh, Yong-Jun [Hanbat National Univ., Daejeon (Korea, Republic of)

2016-07-15

Au-Pd bimetallic nanoparticles (NPs) have received a lot of attention in the fields of catalysts and hydrogen sensors. In this study, Au-Pd core-shell NP arrays were successfully fabricated using two steps: formation of the ordered array of Au NPs cores via solid-state dewetting of a Au thin film on a topographic silica substrate, and Pd shell formation via chemical synthesis using two different surfactants (CTAB and CTAC). Using the CTAB surfactant in particular, a 2-D composite structure comprised of an ordered array of Au-Pd NPs, with smaller Pd NPs on the nanoscopic gaps between the Au-Pd NPs, could be formed. This structure is expected to have potential application in resistance-base hydrogen sensors.

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins

Directory of Open Access Journals (Sweden)

Lia Carolina Soares Medeiros

2017-11-01

Full Text Available Trypanosomatids (order Kinetoplastida, including the human pathogens Trypanosoma cruzi (agent of Chagas disease, Trypanosoma brucei, (African sleeping sickness, and Leishmania (leishmaniasis, affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9 technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9, but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9, and in vitro-transcribed single guide RNAs (sgRNAs results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi, as well as in T. brucei and Leishmania major. RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens.

The point-defect of carbon nanotubes anchoring Au nanoparticles

DEFF Research Database (Denmark)

Lv, Y. A.; Cui, Y. H.; Li, X. N.

2010-01-01

The understanding of the interaction between Au and carbon nanotubes (CNTs) is very important since Au/CNTs composites have wide applications in many fields. In this study, we investigated the dispersion of Au nanoparticles on the CNTs by transmission electron microscopy and the bonding mechanism...

Electrogenerated Chemiluminescence Behavior of Au nanoparticles-hybridized Pb (II) metal-organic framework and its application in selective sensing hexavalent chromium.

Science.gov (United States)

Ma, Hongmin; Li, Xiaojian; Yan, Tao; Li, Yan; Liu, Haiyang; Zhang, Yong; Wu, Dan; Du, Bin; Wei, Qin

2016-02-23

In this work, a novel electrochemiluminescence (ECL) sensor based on Au nanoparticles-hybridized Pb (II)-β-cyclodextrin (Pb-β-CD) metal-organic framework for detecting hexavalent chromium (Cr(VI)) was developed. Pb-β-CD shows excellent ECL behavior and unexpected reducing ability towards Au ions. Au nanoparticles could massively form on the surface of Pb-β-CD (Au@Pb-β-CD) without use of any additional reducing agent. In the presence of coreactant K2S2O8, the ECL emission of Pb-β-CD was enhanced by the formation of Au nanoparticles. Cr(VI) can collisionally quench the ECL behavior of Au@Pb-β-CD/S2O8(2-) system and the detection mechanism was investigated. This ECL sensor is found to have a linear response in the range of 0.01-100 μM and a low detection limit of 3.43 nM (S/N = 3) under the optimal conditions. These results suggest that metal-organic framework Au@Pb-β-CD has great potential in extending the application in the ECL field as an efficient luminophore.

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

Science.gov (United States)

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

CRISPR/Cas9-mediated viral interference in plants

KAUST Repository

Ali, Zahir

2015-11-11

Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

The use of notebooks in mathematics instruction. What is manageable? What should be avoided? A field report after 10 years of CAS-application

OpenAIRE

Hofbauer, Peter

2012-01-01

Computer Algebra Systems (CAS) have been changing the mathematics instruction requirements for many years. Since the tendency of using CAS in mathematics instruction has been rising for decades and reports have often been positive, the implementation of notebook classes seems to be the consequent next step of mathematics instruction supported by computers. Experiences that have been made with the use of CAS in PC-rooms can be transformed directly into the classroom. Hence the use of CAS is no...

The expanding footprint of CRISPR/Cas9 in the plant sciences

Science.gov (United States)

CRISPR/Cas9 has evolved and transformed the field of biology at an unprecedented pace. From the initial purpose of introducing a site specific mutation within a genome of choice, this technology has morphed into enabling a wide array of molecular applications, including site-specific transgene inser...

Editing plants for virus resistance using CRISPR-Cas.

Science.gov (United States)

Green, J C; Hu, J S

This minireview summarizes recent advancements using the clustered regularly interspaced palindromic repeats-associated nuclease systems (CRISPR-Cas) derived from prokaryotes to breed plants resistant to DNA and RNA viruses. The CRISPR-Cas system represents a powerful tool able to edit and insert novel traits into plants precisely at chosen loci offering enormous advantages to classical breeding. Approaches to engineering plant virus resistance in both transgenic and non-transgenic plants are discussed. Iterations of the CRISPR-Cas system, FnCas9 and C2c2 capable of editing RNA in eukaryotic cells offer a particular advantage for providing resistance to RNA viruses which represent the great majority of known plant viruses. Scientists have obtained conflicting results using gene silencing technology to produce transgenic plants resistant to geminiviruses. CRISPR-Cas systems engineered in plants to target geminiviruses have consistently reduced virus accumulation providing increased resistance to virus infection. CRISPR-Cas may provide novel and reliable approaches to control geminiviruses and other ssDNA viruses such as Banana bunchy top virus (BBTV).

Controlled electrodeposition of Au monolayer film on ionic liquid

Energy Technology Data Exchange (ETDEWEB)

Ma, Qiang; Pang, Liuqing; Li, Man; Zhang, Yunxia; Ren, Xianpei [Key Laboratory of Applied Surface and Colloid Chemistry, National Ministry of Education, Shaanxi Engineering Lab for Advanced Energy Technology, School of Materials Science and Engineering, Shaanxi Normal University, Xi’an 710062 (China); Liu, Shengzhong Frank, E-mail: szliu@dicp.ac.cn [Key Laboratory of Applied Surface and Colloid Chemistry, National Ministry of Education, Shaanxi Engineering Lab for Advanced Energy Technology, School of Materials Science and Engineering, Shaanxi Normal University, Xi’an 710062 (China); Dalian Institute of Chemical Physics, Dalian National Laboratory for Clean Energy, Chinese Academy of Sciences, Dalian 116023 (China)

2016-05-15

Highlights: • We fabricate Au monolayer film on Ionic liquid substrate using an electrochemical deposition technique. • Au monolayer film was deposited on a “soft substrate” for the first time. • Au monolayer film can contribute extra Raman enhancement. - Abstract: Gold (Au) nanoparticles have been attractive for centuries for their vibrant appearance enhanced by their interaction with sunlight. Nowadays, there have been tremendous research efforts to develop them for high-tech applications including therapeutic agents, sensors, organic photovoltaics, medical applications, electronics and catalysis. However, there remains to be a challenge to fabricate a monolayer Au coating with complete coverage in controlled fashion. Here we present a facile method to deposit a uniform Au monolayer (ML) film on the [BMIM][PF{sub 6}] ionic liquid substrate using an electrochemical deposition process. It demonstrates that it is feasible to prepare a solid phase coating on the liquid-based substrate. Moreover, the thickness of the monolayer coating can be controlled to a layer-by-layer accuracy.

CAS - CERN Accelerator School: Specialised course on Magnets

CERN Document Server

CAS 2009

2010-01-01

These proceedings present the lectures given at the twenty-third specialized course organized by the CERN Accelerator School (CAS), the topic being 'Magnets'. The course was held in Bruges, Belgium, from 16 to 25 June 2009. This is the first time this topic has been selected for a specialized course. Taking into account the number of related applications currently in use in accelerators around the world, but, even more important, the worrying decrease in the corresponding expertise in the different laboratories, it was recognized that such a topic should definitively be incorporated into the CAS series of specialized courses. The specific aim of the course was to introduce the participants to the basics of resistive magnet design and its underlying theoretical concepts. The first part of the school dealt with basic introductory courses such as Maxwell's equations for magnets, beam optics, physics and measurement of magnetic materials, the different types of resistive magnets and their respective performance, ...

La Cyberdépendance: Cas de l’addiction au réseau social Facebook

Directory of Open Access Journals (Sweden)

Houssem Edine Nasr

2015-09-01

Full Text Available La démocratisation de l’internet et la forte pénétration des réseaux sociaux dans la vie de tous les jours des internautes a montré un comportement d’addiction au réseau social Facebook. Malgré l’importance de ce phénomène, très peu de recherches en marketing se sont intéressées à son étude. La présente recherche a pour objectif de mesurer l’addiction des internautes aux réseaux sociaux et d’en déterminer les antécédents. La revue de la littérature relative à l’internet a mis en relief que l’estime de soi et la personnalité sont des antécédents psychologiques de l’addiction. L’enquête par questionnaire que nous avons menée sur Facebook auprès d’un échantillon de convenance de 252 internautes a révélé que trois dimensions sur cinq de la personnalité à savoir l’ouverture aux expériences, l’agréabilité et le névrosisme, sont en relation faible avec la cyberdépendance au réseau social Facebook. Par ailleurs, la variable âge et le nombre d’amis sur Facebook sont en corrélation avec la cyberdépendance à ce réseau social.

Programmable RNA recognition and cleavage by CRISPR/Cas9.

Science.gov (United States)

O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

2014-12-11

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

Throne of Glass, Celaena Sardothien et la violence au féminin

Directory of Open Access Journals (Sweden)

Fanie Demeule

2017-05-01

Full Text Available En analysant le cas de l’héroïne de la série de fantasy jeunesse Throne of Glass de Sarah J. Maas, cet article désire réfléchir sur la normativité des représentations de la violence physique chez un personnage littéraire de sexe féminin. Pour ce faire, nous examinerons dans un premier temps les stratégies de négociation de la violence du personnage de Celaena Sardothien à travers le dispositif textuel, puis nous observerons la polémique entourant sa réception au sein du lectorat.

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System.

Science.gov (United States)

Nandal, Anjali; Mallon, Barbara; Telugu, Bhanu P

2017-11-08

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide "NGG" protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

Epithelioma Spinocellulaire sur Cicatrice de Brulure (a Propos de Cinq Cas)

Science.gov (United States)

Tourabi, K.; Mejjati, H.; Ribqg, Y.; Achbouk, A.; Arrob, A.; Moussaoui, A.; Ihrai, H.

2009-01-01

Summary Pour étudier l'ulcère de Marjolin, tous les dossiers des patients qui se sont présentés pendant une période quinquennale à un Service de Chirurgie Plastique et des Brûlés au Maroc ayant un cancer sur cicatrice de brûlure, objectivé par un examen histopathologique, ont été inclus dans une fiche de recueil de données comprenant des paramètres liés à l'identité du malade, à l'inventaire préthérapeutique, au traitement et à l'évolution du cas. Les Auteurs, après avoir présenté les données des cinq patients inclus dans l'étude, considèrent les problèmes posés par les épithéliomas spinocellulaires sur cicatrice de brûlure, qui sont des affections graves et rares. La dégénérescence des cicatrices de brûlures est une évolution dramatique, causée par la négligence. Le traitement préventif par exérèse systématique de toute lésion suspecte doit être fortement souligné puisqu'il garantit la guérison quasi certaine. PMID:21991185

Inconclusive studies on possible CRISPR-Cas off-targets should ...

Indian Academy of Sciences (India)

Sandeep Chakraborty

Published online: 30 April 2018. Keywords. ... in gene-editing technologies have resulted from the simplicity of the single effector (Cas9) class 2 CRISPR-Cas ... a Cas9/gRNA concentration dependence on off-target activity (Pattanayak et al.

Traumatismes Du Rachis Chez L\\'enfant | Diop | African Journal of ...

African Journals Online (AJOL)

Les explorations neuroradiologiques avaient montré au niveau dorsolombaire un cas de fracture, un cas de luxation, un cas de fracture luxation, et au niveau cervical une entorse C1-C2. Dans un cas, le diagnostic de SCIWORA (« Spinal Cord Injury Without Radiological Abnormalities » c\\'est-à-dire traumatisme médullaire ...

Optimizing the planar structure of (1 1 1) Au/Co/Au trilayers

International Nuclear Information System (INIS)

Kumah, D P; Cebollada, A; Clavero, C; Garcia-MartIn, J M; Skuza, J R; Lukaszew, R A; Clarke, R

2007-01-01

Au/Co/Au trilayers are interesting for a range of applications which exploit their unusual optical and electronic transport behaviour in a magnetic field. Here we present a comprehensive structural and morphological study of a series of trilayers with 0-7 nm Co layer thickness fabricated on glass by ultrahigh vacuum vapour deposition. We use a combination of in situ electron diffraction, atomic force microscopy and x-ray scattering to determine the optimum deposition conditions for highly textured, flat and continuous layered structures. The 16 nm Au-on-glass buffer layer, deposited at ambient temperature, is found to develop a smooth (1 1 1) texture on annealing at 350 deg. C for 10 min. Subsequent growth of the Co layer at 150 deg. C produces a (1 1 1) textured film with lateral grain size of ∼150 nm in the 7 nm-thick Co layer. A simultaneous in-plane and out-of-plane Co lattice expansion is observed for the thinnest Co layers, converging to bulk values for the thickest films. The roughness of the Co layer is similar to that of the Au buffer layer, indicative of conformal growth. The 6 nm Au capping layer smoothens the trilayer surface, resulting in a surface roughness independent of the Co layer thickness

Developing an aqueous approach for synthesizing Au and M@Au (M = Pd, CuPt) hybrid nanostars with plasmonic properties

OpenAIRE

Du, Jingshan; Yu, Junjie; Xiong, Yalin; Lin, Zhuoqing; Zhang, Hui; Yang, Deren

2014-01-01

Anisotropic Au nanoparticles show unique localized surface plasmon resonance (LSPR) properties, which make it attractive in optical, sensing, and biomedical applications. In this contribution, we report a general and facile strategy towards aqueous synthesis of Au and M@Au (M = Pd, CuPt) hybrid nanostars by reducing HAuCl4 with ethanolamine in the presence of cetyltrimethylammonium bromide (CTAB). According to electron microscopic observation and spectral monitoring, we found that the layered...

Application of 198Au colloid in the treatment of chronic knee synovial effusions

International Nuclear Information System (INIS)

Buril, J.; Zak, J.

1979-01-01

25 cases of chronic synovial exudates of the knee joint were treated with the use of 198 Au colloid (5mCi). Improvement was noted in two thirds of the patients after different numbers of applications (one to five). No improvement was seen in four cases of progressive polyarthritis or in two patients with gonarthrosis. This was to be expected though the number of the cases of the disease treated was very low. (author)

CRISPR-Cas

NARCIS (Netherlands)

Jackson, Simon A.; McKenzie, Rebecca E.; Fagerlund, Robert D.; Kieper, Sebastian N.; Fineran, Peter C.; Brouns, Stan J.J.

2017-01-01

Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective

Ethos: pour une mise au point conceptuelle et méthodologique

OpenAIRE

Dhondt, Reindert; Vanacker, Beatrijs

2013-01-01

En guise de préambule théorique aux études de cas réunies dans ce numéro, le présent article se veut une mise au point du concept d’ethos et de ses concepts voisins, tout en formulant quelques-uns des défis, tant définitionnels que méthodologiques, qui en résultent. Aussi cette contribution adopte-t-elle une double démarche, proposant d’abord un regard rétrospectif, qui résume le parcours historique de la notion d’ethos et de la figure de l’auteur en général, avant de formuler quelques pistes...

The role of Cas8 in type I CRISPR interference.

Science.gov (United States)

Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

2015-05-05

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. © 2015 Authors.

Exploiting CRISPR-Cas to manipulate Enterococcus faecalis populations.

Science.gov (United States)

Hullahalli, Karthik; Rodrigues, Marinelle; Palmer, Kelli L

2017-06-23

CRISPR-Cas provides a barrier to horizontal gene transfer in prokaryotes. It was previously observed that functional CRISPR-Cas systems are absent from multidrug-resistant (MDR) Enterococcus faecalis , which only possess an orphan CRISPR locus, termed CRISPR2, lacking cas genes. Here, we investigate how the interplay between CRISPR-Cas genome defense and antibiotic selection for mobile genetic elements shapes in vitro E. faecalis populations. We demonstrate that CRISPR2 can be reactivated for genome defense in MDR strains. Interestingly, we observe that E. faecalis transiently maintains CRISPR targets despite active CRISPR-Cas systems. Subsequently, if selection for the CRISPR target is present, toxic CRISPR spacers are lost over time, while in the absence of selection, CRISPR targets are lost over time. We find that forced maintenance of CRISPR targets induces a fitness cost that can be exploited to alter heterogeneous E. faecalis populations.

Properties of ordered titanium templates covered with Au thin films for SERS applications

Energy Technology Data Exchange (ETDEWEB)

Grochowska, Katarzyna, E-mail: kgrochowska@imp.gda.pl [Centre for Plasma and Laser Engineering, Szewalski Institute of Fluid-Flow Machinery, Polish Academy of Sciences, Fiszera 14 St., 80-231 Gdańsk (Poland); Siuzdak, Katarzyna [Centre for Plasma and Laser Engineering, Szewalski Institute of Fluid-Flow Machinery, Polish Academy of Sciences, Fiszera 14 St., 80-231 Gdańsk (Poland); Sokołowski, Michał; Karczewski, Jakub [Faculty of Applied Physics and Mathematics, Gdańsk University of Technology, Narutowicza 11/12 St., 80-233 Gdańsk (Poland); Szkoda, Mariusz [Centre for Plasma and Laser Engineering, Szewalski Institute of Fluid-Flow Machinery, Polish Academy of Sciences, Fiszera 14 St., 80-231 Gdańsk (Poland); Faculty of Chemistry, Gdańsk University of Technology, Narutowicza 11/12 St., 80-233 Gdańsk (Poland); Śliwiński, Gerard [Centre for Plasma and Laser Engineering, Szewalski Institute of Fluid-Flow Machinery, Polish Academy of Sciences, Fiszera 14 St., 80-231 Gdańsk (Poland)

2016-12-01

Graphical abstract: - Highlights: • Dimpled Ti substrates prepared via anodization followed by etching. • Highly ordered nano-patterned titanium templates covered with thin Au films. • Enhanced Raman signal indicates on promising sensing material. - Abstract: Currently, roughened metal nanostructures are widely studied as highly sensitive Raman scattering substrates that show application potential in biochemistry, food safety or medical diagnostic. In this work the structural properties and the enhancement effect due to surface enhanced Raman scattering (SERS) of highly ordered nano-patterned titanium templates covered with thin (5–20 nm) gold films are reported. The templates are formed by preparation of a dense structure of TiO{sub 2} nanotubes on a flat Ti surface (2 × 2 cm{sup 2}) and their subsequent etching down to the substrate. SEM images reveal the formation of honeycomb nanostructures with the cavity diameter of 80 nm. Due to the strongly inhomogeneous distribution of the electromagnetic field in the vicinity of the Au film discontinuities the measured average enhancement factor (10{sup 7}–10{sup 8}) is markedly higher than observed for bare Ti templates. The enhancement factor and Raman signal intensity can be optimized by adjusting the process conditions and thickness of the deposited Au layer. Results confirm that the obtained structures can be used in surface enhanced sensing.

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

Science.gov (United States)

Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

2012-05-01

The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

Control of gene expression by CRISPR-Cas systems

Science.gov (United States)

2013-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this review, we summarize the role of these loci in the regulation of gene expression as well as the recent development of synthetic gene regulation using engineered CRISPR-Cas systems. PMID:24273648

Intoxication aiguë sévère par les pesticides organophosphorés: à propos de 28 cas

Directory of Open Access Journals (Sweden)

Ali Derkaoui

2011-03-01

Full Text Available Les pesticides organophosphorés (POP sont des pesticides organiques de synthèse, largement utilisés en agriculture essentiellement comme insecticide, nemacide ou acaricide. Ce sont les produits agricoles, les plus incriminés dans les intoxications dans notre contexte. L’objectif de ce travail était de déterminer les caractéristiques cliniques, paracliniques, et évolutives de cette intoxication en milieu de réanimation. étude rétrospective portant sur les cas admis en réanimation (2003-2010. Les critères d’inclusion étaient d’ordre clinique, para clinique, thérapeutique et évolutif. 28 cas ont été recensés : 19 femmes et 9 hommes, âge moyen = 24,5 plus or minus 11 ans. La tentative de suicide était le principal motif d’intoxication (19cas. Le Glasgow coma score était en moyenne de 11 plus or minus 4. Le syndrome central, était présent chez 78% de nos patients, suivi du syndrome muscarinique 71% et le syndrome nicotinique dans 53% des cas. La prise en charge thérapeutique a consisté à la ventilation mécanique dans 50% des cas, l’usage des drogues vasoactives dans 14% des cas et l’administration d’un traitement antidotique dans 64% des cas. La mortalité globale était de 25%. Les pesticides organophosphorés sont les toxiques agricoles, le plus souvent incriminés dans notre contexte. Les symptômes résultent d’une importante accumulation d’Acétyle-choline (Ach dans l’organisme ; responsable de l’apparition des trois syndromes caractéristiques. Le diagnostic biologique se fait par le dosage de l’activité cholinestérasique dans le plasma. Le traitement associe des mesures symptomatiques qui reposent essentiellement sur la réanimation respiratoire et neurologique au traitement antidotique. L’évolution clinique dans ce type d’intoxication, est généralement favorable sous traitement avec régression des signes en quelques jours. Le décès est essentiellement, le fait d�

Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9.

Science.gov (United States)

Raper, Austin T; Stephenson, Anthony A; Suo, Zucai

2018-02-28

The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

CRISPR/Cas9 Technology as an Emerging Tool for Targeting Amyotrophic Lateral Sclerosis (ALS).

Science.gov (United States)

Kruminis-Kaszkiel, Ewa; Juranek, Judyta; Maksymowicz, Wojciech; Wojtkiewicz, Joanna

2018-03-19

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) is a genome editing tool that has recently caught enormous attention due to its novelty, feasibility, and affordability. This system naturally functions as a defense mechanism in bacteria and has been repurposed as an RNA-guided DNA editing tool. Unlike zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 takes advantage of an RNA-guided DNA endonuclease enzyme, Cas9, which is able to generate double-strand breaks (DSBs) at specific genomic locations. It triggers cellular endogenous DNA repair pathways, contributing to the generation of desired modifications in the genome. The ability of the system to precisely disrupt DNA sequences has opened up new avenues in our understanding of amyotrophic lateral sclerosis (ALS) pathogenesis and the development of new therapeutic approaches. In this review, we discuss the current knowledge of the principles and limitations of the CRISPR/Cas9 system, as well as strategies to improve these limitations. Furthermore, we summarize novel approaches of engaging the CRISPR/Cas9 system in establishing an adequate model of neurodegenerative disease and in the treatment of SOD1-linked forms of ALS. We also highlight possible applications of this system in the therapy of ALS, both the inherited type as well as ALS of sporadic origin.

CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.

Science.gov (United States)

Veeranagouda, Yaligara; Debono-Lagneaux, Delphine; Fournet, Hamida; Thill, Gilbert; Didier, Michel

2018-01-16

The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption

NARCIS (Netherlands)

Johansen, Anne Katrine; Molenaar, Bas; Versteeg, Danielle; Leitoguinho, Ana Rita; Demkes, Charlotte; Spanjaard, Bastiaan; de Ruiter, Hesther; Akbari Moqadam, Farhad; Kooijman, Lieneke; Zentilin, Lorena; Giacca, Mauro; van Rooij, Eva

2017-01-01

RATIONALE: CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)-based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in

Mise au point

African Journals Online (AJOL)

dont 1,96% de cas de rhinoplastie, 2,17%des cas de chi- ... total thyroidectomy and histological exam. nine-month treatment with anti- .... 5. Lecerf j.m. Les thyroïdites. La gazette méd 1993 ; 101 : 10-14. 6. el malki HO, mohsine r, Benkhraba k.

Interference activity of a minimal Type I CRISPR–Cas system from Shewanella putrefaciens

Science.gov (United States)

Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

2015-01-01

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. PMID:26350210

Some applications of neutron activation analysis in plant biology and agronomy; Quelques applications de l'analyse par radioactivation neutronique en biologie vegetale et en agronomie

Energy Technology Data Exchange (ETDEWEB)

Fourcy, A [Commissariat a l' Energie Atomique, Grenoble (France). Centre d' Etudes Nucleaires

1966-06-01

Plants materials are not so commonly analysed by radioactivation than biological extracts of medical importance. With help of concrete examples, applications of neutrons activation analysis to the determination of some metals (Mn, Cu, Co, Fe, Zn, and K) in plant materials, are proposed. Samples are activated in a swimming-pool reactor at the thermal flux of 5.10{sup 12} n.cm{sup -2}s{sup -1} for a time varying between few minutes and several days according to the element being analysed. The induced radioactivity is measured by spectrometry, with radiochemical separation ( Cu, Co, Fe, Zn and K) or without separation in best cases (Mn,Cu, K). Described dosages are related to: manganese in a graminaceous plant, copper in vine treatments, cobalt, iron and zinc in animal feeding, potassium in a radiological experiment. (author) [French] Les produits vegetaux sont beaucoup moins souvent soumis a l'analyse par activation que les extraits biologiques d'interet medical. En s'appuyant sur des cas concrets, nous proposons des applications de l'analyse par activation neutronique au dosage de differents metaux (Mn, Cu, Co, Fe, Zn, et K) dans la matiere vegetale. Les echantillons sont actives en pile piscine au flux thermique de 5.10{sup 12} n.cm{sup -2} s{sup -1} pendant des temps variant de quelques minutes a plusieurs jours selon les elements a analyser. le comptage de la radioactivite induite est effectue par spectrometrie gamma apres separation radiochimique (Cu, Co, Fe, Zn, et K) ou sans separation dans les cas favorables (Mn, Cu, K). Les dosages decrits concernent: le manganese dans une graminee, le cuivre en viticulture, le cobalt, le fer, et le zinc dans un aliment du betail, le potassium dans une experience de radioecologie. (auteur)

Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

Energy Technology Data Exchange (ETDEWEB)

Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong (Yale); (Cornell); (Tsinghua)

2012-10-10

Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.

Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

Science.gov (United States)

Nakajima, Osamu; Nishimaki-Mogami, Tomoko; Kondo, Kazunari

2016-01-01

Genome editing has undergone rapid development during the last three years. It is anticipated that genetically modified organisms (GMOs) for food purposes will be widely produced using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system in the near future. However, the Cas9 gene may then enter the genomes of GMOs for food if the breeding process is not strictly managed, which could lead to the Cas9 protein or associated peptides being produced within these organisms. A variety of peptides could theoretically be produced from the Cas9 gene by using open reading frames different from that of Cas9 in the GMOs. In this study, Cas9 and the peptides potentially encoded by Cas9 genes were studied regarding their immunogenicity, in terms of the digestibility of Cas9 and the homology of the peptides to food allergens. First, the digestibility and thermal stability of Cas9 were studied. Digestibility was tested with natural or heat-denatured Cas9 in simulated gastric fluid in vitro. The two types of Cas9 were digested rapidly. Cas9 was also gradually degraded during heat treatment. Second, the peptides potentially encoded by Cas9 genes were examined for their homology to food allergens. Specifically, an 8-mer exact match search and a sliding 80-mer window search were performed using allergen databases. One of the peptides was found to have homology with a food allergen.

Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

Science.gov (United States)

Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott; Du, Mingjian; Rollins, MaryClare F; Hidalgo-Reyes, Yurima; Wiedenheft, Blake; Maxwell, Karen L; Davidson, Alan R

2015-10-01

The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens.

Science.gov (United States)

Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

2015-10-15

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering.

Science.gov (United States)

Watakabe, Ikuko; Hashimoto, Hisashi; Kimura, Yukiko; Yokoi, Saori; Naruse, Kiyoshi; Higashijima, Shin-Ichi

2018-01-01

Medaka ( Oryzias latipes ) is a popular animal model used in vertebrate genetic analysis. Recently, an efficient (~ 30%) knock-in system via non-homologous end joining (NHEJ) was established in zebrafish using the CRISPR/Cas9 system. If the same technique were applicable in medaka, it would greatly expand the usefulness of this model organism. The question of the applicability of CRISPR/Cas9 in medaka, however, has yet to be addressed. We report the highly efficient generation of knock-in transgenic medaka via non-homologous end joining (NHEJ). Donor plasmid containing a heat-shock promoter and a reporter gene was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, an sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. Broad transgene expression in the expression domain of a target gene was observed in approximately 25% of injected embryos. By raising these animals, we established stable knock-in transgenic fish with several different constructs for five genetic loci, obtaining transgenic founders at efficiencies of > 50% for all five loci. Further, we show that the method is useful for obtaining mutant alleles. In the experiments where transgene integrations were targeted between the transcription start site and the initiation methionine, the resultant transgenic fish became mutant alleles. With its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in via NHEJ will become a standard method for the generation of transgenic and mutant medaka.

Cas Wepener

African Journals Online (AJOL)

Owner

Dubbelfoto is die eerste kortverhaalbundel van die teoloog Cas Wepener wat tot dusver veral akademiese artikels en godsdienstige boeke geskryf het. Die bundel se titel gee besondere prominensie aan die gegewe van die foto ter- wyl die motto wat gehaal is uit Roland Barthes se Camera Lucida die aandag vestig op die ...

Simultaneous AuIII Extraction and In Situ Formation of Polymeric Membrane-Supported Au Nanoparticles: A Sustainable Process with Application in Catalysis.

Science.gov (United States)

Mora-Tamez, Lucía; Esquivel-Peña, Vicente; Ocampo, Ana L; Rodríguez de San Miguel, Eduardo; Grande, Daniel; de Gyves, Josefina

2017-04-10

A polymeric membrane-supported catalyst with immobilized gold nanoparticles (AuNPs) was prepared through the extraction and in situ reduction of Au III salts in a one-step strategy. Polymeric inclusion membranes (PIMs) and polymeric nanoporous membranes (PNMs) were tested as different membrane-support systems. Transport experiments indicated that PIMs composed of cellulose triacetate, 2-nitrophenyloctyl ether, and an aliphatic tertiary amine (Adogen 364 or Alamine 336) were the most efficient supports for Au III extraction. The simultaneous extraction and reduction processes were proven to be the result of a synergic phenomenon in which all the membrane components were involved. Scanning electron microscopy characterization of cross-sectional samples suggested a distribution of AuNPs throughout the membrane. Transmission electron microscopy characterization of the AuNPs indicated average particle sizes of 36.7 and 2.9 nm for the PIMs and PNMs, respectively. AuNPs supported on PIMs allowed for >95.4 % reduction of a 0.05 mmol L -1 4-nitrophenol aqueous solution with 10 mmol L -1 NaBH 4 solution within 25 min. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tentative d'évaluation du systéme d'information marketing au niveau des banques publiques Algériennes

OpenAIRE

Hassaine-kazi-tani, Amel

2014-01-01

Le premier chapitre présente les fondements théoriques relatifs au marketing, marketing des services et notamment le marketing bancaire.dans le deuxiéme chapitre seront développées les notions de base du concept d'information et celui de systéme d'information et puis le systéme d'information marketing.le troisiéme chapitre pratique présente une étude de cas auprés des banques publiques Algériennes.

In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9

International Nuclear Information System (INIS)

Zhen, Shuai; Hua, Ling; Takahashi, Y.; Narita, S.; Liu, Yun-Hui; Li, Yan

2014-01-01

Highlights: • Established CRISPR/Cas9 targeting promoter of HPV 16 and targeting E6, E7 transcript. • CRISPR/Cas9 resulted in accumulation of p53 and p21, reduced the proliferation of cervical cancer cells. • Finding inhibited tumorigenesis and growth of mice incubated by cells with CRISPR/Cas9. • CRISPR/Cas9 will be a new treatment strategy, in cervical and other HPV-associated cancer therapy. - Abstract: Deregulated expression of high-risk human papillomavirus oncogenes (E6 and E7) is a pivotal event for pathogenesis and progression in cervical cancer. Both viral oncogenes are therefore regarded as ideal therapeutic targets. In the hope of developing a gene-specific therapy for HPV-related cancer, we established CRISPR/Cas9 targeting promoter of HPV 16 E6/E7 and targeting E6, E7 transcript, transduced the CRISPR/Cas9 into cervical HPV-16-positive cell line SiHa. The results showed that CRISPR/Cas9 targeting promoter, as well as targeting E6 and E7 resulted in accumulation of p53 and p21 protein, and consequently remarkably reduced the abilities of proliferation of cervical cancer cells in vitro. Then we inoculated subcutaneously cells into nude mice to establish the transplanted tumor animal models, and found dramatically inhibited tumorigenesis and growth of mice incubated by cells with CRISPR/Cas9 targeting (promoter+E6+E7)-transcript. Our results may provide evidence for application of CRISPR/Cas9 targeting HR-HPV key oncogenes, as a new treatment strategy, in cervical and other HPV-associated cancer therapy

In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9

Energy Technology Data Exchange (ETDEWEB)

Zhen, Shuai [Baoji Maternal and Child Health Hospital, 2 Xinjian Road East, WeiBin District, Baoji City, 721000, Shanxi Province (China); Xijing Hospital, Fourth Military Medical University, Xi’an (China); Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Kyoto University, Kyoto 606-8507 (Japan); Hua, Ling [Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Takahashi, Y.; Narita, S. [Kyoto University, Kyoto 606-8507 (Japan); Liu, Yun-Hui [Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Li, Yan [Baoji Hospital of Traditional Chinese Medicine, No 43, BaoFu Road, Baoji City, Shanxi Province (China)

2014-08-08

Highlights: • Established CRISPR/Cas9 targeting promoter of HPV 16 and targeting E6, E7 transcript. • CRISPR/Cas9 resulted in accumulation of p53 and p21, reduced the proliferation of cervical cancer cells. • Finding inhibited tumorigenesis and growth of mice incubated by cells with CRISPR/Cas9. • CRISPR/Cas9 will be a new treatment strategy, in cervical and other HPV-associated cancer therapy. - Abstract: Deregulated expression of high-risk human papillomavirus oncogenes (E6 and E7) is a pivotal event for pathogenesis and progression in cervical cancer. Both viral oncogenes are therefore regarded as ideal therapeutic targets. In the hope of developing a gene-specific therapy for HPV-related cancer, we established CRISPR/Cas9 targeting promoter of HPV 16 E6/E7 and targeting E6, E7 transcript, transduced the CRISPR/Cas9 into cervical HPV-16-positive cell line SiHa. The results showed that CRISPR/Cas9 targeting promoter, as well as targeting E6 and E7 resulted in accumulation of p53 and p21 protein, and consequently remarkably reduced the abilities of proliferation of cervical cancer cells in vitro. Then we inoculated subcutaneously cells into nude mice to establish the transplanted tumor animal models, and found dramatically inhibited tumorigenesis and growth of mice incubated by cells with CRISPR/Cas9 targeting (promoter+E6+E7)-transcript. Our results may provide evidence for application of CRISPR/Cas9 targeting HR-HPV key oncogenes, as a new treatment strategy, in cervical and other HPV-associated cancer therapy.

Novel electrochemical redox-active species: one-step synthesis of polyaniline derivative-Au/Pd and its application for multiplexed immunoassay

Science.gov (United States)

Wang, Liyuan; Feng, Feng; Ma, Zhanfang

2015-11-01

Electrochemical redox-active species play crucial role in electrochemically multiplexed immunoassays. A one-pot method for synthesizing four kinds of new electrochemical redox-active species was reported using HAuCl4 and Na2PdCl4 as dual oxidating agents and aniline derivatives as monomers. The synthesized polyaniline derivative-Au/Pd composites, namely poly(N-methyl-o-benzenediamine)-Au/Pd, poly(N-phenyl-o-phenylenediamine)-Au/Pd, poly(N-phenyl-p-phenylenediamine)-Au/Pd and poly(3,3’,5,5’-tetramethylbenzidine)-Au/Pd, exhibited electrochemical redox activity at -0.65 V, -0.3 V, 0.12 V, and 0.5 V, respectively. Meanwhile, these composites showed high H2O2 electrocatalytic activity because of the presence of Au/Pd. The as-prepared composites were used as electrochemical immunoprobes in simultaneous detection of four tumor biomarkers (carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA199), carbohydrate antigen 72-4 (CA724), and alpha fetoprotein (AFP)). This immunoassay shed light on potential applications in simultaneous gastric cancer (related biomarkers: CEA, CA199, CA724) and liver cancer diagnosis (related biomarkers: CEA, CA199, AFP). The present strategy to the synthesize redox species could be easily extended to other polymers such as polypyrrole derivatives and polythiophene derivatives. This would be of great significance in the electrochemical detection of more analytes.

Electrochemical Properties of Alkanethiol Monolayers Adsorbed on Nanoporous Au Surfaces

International Nuclear Information System (INIS)

Chu, Yeon Yi; Seo, Bora; Kim, Jong Won

2010-01-01

We investigated the electrochemical properties of alkanethiol monolayers adsorbed on NPG surfaces by cyclic voltammetry and electrochemical impedance spectroscopy, and the results are compared to those on flat Au surfaces. The reductive desorption of alkanethiols on NPG surfaces is observed in more negative potential regions than that on flat Au surfaces due the stronger S-Au interaction on NPG surfaces. While the electron transfer through alkanethiol monolayers on flat Au surfaces occurs via a tunneling process through the monolayer films, the redox species can permeate through the monolayers on NPG surfaces to transfer the electrons to the Au surfaces. The results presented here will help to elucidate the intrinsic electrochemical properties of alkanethiol monolayers adsorbed on curved Au surfaces, particularly on the surface of AuNPs. Self-assembled monolayers (SAMs) of thiolate molecules on Au surfaces have been the subject of intensive research for the last few decades due to their unique physical and chemical properties. The well-organized surface structures of thiolate SAMs with various end-group functionalities can be further utilized for many applications in biology and nanotechnology. In addition to the practical applications, SAMs of thiolate molecules on Au surfaces also provide unique opportunities to address fundamental issues in surface chemistry such as self-organized surface structures, electron transfer behaviors, and moleculesubstrate interactions. Although there have been numerous reports on the fundamental physical and chemical properties of thiolate SAMs on Au surfaces, most of them were investigated on flat Au surfaces, typically on well-defined Au(111) surfaces

CRISPR/Cas9 Inhibits Multiple Steps of HIV-1 Infection.

Science.gov (United States)

Yin, Lijuan; Hu, Siqi; Mei, Shan; Sun, Hong; Xu, Fengwen; Li, Jian; Zhu, Weijun; Liu, Xiaoman; Zhao, Fei; Zhang, Di; Cen, Shan; Liang, Chen; Guo, Fei

2018-05-09

CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.

CAS - CERN Accelerator School: Course on Digital Signal Processing

CERN Document Server

Digital Signal Processing; CAS 2007

2008-01-01

These proceedings present the lectures given at the twenty-first specialized course organized by the CERN Accelerator School (CAS), the topic being Digital Signal Processing. The course was held in Sigtuna, Sweden, from 31 May–9 June 2007. This is the first time this topic has been selected for a specialized course. Taking into account the number of related applications currently in use in accelerators around the world, it was recognized that such a topic should definitively be incorporated into the CAS series of specialized courses. The specific aim of the course was to introduce the participants to the use and programming of Digital Signal Processors (DSPs) and Field Programmable Gate Arrays (FPGAs) evaluation boards. The course consisted of lectures in the mornings covering fundamental background knowledge in mathematics, controls theory, design tools, programming hardware platforms, and implementation details. In the afternoons the students split into two groups with people working in pairs. One group w...

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

Science.gov (United States)

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

Science.gov (United States)

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Large-scale image-based profiling of single-cell phenotypes in arrayed CRISPR-Cas9 gene perturbation screens.

Science.gov (United States)

de Groot, Reinoud; Lüthi, Joel; Lindsay, Helen; Holtackers, René; Pelkmans, Lucas

2018-01-23

High-content imaging using automated microscopy and computer vision allows multivariate profiling of single-cell phenotypes. Here, we present methods for the application of the CISPR-Cas9 system in large-scale, image-based, gene perturbation experiments. We show that CRISPR-Cas9-mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image-based phenotyping. We developed a pipeline to construct a large-scale arrayed library of 2,281 sequence-verified CRISPR-Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine-learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in-depth characterization of gene perturbation effects. This approach enables genome-scale image-based multivariate gene perturbation profiling using CRISPR-Cas9. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Diaspora et économie internationale: le cas des Sud-Africains d’origine indienne

Directory of Open Access Journals (Sweden)

Frédéric Landy

2003-03-01

Full Text Available L’Inde rêve que la diaspora indienne joue un rôle économique comparable à celui de la diaspora chinoise pour la Chine. En comparant trois cartes de la répartition mondiale des personnes d’origine indienne et des investissements internationaux, on constate que les pays à forte communauté indienne ne sont pas toujours des partenaires économiques majeurs de l’Inde. Celle-ci demeure une référence forte pour la diaspora, parfois très ancienne. Le cas de l’Afrique du Sud illustre bien la dissolution des liens matériels entre les personnes d’origine indienne et l’Inde, aggravée par la coupure de l’apartheid: la carte de l’Inde a souvent disparu des esprits, au profit d’une Inde bien plus abstraite.

A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum.

Science.gov (United States)

Wang, Bo; Hu, Qitiao; Zhang, Yu; Shi, Ruilin; Chai, Xin; Liu, Zhe; Shang, Xiuling; Zhang, Yun; Wen, Tingyi

2018-04-23

Extensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR-Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation. Here, we developed a RecET-assisted CRISPR-Cas9 genome editing method using a chromosome-borne Cas9-RecET and a single plasmid harboring sgRNA and repair templates. The inducible expression of chromosomal RecET promoted the frequencies of homologous recombination, and increased the efficiency for gene deletion. Due to the high transformation efficiency of a single plasmid, this method enabled 10- and 20-kb region deletion, 2.5-, 5.7- and 7.5-kb expression cassette insertion and precise site-specific mutation, suggesting a versatility of this method. Deletion of argR and farR regulators as well as site-directed mutation of argB and pgi genes generated the mutant capable of accumulating L-arginine, indicating the stability of chromosome-borne Cas9 for iterative genome editing. Using this method, the model-predicted target genes were modified to redirect metabolic flux towards 1,2-propanediol biosynthetic pathway. The final engineered strain produced 6.75 ± 0.46 g/L of 1,2-propanediol that is the highest titer reported in C. glutamicum. Furthermore, this method is available for Corynebacterium pekinense 1.563, suggesting its universal applicability in other Corynebacterium species. The RecET-assisted CRISPR-Cas9 genome editing method will facilitate engineering of metabolic networks for the synthesis of interested bio-based products from renewable

The Impact of CRISPR/Cas9 Technology on Cardiac Research: From Disease Modelling to Therapeutic Approaches

Science.gov (United States)

Pramstaller, Peter P.; Hicks, Andrew A.; Rossini, Alessandra

2017-01-01

Genome-editing technology has emerged as a powerful method that enables the generation of genetically modified cells and organisms necessary to elucidate gene function and mechanisms of human diseases. The clustered regularly interspaced short palindromic repeats- (CRISPR-) associated 9 (Cas9) system has rapidly become one of the most popular approaches for genome editing in basic biomedical research over recent years because of its simplicity and adaptability. CRISPR/Cas9 genome editing has been used to correct DNA mutations ranging from a single base pair to large deletions in both in vitro and in vivo model systems. CRISPR/Cas9 has been used to increase the understanding of many aspects of cardiovascular disorders, including lipid metabolism, electrophysiology and genetic inheritance. The CRISPR/Cas9 technology has been proven to be effective in creating gene knockout (KO) or knockin in human cells and is particularly useful for editing induced pluripotent stem cells (iPSCs). Despite these progresses, some biological, technical, and ethical issues are limiting the therapeutic potential of genome editing in cardiovascular diseases. This review will focus on various applications of CRISPR/Cas9 genome editing in the cardiovascular field, for both disease research and the prospect of in vivo genome-editing therapies in the future. PMID:29434642

Engineering Plant Immunity via CRISPR/Cas13a System

KAUST Repository

Aljedaani, Fatimah R.

2018-05-01

Viral diseases constitute a major threat to the agricultural production and food security throughout the world. Plants cope with the invading viruses by triggering immune responses and small RNA interference (RNAi) systems. In prokaryotes, CRISPR/Cas systems function as an adaptive immune system to provide bacteria with resistance against invading phages and conjugative plasmids. Interestingly, CRISPR/Cas9 system was shown to interfere with eukaryotic DNA viruses and confer resistance against plant DNA viruses. The majority of the plant viruses have RNA genomes. The aim of this study is to test the ability of the newly discovered CRISPR/Cas13a immune system, that targets and cleaves single stranded RNA (ssRNA) in prokaryotes, to provide resistance against RNA viruses in plants. Here, I employ the CRISPR/Cas13a system for molecular interference against Turnip Mosaic Virus (TuMV), a plant RNA virus. The results of this study established the CRISPR/Cas13a as a molecular interference machinery against RNA viruses in plants. Specifically, my data show that the CRISPR/Cas13a machinery is able to interfere with and degrade the TuMV (TuMV-GFP) RNA genome. In conclusion, these data indicate that the CRISPR/Cas13 systems can be employed for engineering interference and durable resistance against RNA viruses in diverse plant species.

Single step production of Cas9 mRNA for zygote injection.

Science.gov (United States)

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris

Energy Technology Data Exchange (ETDEWEB)

Samai, Poulami; Smith, Paul; Shuman, Stewart [Molecular Biology Program, Sloan-Kettering Institute for Cancer Research (United States)

2010-12-01

A 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. CRISPRs (clustered regularly interspaced short palindromic repeats) provide bacteria and archaea with RNA-guided acquired immunity to invasive DNAs. CRISPR-associated (Cas) proteins carry out the immune effector functions. Cas2 is a universal component of the CRISPR system. Here, a 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. DvuCas2 is a homodimer, with each protomer consisting of an N-terminal βαββαβ ferredoxin fold (amino acids 1–78) to which is appended a C-terminal segment (amino acids 79–102) that includes a short 3{sub 10}-helix and a fifth β-strand. The β5 strands align with the β4 strands of the opposite protomers, resulting in two five-stranded antiparallel β-sheets that form a sandwich at the dimer interface. The DvuCas2 dimer is stabilized by a distinctive network of hydrophilic cross-protomer side-chain interactions.

Structure of a CRISPR-associated protein Cas2 from Desulfovibrio vulgaris

International Nuclear Information System (INIS)

Samai, Poulami; Smith, Paul; Shuman, Stewart

2010-01-01

A 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. CRISPRs (clustered regularly interspaced short palindromic repeats) provide bacteria and archaea with RNA-guided acquired immunity to invasive DNAs. CRISPR-associated (Cas) proteins carry out the immune effector functions. Cas2 is a universal component of the CRISPR system. Here, a 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. DvuCas2 is a homodimer, with each protomer consisting of an N-terminal βαββαβ ferredoxin fold (amino acids 1–78) to which is appended a C-terminal segment (amino acids 79–102) that includes a short 3 10 -helix and a fifth β-strand. The β5 strands align with the β4 strands of the opposite protomers, resulting in two five-stranded antiparallel β-sheets that form a sandwich at the dimer interface. The DvuCas2 dimer is stabilized by a distinctive network of hydrophilic cross-protomer side-chain interactions

Crystallographic investigation of Au nanoparticles embedded in a SrTiO{sub 3} thin film for plasmonics applications by means of synchrotron radiation

Energy Technology Data Exchange (ETDEWEB)

Pincini, Davide, E-mail: davide.pincini@mail.polimi.it [European Synchrotron Radiation Facility, CS 40220, 71, avenue des Martyrs, F-38043 Grenoble Cedex 9 (France); Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo Da Vinci 32, 20133 Milano (Italy); Mazzoli, Claudio [Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo Da Vinci 32, 20133 Milano (Italy); Bernhardt, Hendrik; Katzer, Christian; Schmidl, Frank [Institut für Festkörperphysik, Friedrich-Schiller-Universität Jena, Helmholtzweg 5, 07743 Jena (Germany); Uschmann, Ingo [Institut für Optik und Quantenelektronik, Friedrich-Schiller-Universität Jena, Max-Wien-Platz 1, 07743 Jena (Germany); Detlefs, Carsten [European Synchrotron Radiation Facility, CS 40220, 71, avenue des Martyrs, F-38043 Grenoble Cedex 9 (France)

2015-03-14

Self-organized monocrystalline Au nanoparticles with potential applications in plasmonics are grown in a SrTiO{sub 3} matrix by a novel two-step deposition process. The crystalline preferred orientation of these Au nanoparticles is investigated by synchrotron hard x-ray diffraction. Nanoparticles preferentially align with the (111) direction along the substrate normal (001), whereas two in-plane orientations are found with [110]{sub SrTiO{sub 3}}∥[110]{sub Au} and [100]{sub SrTiO{sub 3}}∥[110]{sub Au}. Additionally, a smaller diffraction signal from nanoparticles with the (001) direction parallel to the substrate normal (001) is observed; once again, two in-plane orientations are found, with [100]{sub SrTiO{sub 3}}∥[100]{sub Au} and [100]{sub SrTiO{sub 3}}∥[110]{sub Au}. The populations of the two in-plane orientations are found to depend on the thickness of the gold film deposited in the first step of the growth.

Semiotic and discursive variables in cas-based didactical engineering

DEFF Research Database (Denmark)

Winsløw, Carl

2003-01-01

CAS, didactical engeneering, Maple, semiotics, undergraduate teaching, mathematics, education, didactics......CAS, didactical engeneering, Maple, semiotics, undergraduate teaching, mathematics, education, didactics...

Chromosomal Targeting by the Type III-A CRISPR-Cas System Can Reshape Genomes in Staphylococcus aureus.

Science.gov (United States)

Guan, Jing; Wang, Wanying; Sun, Baolin

2017-01-01

trinucleotide pairings between a selected target and the 5' tag of crRNA can block targeting. These findings add to our understanding of the molecular mechanisms of the type III-A CRISPR-Cas system and provide a novel strategy for the exploitation of engineered CRISPR immunity against integrated MGEs in bacteria for clinical and industrial applications.

Les Lesions Oculaires Par Jeu de Feu au Cours de Achoura

Science.gov (United States)

El Ketani, A.; Amir, F.; Ali, T.B.; Hamdani, M.; Zaghloul, K.

2006-01-01

Summary Les enfants célèbrent la fête de Achoura au Maroc par des jeux de feu, ce qui occasionne des blessures oculaires plus au moins graves. Nous rapportons 15 observations de malades traités au Service d'Ophtalmologie Pédiatrique de l'Hôpital 20 Août 1953 de Casablanca. L'âge moyen des patients était de 12 ans et demi, avec des extrêmes de 3 et 25 ans. Les pétards, la première cause des accidents (50%), ont occasionné des contusions oculaires avec parfois un oedème de Berlin (deux cas). L'atteinte oculaire par fusée a occasionné un éclatement de globe et une plaie de paupière. Les bombes de carbone ont été responsables de brûlures de deuxième degré palpébrales et conjonctivo-cornéennes avec de multiples corps étrangers cornéens profonds. Les «étoiles» et la limaille de fer ont provoqué des brûlures cornéennes moins graves avec des corps étrangers superficiels. Les pistolets à bille ont été responsables de contusions oculaires. La réglementation de vente des jeux de feu et la sensibilisation du grand public par les moyens audiovisuels permettraient de prévenir ces blessures oculaires. PMID:21991053

Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae.

Science.gov (United States)

Ostria-Hernández, Martha Lorena; Sánchez-Vallejo, Carlos Javier; Ibarra, J Antonio; Castro-Escarpulli, Graciela

2015-08-04

In recent years the emergence of multidrug resistant Klebsiella pneumoniae strains has been an increasingly common event. This opportunistic species is one of the five main bacterial pathogens that cause hospital infections worldwide and multidrug resistance has been associated with the presence of high molecular weight plasmids. Plasmids are generally acquired through horizontal transfer and therefore is possible that systems that prevent the entry of foreign genetic material are inactive or absent. One of these systems is CRISPR/Cas. However, little is known regarding the clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) system in K. pneumoniae. The adaptive immune system CRISPR/Cas has been shown to limit the entry of foreign genetic elements into bacterial organisms and in some bacteria it has been shown to be involved in regulation of virulence genes. Thus in this work we used bioinformatics tools to determine the presence or absence of CRISPR/Cas systems in available K. pneumoniae genomes. The complete CRISPR/Cas system was identified in two out of the eight complete K. pneumoniae genomes sequences and in four out of the 44 available draft genomes sequences. The cas genes in these strains comprises eight cas genes similar to those found in Escherichia coli, suggesting they belong to the type I-E group, although their arrangement is slightly different. As for the CRISPR sequences, the average lengths of the direct repeats and spacers were 29 and 33 bp, respectively. BLAST searches demonstrated that 38 of the 116 spacer sequences (33%) are significantly similar to either plasmid, phage or genome sequences, while the remaining 78 sequences (67%) showed no significant similarity to other sequences. The region where the CRISPR/Cas systems were located is the same in all the Klebsiella genomes containing it, it has a syntenic architecture, and is located among genes encoding for proteins likely involved in

Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption.

Science.gov (United States)

Johansen, Anne Katrine; Molenaar, Bas; Versteeg, Danielle; Leitoguinho, Ana Rita; Demkes, Charlotte; Spanjaard, Bastiaan; de Ruiter, Hesther; Akbari Moqadam, Farhad; Kooijman, Lieneke; Zentilin, Lorena; Giacca, Mauro; van Rooij, Eva

2017-10-27

CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)-based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in postnatal gene editing remains incompletely characterized. To evaluate the feasibility of CRISPR/Cas9-based cardiac genome editing in vivo in postnatal mice. We generated cardiomyocyte-specific Cas9 mice and demonstrated that Cas9 expression does not affect cardiac function or gene expression. As a proof-of-concept, we delivered short guide RNAs targeting 3 genes critical for cardiac physiology, Myh6 , Sav1 , and Tbx20 , using a cardiotropic adeno-associated viral vector 9. Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of short guide RNA exposure or the level of Cas9 expression. DNA sequencing analysis revealed target-dependent mutations that were highly reproducible across mice resulting in differential rates of in- and out-of-frame mutations. Finally, we applied a dual short guide RNA approach to effectively delete an important coding region of Sav1 , which increased the editing efficiency. Our results indicate that the effect of postnatal CRISPR/Cas9-based cardiac gene editing using adeno-associated virus serotype 9 to deliver a single short guide RNA is target dependent. We demonstrate a mosaic pattern of gene disruption, which hinders the application of the technology to study gene function. Further studies are required to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo. © 2017 American Heart Association, Inc.

Stabilizing ultrasmall Au clusters for enhanced photoredox catalysis.

Science.gov (United States)

Weng, Bo; Lu, Kang-Qiang; Tang, Zichao; Chen, Hao Ming; Xu, Yi-Jun

2018-04-18

Recently, loading ligand-protected gold (Au) clusters as visible light photosensitizers onto various supports for photoredox catalysis has attracted considerable attention. However, the efficient control of long-term photostability of Au clusters on the metal-support interface remains challenging. Herein, we report a simple and efficient method for enhancing the photostability of glutathione-protected Au clusters (Au GSH clusters) loaded on the surface of SiO 2 sphere by utilizing multifunctional branched poly-ethylenimine (BPEI) as a surface charge modifying, reducing and stabilizing agent. The sequential coating of thickness controlled TiO 2 shells can further significantly improve the photocatalytic efficiency, while such structurally designed core-shell SiO 2 -Au GSH clusters-BPEI@TiO 2 composites maintain high photostability during longtime light illumination conditions. This joint strategy via interfacial modification and composition engineering provides a facile guideline for stabilizing ultrasmall Au clusters and rational design of Au clusters-based composites with improved activity toward targeting applications in photoredox catalysis.

Properties of ordered titanium templates covered with Au thin films for SERS applications

Science.gov (United States)

Grochowska, Katarzyna; Siuzdak, Katarzyna; Sokołowski, Michał; Karczewski, Jakub; Szkoda, Mariusz; Śliwiński, Gerard

2016-12-01

Currently, roughened metal nanostructures are widely studied as highly sensitive Raman scattering substrates that show application potential in biochemistry, food safety or medical diagnostic. In this work the structural properties and the enhancement effect due to surface enhanced Raman scattering (SERS) of highly ordered nano-patterned titanium templates covered with thin (5-20 nm) gold films are reported. The templates are formed by preparation of a dense structure of TiO2 nanotubes on a flat Ti surface (2 × 2 cm2) and their subsequent etching down to the substrate. SEM images reveal the formation of honeycomb nanostructures with the cavity diameter of 80 nm. Due to the strongly inhomogeneous distribution of the electromagnetic field in the vicinity of the Au film discontinuities the measured average enhancement factor (107-108) is markedly higher than observed for bare Ti templates. The enhancement factor and Raman signal intensity can be optimized by adjusting the process conditions and thickness of the deposited Au layer. Results confirm that the obtained structures can be used in surface enhanced sensing.

Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System

Directory of Open Access Journals (Sweden)

Zhanqi Dong

2018-02-01

Full Text Available The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV immediate early-1 (ie-1 gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-/sgRNA(-, Cas9(+/sgRNA(-, Cas9(-/sgRNA(+, and Cas9(+/sgRNA(+. We demonstrated that the Cas9(+/sgRNA(+ transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+/sgRNA(+ transgenic lines shows that the median lethal dose (LD50 is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+/sgRNA(+ transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System.

Science.gov (United States)

Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

2018-01-01

The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 ( ie-1 ) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

NARCIS (Netherlands)

Kieper, Sebastian N.; Almendros, Cristóbal; Behler, Juliane; McKenzie, Rebecca E.; Nobrega, Franklin L.; Haagsma, Anna C.; Vink, Jochem N.A.; Hess, Wolfgang R.; Brouns, Stan J.J.

2018-01-01

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II

PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens.

Science.gov (United States)

Spahn, Philipp N; Bath, Tyler; Weiss, Ryan J; Kim, Jihoon; Esko, Jeffrey D; Lewis, Nathan E; Harismendy, Olivier

2017-11-20

Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service. PinAPL-Py implements state-of-the-art tools and statistical models, assembled in a comprehensive workflow covering sequence quality control, automated sgRNA sequence extraction, alignment, sgRNA enrichment/depletion analysis and gene ranking. The workflow is set up to use a variety of popular sgRNA libraries as well as custom libraries that can be easily uploaded. Various analysis options are offered, suitable to analyze a large variety of CRISPR/Cas9 screening experiments. Analysis output includes ranked lists of sgRNAs and genes, and publication-ready plots. PinAPL-Py helps to advance genome-wide screening efforts by combining comprehensive functionality with user-friendly implementation. PinAPL-Py is freely accessible at http://pinapl-py.ucsd.edu with instructions and test datasets.

Heterojunction metal-oxide-metal Au-Fe{sub 3}O{sub 4}-Au single nanowire device for spintronics

Energy Technology Data Exchange (ETDEWEB)

Reddy, K. M., E-mail: mrkongara@boisestate.edu; Punnoose, Alex; Hanna, Charles [Department of Physics, Boise State University, Boise, Idaho 83725 (United States); Padture, Nitin P. [School of Engineering, Brown University, Providence, Rhode Island 02912 (United States)

2015-05-07

In this report, we present the synthesis of heterojunction magnetite nanowires in alumina template and describe magnetic and electrical properties from a single nanowire device for spintronics applications. Heterojunction Au-Fe-Au nanowire arrays were electrodeposited in porous aluminum oxide templates, and an extensive and controlled heat treatment process converted Fe segment to nanocrystalline cubic magnetite phase with well-defined Au-Fe{sub 3}O{sub 4} interfaces as confirmed by the transmission electron microscopy. Magnetic measurements revealed Verwey transition shoulder around 120 K and a room temperature coercive field of 90 Oe. Current–voltage (I-V) characteristics of a single Au-Fe{sub 3}O{sub 4}-Au nanowire have exhibited Ohmic behavior. Anomalous positive magnetoresistance of about 0.5% is observed on a single nanowire, which is attributed to the high spin polarization in nanowire device with pure Fe{sub 3}O{sub 4} phase and nanocontact barrier. This work demonstrates the ability to preserve the pristine Fe{sub 3}O{sub 4} and well defined electrode contact metal (Au)–magnetite interface, which helps in attaining high spin polarized current.

Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona.

Science.gov (United States)

Gandhi, Shashank; Haeussler, Maximilian; Razy-Krajka, Florian; Christiaen, Lionel; Stolfi, Alberto

2017-05-01

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos. We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona. Copyright © 2017 Elsevier Inc. All rights reserved.

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

NARCIS (Netherlands)

Kieper, S.N.; Almendros, Cristóbal; Behler, Juliane; McKenzie, R.E.; Luzia De Nóbrega, F.; van Eijkeren-Haagsma, A.C.; Vink, J.N.A.; Hess, Wolfgang R.; Brouns, S.J.J.

2018-01-01

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II

Cancers attribuables à l’excès de poids au Canada en 2010

Directory of Open Access Journals (Sweden)

Dianne Zakaria

2017-01-01

Full Text Available Introduction : L’excès de poids (indice de masse corporelle [IMC] de 25,00 kg/m2 ou plus est un facteur de risque bien connu de diabète, d’hypertension et de maladie cardiovasculaire, mais on en sait moins sur son lien avec le cancer. Dans cette étude, nous avons utilisé le risque attribuable dans la population (RAP pour estimer le fardeau des cancers attribuables à l’excès de poids chez les adultes canadiens (de 25 ans ou plus en 2010. Méthodologie : Nous avons estimé les RAP en utilisant des estimations du risque relatif (RR tirées du Continuous Update Project du World Cancer Research Fund International, des estimations du surpoids et de l’obésité fondées sur l’IMC tirées de l’Enquête sur la santé dans les collectivités canadiennes de 2000-2001 (surpoids : 25,00 à 29,99 kg/m2; obésité : 30,00 kg/m2 et plus et nous avons utilisé les nombres de cas de cancer figurant dans le Registre canadien du cancer. Les RAP ont été fondés sur des IMC corrigés pour tenir compte du biais associé à l’autodéclaration de la taille et du poids. Résultats : Au Canada, en 2010, on peut attribuer environ 9 645 cas de cancer à un excès de poids, ce qui représente 5,7 % de tous les cas de cancer (hommes : 4,9 %; femmes : 6,5 %. En limitant l’analyse aux types de cancer associés à un IMC élevé, le RAP augmente à 14,9 % (hommes : 17,5 %; femmes : 13,3 %. Les types de cancer pour lesquels le RAP était le plus élevé étaient l’adénocarcinome de l’oesophage (42,2 %, le cancer du rein (25,4 %, le cancer du cardia (20,7 %, le cancer du foie (20,5 %, le cancer du côlon (20,5 % et le cancer de la vésicule biliaire (20,2 % chez les hommes, et l’adénocarcinome de l’oesophage (36,1 %, le cancer de l’utérus (35,2 %, le cancer de la vésicule biliaire (23,7 % et le cancer du rein (23,0 % chez les femmes. Les types de cancer pour lesquels le nombre de cas attribuables était le plus élevé étaient le cancer du c

Modification of ethylene sensitivity in ornamental plants using CRISPR/Cas9

DEFF Research Database (Denmark)

Kemp, Oliver; Favero, Bruno Trevenzoli; Hegelund, Josefine Nymark

2017-01-01

; the Clustered Regularly Interspaced Palindromic Repeats (CRISPR) RNA guided Cas9 DNA nuclease (CRISPR/Cas9). CRISPR/Cas9 may be employed to introduce targeted double-stranded breaks (DSBs) at desired sites in the host genome. The DSBs will be repaired by the non-homologous end-joining (NHEJ) repair mechanism...... which often results in small indels and consequently gene knockout. The CRISPR/Cas9 system consists of a protein DNA nuclease (Cas9) which is guided to the target sequence by a small RNA molecule (sgRNA) that recognizes a 20 bp target sequence in the genome situated immediately downstream of a 3 bp...... protospacer adjacent motif (PAM). The sgRNA confers the sequence specificity of the CRISPR/Cas9 complex and may thus be designed to target virtually any sequence, a feature that has made it the method of choice within precise genetic engineering. Although most research with CRISPR/Cas9 has been conducted...

Cluster-to-cluster transformation among Au6, Au8 and Au11 nanoclusters.

Science.gov (United States)

Ren, Xiuqing; Fu, Junhong; Lin, Xinzhang; Fu, Xuemei; Yan, Jinghui; Wu, Ren'an; Liu, Chao; Huang, Jiahui

2018-05-22

We present the cluster-to-cluster transformations among three gold nanoclusters, [Au6(dppp)4]2+ (Au6), [Au8(dppp)4Cl2]2+ (Au8) and [Au11(dppp)5]3+ (Au11). The conversion process follows a rule that states that the transformation of a small cluster to a large cluster is achieved through an oxidation process with an oxidizing agent (H2O2) or with heating, while the conversion of a large cluster to a small one occurs through a reduction process with a reducing agent (NaBH4). All the reactions were monitored using UV-Vis spectroscopy and ESI-MS. This work may provide an alternative approach to the synthesis of novel gold nanoclusters and a further understanding of the structural transformation relationship of gold nanoclusters.

The Preparation of Au@TiO2 Yolk-Shell Nanostructure and its Applications for Degradation and Detection of Methylene Blue.

Science.gov (United States)

Wan, Gengping; Peng, Xiange; Zeng, Min; Yu, Lei; Wang, Kan; Li, Xinyue; Wang, Guizhen

2017-09-18

This paper reports the synthesis of a new type of Au@TiO 2 yolk-shell nanostructures by integrating ion sputtering method with atomic layer deposition (ALD) technique and its applications as visible light-driven photocatalyst and surface-enhanced Raman spectroscopy (SERS) substrate. Both the size and amount of gold nanoparticles confined in TiO 2 nanotubes could be facilely controlled via properly adjusting the sputtering time. The unique structure and morphology of the resulting Au@TiO 2 samples were investigated by using various spectroscopic and microscopic techniques in detail. It is found that all tested samples can absorb visible light with a maximum absorption at localized surface plasmon resonance (LSPR) wavelengths (550-590 nm) which are determined by the size of gold nanoparticles. The Au@TiO 2 yolk-shell composites were used as the photocatalyst for the degradation of methylene blue (MB). As compared with pure TiO 2 nanotubes, Au@TiO 2 composites exhibit improved photocatalytic properties towards the degradation of MB. The SERS effect of Au@TiO 2 yolk-shell composites was also performed to investigate the detection sensitivity of MB.

The Preparation of Au@TiO2 Yolk-Shell Nanostructure and its Applications for Degradation and Detection of Methylene Blue

Science.gov (United States)

Wan, Gengping; Peng, Xiange; Zeng, Min; Yu, Lei; Wang, Kan; Li, Xinyue; Wang, Guizhen

2017-09-01

This paper reports the synthesis of a new type of Au@TiO2 yolk-shell nanostructures by integrating ion sputtering method with atomic layer deposition (ALD) technique and its applications as visible light-driven photocatalyst and surface-enhanced Raman spectroscopy (SERS) substrate. Both the size and amount of gold nanoparticles confined in TiO2 nanotubes could be facilely controlled via properly adjusting the sputtering time. The unique structure and morphology of the resulting Au@TiO2 samples were investigated by using various spectroscopic and microscopic techniques in detail. It is found that all tested samples can absorb visible light with a maximum absorption at localized surface plasmon resonance (LSPR) wavelengths (550-590 nm) which are determined by the size of gold nanoparticles. The Au@TiO2 yolk-shell composites were used as the photocatalyst for the degradation of methylene blue (MB). As compared with pure TiO2 nanotubes, Au@TiO2 composites exhibit improved photocatalytic properties towards the degradation of MB. The SERS effect of Au@TiO2 yolk-shell composites was also performed to investigate the detection sensitivity of MB.

Mr.CAS-A minimalistic (pure) Ruby CAS for fast prototyping and code generation

Science.gov (United States)

Ragni, Matteo

There are Computer Algebra System (CAS) systems on the market with complete solutions for manipulation of analytical models. But exporting a model that implements specific algorithms on specific platforms, for target languages or for particular numerical library, is often a rigid procedure that requires manual post-processing. This work presents a Ruby library that exposes core CAS capabilities, i.e. simplification, substitution, evaluation, etc. The library aims at programmers that need to rapidly prototype and generate numerical code for different target languages, while keeping separated mathematical expression from the code generation rules, where best practices for numerical conditioning are implemented. The library is written in pure Ruby language and is compatible with most Ruby interpreters.

Les tuberculomes intracraniens: à propos de 125 cas

Science.gov (United States)

Moufid, Faycal; Oulali, Noureddine; El Fatemi, Nizare; Gana, Rachid; Maaqili, Rachid; Bellakhdar, Fouad

2012-01-01

Les tuberculomes intracrâniens représentent l'une des localisations les plus graves de la tuberculose, leur incidence varie en fonction du contexte représentant 0,2% des processus intracrâniens dans les pays occidentaux et 5 à 10% des masses intracrâniennes dans les pays en voie de développement. Nous rapportons une étude rétrospective de 125 cas. L'hypertension intracrânienne (45%) et le déficit neurologique (36%) sont les signes cliniques les plus fréquents. La lésion était localisée dans 60% des cas en sus-tentoriel et dans 40% des cas en sous-tentoriel. L'approche thérapeutique a consisté en un abord direct du tuberculome dans 67 cas (53%), une biopsie stéréotaxique dans 32 cas (25%), le traitement médical en première intention sans confirmation histologique dans 26 cas (20%). Avant 1993 notre service ne disposait pas de cadre de stéréotaxie, notre attitude thérapeutique consistait soit en un abord direct de la lésion dans 70% des cas, soit un traitement antituberculeux en première intention sans confirmation histologique (30%). Cette attitude était corrélée à une mortalité et morbidité non négligeables respectivement 3% et 10%. Après 1993; le taux d'abords direct a chuté a 38%, avec 47% de biopsies stéréotaxiques et seulement 13% des patients traités par antibacillaires sans preuve histologique. Ceci s'est accompagné d'une réduction significative de mortalité a 1,4% (p = 0,0003) et de morbidité a 2% (p = 0,0027). PMID:22937196

Coaching and engaging. Developing teaching with CAS in High School

DEFF Research Database (Denmark)

Bang, Henrik Peter; Grønbæk, Niels; Larsen, Claus Richard

The extensive use of CAS at upper secondary school in Denmark provides a laboratory for research on the development of standards for CAS teaching. The poster focuses on action research into teachers developing lessons and student activities in an ongoing collaboration between university and high ...... schools on use of CAS in mathematics teaching. Coaches1 mediate design processes, reflection and documentation, and enable sharing. We discuss coaching as a valuable part of action research, and how to draw findings from the collaboration.......The extensive use of CAS at upper secondary school in Denmark provides a laboratory for research on the development of standards for CAS teaching. The poster focuses on action research into teachers developing lessons and student activities in an ongoing collaboration between university and high...

Class 2 CRISPR-Cas RNA-guided endonucleases

DEFF Research Database (Denmark)

Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

2017-01-01

CRISPR-Cas is a bacterial defense system against phage infection and nucleic acid invasion. Class 2 type II CRISPR-Cas9 has also been widely used for genome engineering. Here, we review novel insights into the CRISPR class 2 type V enzymes, specifically Cpf1 and C2c1, which display different DNA-...

Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

Directory of Open Access Journals (Sweden)

Li-Fang JIN

2016-07-01

Full Text Available With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches.

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli.

Science.gov (United States)

Díez-Villaseñor, César; Guzmán, Noemí M; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J M

2013-05-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

Science.gov (United States)

Díez-Villaseñor, César; Guzmán, Noemí M.; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J.M.

2013-01-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism. PMID:23445770

Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures.

Science.gov (United States)

Tsunematsu, Hiroto; Uyeda, Akiko; Yamamoto, Nobuhiko; Sugo, Noriyuki

2017-08-01

CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

Efficient CRISPR/Cas9-based gene knockout in watermelon.

Science.gov (United States)

Tian, Shouwei; Jiang, Linjian; Gao, Qiang; Zhang, Jie; Zong, Mei; Zhang, Haiying; Ren, Yi; Guo, Shaogui; Gong, Guoyi; Liu, Fan; Xu, Yong

2017-03-01

CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

Developing an aqueous approach for synthesizing Au and M@Au (M = Pd, CuPt) hybrid nanostars with plasmonic properties

Science.gov (United States)

Du, Jingshan; Yu, Junjie; Xiong, Yalin; Lin, Zhuoqing; Zhang, Hui; Yang, Deren

Anisotropic Au nanoparticles show unique localized surface plasmon resonance (LSPR) properties, which make it attractive in optical, sensing, and biomedical applications. In this contribution, we report a general and facile strategy towards aqueous synthesis of Au and M@Au (M = Pd, CuPt) hybrid nanostars by reducing HAuCl4 with ethanolamine in the presence of cetyltrimethylammonium bromide (CTAB). According to electron microscopic observation and spectral monitoring, we found that the layered epitaxial growth mode (i.e., Frank-van der Merwe mechanism) contributes to the enlargement of the core, while, the random attachment of Au nanoclusters onto the cores accounts for the formation of the branches. Both of them are indispensable for the formation of the nanostars. The LSPR properties of the Au nanoparticles have been well investigated with morphology control via precursor amount and growth temperature. The Au nanostars showed improved surface-enhanced Raman spectroscopy (SERS) performance for rhodamine 6G due to their sharp edges and tips, which were therefore confirmed as good SERS substrate to detect trace amount of molecules.

Molecular beam epitaxy of GaAs nanowires and their sustainability for optoelectronic applications. Comparing Au- and self-assisted growth methods

Energy Technology Data Exchange (ETDEWEB)

Breuer, Steffen

2011-09-28

In this work the synthesis of GaAs nanowires by molecular beam epitaxy (MBE) using the vapour-liquid-solid (VLS) mechanism is investigated. A comparison between Au- and self-assisted VLS growth is at the centre of this thesis. While the Au-assisted method is established as a versatile tool for nanowire growth, the recently developed self-assisted variation results from the exchange of Au by Ga droplets and thus eliminates any possibility of Au incorporation. By both methods, we achieve nanowires with epitaxial alignment to the Si(111) substrates. Caused by differences during nanowire nucleation, a parasitic planar layer grows between the nanowires by the Au-assisted method, but can be avoided by the self-assisted method. Au-assisted nanowires grow predominantly in the metastable wurtzite crystal structure, while their self-assisted counterparts have the zincblende structure. All GaAs nanowires are fully relaxed and the strain arising from the lattice mismatch between GaAs and Si of 4.1 % is accommodated by misfit dislocations at the interface. Self-assisted GaAs nanowires are generally found to have vertical and non-polar side facets, while tilted and polar nanofacets were described for Au-assisted GaAs nanowires. We employ VLS nucleation theory to understand the effect of the droplet material on the lateral facets. Optoelectronic applications require long minority carrier lifetimes at room temperature. We fabricate GaAs/(Al,Ga)As core-shell nanowires and analyse them by transient photoluminescence (PL) spectroscopy. The results are 2.5 ns for the self-assisted nanowires as well as 9 ps for the Au-assisted nanowires. By temperature-dependent PL measurements we find a characteristic activation energy of 77 meV that is present only in the Au-assisted nanowires. We conclude that most likely Au is incorporated from the droplets into the GaAs nanowires and acts as a deep, non-radiative recombination centre.

Design and Validation of CRISPR/Cas9 Systems for Targeted Gene Modification in Induced Pluripotent Stem Cells.

Science.gov (United States)

Lee, Ciaran M; Zhu, Haibao; Davis, Timothy H; Deshmukh, Harshahardhan; Bao, Gang

2017-01-01

The CRISPR/Cas9 system is a powerful tool for precision genome editing. The ability to accurately modify genomic DNA in situ with single nucleotide precision opens up new possibilities for not only basic research but also biotechnology applications and clinical translation. In this chapter, we outline the procedures for design, screening, and validation of CRISPR/Cas9 systems for targeted modification of coding sequences in the human genome and how to perform genome editing in induced pluripotent stem cells with high efficiency and specificity.

Progress Scored in Management Information System at CAS

Institute of Scientific and Technical Information of China (English)

无

2006-01-01

@@ CAS initiative to upgrade its management information system (MIS) is making significant progress. Recently, 116 CAS subordinates have completed their online trial operation of a MIS project at the Academy, called Academia Resource Planning (ARP), marking an important phased achievement of the initiative.

Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung.

Science.gov (United States)

Ren, Lu; Deng, Lin-Hua; Zhang, Ri-Peng; Wang, Cheng-Dong; Li, De-Sheng; Xi, Li-Xin; Chen, Zhen-Rong; Yang, Rui; Huang, Jie; Zeng, Yang-Ru; Wu, Hong-Lin; Cao, San-Jie; Wu, Rui; Huang, Yong; Yan, Qi-Gui

2017-02-01

To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin-clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance.

Comparative analysis of CRISPR-Cas systems in Klebsiella genomes.

Science.gov (United States)

Shen, Juntao; Lv, Li; Wang, Xudong; Xiu, Zhilong; Chen, Guoqiang

2017-04-01

Prokaryotic CRISPR-Cas system provides adaptive immunity against invasive genetic elements. Bacteria of the genus Klebsiella are important nosocomial opportunistic pathogens. However, information of CRISPR-Cas system in Klebsiella remains largely unknown. Here, we analyzed the CRISPR-Cas systems of 68 complete genomes of Klebsiella representing four species. All the elements for CRISPR-Cas system (cas genes, repeats, leader sequences, and PAMs) were characterized. Besides the typical Type I-E and I-F CRISPR-Cas systems, a new Subtype I system located in the ABC transport system-glyoxalase region was found. The conservation of the new subtype CRISPR system between different species showed new evidence for CRISPR horizontal transfer. CRISPR polymorphism was strongly correlated both with species and multilocus sequence types. Some results indicated the function of adaptive immunity: most spacers (112 of 124) matched to prophages and plasmids and no matching housekeeping genes; new spacer acquisition was observed within the same sequence type (ST) and same clonal complex; the identical spacers were observed only in the ancient position (far from the leader) between different STs and clonal complexes. Interestingly, a high ratio of self-targeting spacers (7.5%, 31 of 416) was found in CRISPR-bearing Klebsiella pneumoniae (61%, 11 of 18). In some strains, there even were multiple full matching self-targeting spacers. Some self-targeting spacers were conserved even between different STs. These results indicated that some unknown mechanisms existed to compromise the function of self-targets of CRISPR-Cas systems in K. pneumoniae. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

Science.gov (United States)

Tu, Zhuchi; Yang, Weili; Yan, Sen; Yin, An; Gao, Jinquan; Liu, Xudong; Zheng, Yinghui; Zheng, Jiezhao; Li, Zhujun; Yang, Su; Li, Shihua; Guo, Xiangyu; Li, Xiao-Jiang

2017-01-01

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing. PMID:28155910

Shocks in magneto-reactive-gas dynamics with application to the piston problem; Sur les chocs dans un milieu magnetodynamique reactif avec application au probleme du piston

Energy Technology Data Exchange (ETDEWEB)

Soubbaramayer, [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1967-03-01

electrique et thermique). Enfin on discute de toutes les solutions possibles du probleme du piston dans un cas simple. Les methodes developpees ici peuvent etre etendues au choc ionisant. (auteur)

Auditing the Functional Part of the CAS Software

Directory of Open Access Journals (Sweden)

Adamyk Oksana V.

2017-11-01

Full Text Available The article is aimed at determining the order and methodology of auditing the functional component of the software for an computer accounting system (CAS. It has been found that software auditing should be performed separately for each of its components. The components of the functional part of the CAS software are the database management system (DBMS and the application software supporting the accountance automation. For auditing of the first component part are used such techniques as general evaluation, subject check of the embedded algorithms of information processing. Auditing the client software algorithms is carried out by means of the control data method, which is reduced to such procedures as creation of another database of test data with imaginary objects and its processing by the client program, as well as introduction in a copy of the real database of imaginary objects (employees, creditors, material values and the formation of reporting. Not only the current methods of calculation or evaluation of accounting objects, but all of the software, are subject to mandatory verification. This will avoid errors if the enterprise accounting policy changes.

Observation of dynamic water microadsorption on Au surface

Energy Technology Data Exchange (ETDEWEB)

Huang, Xiaokang, E-mail: xiaokang.huang@tqs.com; Gupta, Gaurav; Gao, Weixiang; Tran, Van; Nguyen, Bang; McCormick, Eric; Cui, Yongjie; Yang, Yinbao; Hall, Craig; Isom, Harold [TriQuint Semiconductor, Inc., 500 W Renner Road, Richardson, Texas 75080 (United States)

2014-05-15

Experimental and theoretical research on water wettability, adsorption, and condensation on solid surfaces has been ongoing for many decades because of the availability of new materials, new detection and measurement techniques, novel applications, and different scales of dimensions. Au is a metal of special interest because it is chemically inert, has a high surface energy, is highly conductive, and has a relatively high melting point. It has wide applications in semiconductor integrated circuitry, microelectromechanical systems, microfluidics, biochips, jewelry, coinage, and even dental restoration. Therefore, its surface condition, wettability, wear resistance, lubrication, and friction attract a lot of attention from both scientists and engineers. In this paper, the authors experimentally investigated Au{sub 2}O{sub 3} growth, wettability, roughness, and adsorption utilizing atomic force microscopy, scanning electron microscopy, reflectance spectrometry, and contact angle measurement. Samples were made using a GaAs substrate. Utilizing a super-hydrophilic Au surface and the proper surface conditions of the surrounding GaAs, dynamic microadsorption of water on the Au surface was observed in a clean room environment. The Au surface area can be as small as 12 μm{sup 2}. The adsorbed water was collected by the GaAs groove structure and then redistributed around the structure. A model was developed to qualitatively describe the dynamic microadsorption process. The effective adsorption rate was estimated by modeling and experimental data. Devices for moisture collection and a liquid channel can be made by properly arranging the wettabilities or contact angles of different materials. These novel devices will be very useful in microfluid applications or biochips.

Targeted viral-mediated plant genome editing using crispr/cas9

KAUST Repository

Mahfouz, Magdy M.; Ali, Zahir

2015-01-01

The present disclosure provides a viral-mediated genome-editing platform that facilitates multiplexing, obviates stable transformation, and is applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and systemically deliver a guide RNA molecules into plants overexpressing Cas9 endonuclease. High genomic modification frequencies were observed in inoculated as well as systemic leaves including the plant growing points. This system facilitates multiplexing and can lead to germinal transmission of the genomic modifications in the progeny, thereby obviating the requirements of repeated transformations and tissue culture. The editing platform of the disclosure is useful in plant genome engineering and applicable across plant species amenable to viral infections for agricultural biotechnology applications.

Targeted viral-mediated plant genome editing using crispr/cas9

KAUST Repository

Mahfouz, Magdy M.

2015-12-17

The present disclosure provides a viral-mediated genome-editing platform that facilitates multiplexing, obviates stable transformation, and is applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and systemically deliver a guide RNA molecules into plants overexpressing Cas9 endonuclease. High genomic modification frequencies were observed in inoculated as well as systemic leaves including the plant growing points. This system facilitates multiplexing and can lead to germinal transmission of the genomic modifications in the progeny, thereby obviating the requirements of repeated transformations and tissue culture. The editing platform of the disclosure is useful in plant genome engineering and applicable across plant species amenable to viral infections for agricultural biotechnology applications.

CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani.

Science.gov (United States)

Zhang, Wen-Wei; Matlashewski, Greg

2015-07-21

The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this

Synthesis of Au@Pt bimetallic nanoparticles with concave Au nanocuboids as seeds and their enhanced electrocatalytic properties in the ethanol oxidation reaction

Science.gov (United States)

Tan, Lingyu; Li, Lidong; Peng, Yi; Guo, Lin

2015-12-01

Herein, a new type of uniform and well-structured Au@Pt bimetallic nanoparticles (BNPs) with highly active concave Au nanocuboids (NCs) as seeds was successfully synthesized by using the classic seed-mediated method. Electrochemical measurements were conducted to demonstrate their greatly enhanced catalytic performance in the ethanol oxidation reaction (EOR). It was found that the electrochemical performance for Au@Pt BNPs with the concave Au NCs as seeds, which were enclosed by {611} high-index facets, could be seven times higher than that of the Au@Pt bimetallic nanoparticles with regular spherical Au NPs as seeds. Furthermore, our findings show that the morphology and electrocatalytic activity of the Au@Pt BNPs can be tuned simply by changing the compositional ratios of the growth solution. The lower the amount of H2PtCl6 used in the growth solution, the thinner the Pt shell grew, and the more high-index facets of concave Au NCs seeds were exposed in Au@Pt BNPs, leading to higher electrochemical activity. These as-prepared concave Au@Pt BNPs will open up new strategies for improving catalytic efficiency and reducing the use of the expensive and scarce resource of platinum in the ethanol oxidation reaction, and are potentially applicable as electrochemical catalysts for direct ethanol fuel cells.

Synthesis of Au@Pt bimetallic nanoparticles with concave Au nanocuboids as seeds and their enhanced electrocatalytic properties in the ethanol oxidation reaction

International Nuclear Information System (INIS)

Tan, Lingyu; Li, Lidong; Peng, Yi; Guo, Lin

2015-01-01

Herein, a new type of uniform and well-structured Au@Pt bimetallic nanoparticles (BNPs) with highly active concave Au nanocuboids (NCs) as seeds was successfully synthesized by using the classic seed-mediated method. Electrochemical measurements were conducted to demonstrate their greatly enhanced catalytic performance in the ethanol oxidation reaction (EOR). It was found that the electrochemical performance for Au@Pt BNPs with the concave Au NCs as seeds, which were enclosed by {611}high-index facets, could be seven times higher than that of the Au@Pt bimetallic nanoparticles with regular spherical Au NPs as seeds. Furthermore, our findings show that the morphology and electrocatalytic activity of the Au@Pt BNPs can be tuned simply by changing the compositional ratios of the growth solution. The lower the amount of H_2PtCl_6 used in the growth solution, the thinner the Pt shell grew, and the more high-index facets of concave Au NCs seeds were exposed in Au@Pt BNPs, leading to higher electrochemical activity. These as-prepared concave Au@Pt BNPs will open up new strategies for improving catalytic efficiency and reducing the use of the expensive and scarce resource of platinum in the ethanol oxidation reaction, and are potentially applicable as electrochemical catalysts for direct ethanol fuel cells. (paper)

Quand l'indéfini négatif pouvait s'employer au pluriel. Les emplois de « aucun(es » au XIXe siècle

Directory of Open Access Journals (Sweden)

Denizot Camille

2014-07-01

Full Text Available L’étude porte sur l’existence paradoxale d’indéfinis négatifs au pluriel dans la langue du XIXe siècle. La situation du XIXe siècle atteste en effet d’un état de langue intermédiaire entre deux systèmes stables : celui de l’ancien français où aucun est un indéfini positif qui peut s’employer au singulier comme au pluriel, et celle du français contemporain où aucun est un indéfini négatif qui ne s’emploie plus qu’au singulier, tandis que le sens positif se survit seulement dans la forme de pronom pluriel d’aucuns. Au XIXe siècle, aucun est donc devenu globalement négatif tout en acceptant encore des emplois du pluriel. Si l’explication diachronique motive ce paradoxe, encore faut-il comprendre comment s’interprètent et se justifient ces pluriels pour des indéfinis de sens négatif, dans la synchronie du XIXe siècle. À travers cette étude de cas nous nous interrogeons sur la cohérence synchronique d’un système instable, au croisement de plusieurs évolutions diachroniques. Dans cette optique, nous tentons de comprendre comment se justifie le pluriel des indéfinis négatifs, et comment ces indéfinis pluriels, dont on conçoit mieux la justification a priori pour un sens positif, peuvent développer dans le même état de langue un sens positif et un sens négatif. Nous montrons ainsi que l’emploi du pluriel dans un grammème négatif ne se limite pas au phénomène de l’accord, même s’il tend à s’y cantonner progressivement, et que la coexistence d’un sens négatif et d’un sens positif pour le même grammème n’est pas source d’ambiguïté en synchronie : l’emploi vivant, négatif, vient concurrencer l’emploi positif, résiduel, uniquement dans les contextes à polarité positive et l’interprétation négative y est construite pragmatiquement.

System-level perturbations of cell metabolism using CRISPR/Cas9

DEFF Research Database (Denmark)

Jakociunas, Tadas; Jensen, Michael Krogh; Keasling, Jay

2017-01-01

CRISPR/Cas9 (clustered regularly interspaced palindromic repeats and the associated protein Cas9) techniques have made genome engineering and transcriptional reprogramming studies more advanced and cost-effective. For metabolic engineering purposes, the CRISPR-based tools have been applied...... previously possible. In this mini-review we highlight recent studies adopting CRISPR/Cas9 for systems-level perturbations and model-guided metabolic engineering....

CRISPR Mediated Genome Engineering and its Application in Industry.

Science.gov (United States)

Kaboli, Saeed; Babazada, Hasan

2018-01-01

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) method has been dramatically changing the field of genome engineering. It is a rapid, highly efficient and versatile tool for precise modification of genome that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This novel RNA-guided genome-editing technique has become a revolutionary tool in biomedical science and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing tool, summarize the recent advances in CRISPR/Cas9 technology to engineer the genomes of a wide variety of organisms, and discuss their applications to treatment of fungal and viral disease. We also discuss advantageous of CRISPR/Cas9 technology to drug design, creation of animal model, and to food, agricultural and energy sciences. Adoption of the CRISPR/Cas9 technology in biomedical and biotechnological researches would create innovative applications of it not only for breeding of strains exhibiting desired traits for specific industrial and medical applications, but also for investigation of genome function.

CAS algorithm-based optimum design of PID controller in AVR system

International Nuclear Information System (INIS)

Zhu Hui; Li Lixiang; Zhao Ying; Guo Yu; Yang Yixian

2009-01-01

This paper presents a novel design method for determining the optimal PID controller parameters of an automatic voltage regulator (AVR) system using the chaotic ant swarm (CAS) algorithm. In the tuning process of parameters, the CAS algorithm is iterated to give the optimal parameters of the PID controller based on the fitness theory, where the position vector of each ant in the CAS algorithm corresponds to the parameter vector of the PID controller. The proposed CAS-PID controllers can ensure better control system performance with respect to the reference input in comparison with GA-PID controllers. Numerical simulations are provided to verify the effectiveness and feasibility of PID controller based on CAS algorithm.

CRISPR/Cas9-mediated targeted mutagenesis in upland cotton (Gossypium hirsutum L.).

Science.gov (United States)

Janga, Madhusudhana R; Campbell, LeAnne M; Rathore, Keerti S

2017-07-01

The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas)9 protein system has emerged as a simple and efficient tool for genome editing in eukaryotic cells. It has been shown to be functional in several crop species, yet there are no reports on the application of this or any other genome editing technologies in the cotton plant. Cotton is an important crop that is grown mainly for its fiber, but its seed also serves as a useful source of edible oil and feed protein. Most of the commercially-grown cotton is tetraploid, thus making it much more difficult to target both sets of homeologous alleles. Therefore, in order to understand the efficacy of the CRISPR/Cas9 system to target a gene within the genome of cotton, we made use of a transgenic cotton line previously generated in our laboratory that had a single copy of the green fluorescent protein (GFP) gene integrated into its genome. We demonstrate, for the first time, the use of this powerful new tool in targeted knockout of a gene residing in the cotton genome. By following the loss of GFP fluorescence, we were able to observe the cells that had undergone targeted mutations as a result of CRISPR/Cas9 activity. In addition, we provide examples of the different types of indels obtained by Cas9-mediated cleavage of the GFP gene, guided by three independent sgRNAs. The results provide useful information that will help us target important native genes in the cotton plant in future.

CRISPR/Cas9 advances engineering of microbial cell factories

DEFF Research Database (Denmark)

Jakociunas, Tadas; Jensen, Michael Krogh; Keasling, Jay D.

2016-01-01

interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing......-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering....

Controlled electrodeposition of Au monolayer film on ionic liquid

Science.gov (United States)

Ma, Qiang; Pang, Liuqing; Li, Man; Zhang, Yunxia; Ren, Xianpei; Liu, Shengzhong Frank

2016-05-01

Gold (Au) nanoparticles have been attractive for centuries for their vibrant appearance enhanced by their interaction with sunlight. Nowadays, there have been tremendous research efforts to develop them for high-tech applications including therapeutic agents, sensors, organic photovoltaics, medical applications, electronics and catalysis. However, there remains to be a challenge to fabricate a monolayer Au coating with complete coverage in controlled fashion. Here we present a facile method to deposit a uniform Au monolayer (ML) film on the [BMIM][PF6] ionic liquid substrate using an electrochemical deposition process. It demonstrates that it is feasible to prepare a solid phase coating on the liquid-based substrate. Moreover, the thickness of the monolayer coating can be controlled to a layer-by-layer accuracy.

CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium.

Science.gov (United States)

Soh, Chew-Li; Huangfu, Danwei

2017-01-01

The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system. This chapter describes protocols for CRISPR/Cas9 genome editing in an inducible Cas9 hPSC-based system, using cells cultured in chemically defined, xeno-free E8 Medium on a recombinant human vitronectin substrate. We detail procedures for the design and transfection of CRISPR guide RNAs, colony selection, and the expansion and validation of clonal mutant lines, all within this fully defined culture condition. These methods may be applied to a wide range of genome-engineering applications in hPSCs, including those that utilize different types of site-specific nucleases such as zinc finger nucleases (ZFNs) and TALENs, and form a closer step towards clinical utility of these cells.

Calcium polysulphide, its applications and emerging risk of environmental pollution-a review article.

Science.gov (United States)

Dahlawi, Saad Mohammed; Siddiqui, Samreen

2017-01-01

Easy availability, preparation technique, and economic value make calcium polysulphide (CaS x ) a very useful inorganic chemical for various field and industrial applications. In this article, disparate applications of CaS x solution have been reviewed to suggest potential and future consolidation. This article also encompasses the physiochemical properties and production of CaS x solution, with critical appraisal on research focusing on CaS x application in agriculture industries and removal of potentially toxic elements (PTEs) from the environment. The kinetics of CaS x , technical issues associated with optimization of its dosage and environmental fate is also discussed in detail. This study covers almost all of the peer-reviewed research that has been performed since 1914. Some of the critiques in this article include the lack of integration between the exposure effect and the efficiency of treatment method, effects of oxidizing environments on the long-term performance of CaS x solution, and kinetics of CaS x solution with the PTEs. The working model of CaS x with PTEs is still system dependent, and therefore cannot be used with other applications. The kinetics of CaS x is described in detail with various phase stoichiometric reactions. Environmental fate is discussed based on applications, government reports, peer-reviewed articles and kinetics of CaS x , which provides a clear picture of emerging contaminants in the environment in relation to the insect resistance and ecotoxicology. Real time, lab based research articles are needed to identify toxicity limits of CaS x in environment in order to describe its effective permissible limit in environmental system. This review article provides a risk assessment of environmental pollution by CaS x based on its physicochemical characteristic, stoichiometry, kinetics, field, and industrial applications.

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites

Directory of Open Access Journals (Sweden)

Thomas J Cradick

2014-01-01

Full Text Available Precise genome editing using engineered nucleases can significantly facilitate biological studies and disease treatment. In particular, clustered regularly interspaced short palindromic repeats (CRISPR with CRISPR-associated (Cas proteins are a potentially powerful tool for modifying a genome by targeted cleavage of DNA sequences complementary to designed guide strand RNAs. Although CRISPR/Cas systems can have on-target cleavage rates close to the transfection rates, they may also have relatively high off-target cleavage at similar genomic sites that contain one or more base pair mismatches, and insertions or deletions relative to the guide strand. We have developed a bioinformatics-based tool, COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions that searches genomes for potential off-target sites (http://crispr.bme.gatech.edu. Based on the user-supplied guide strand and input parameters, COSMID identifies potential off-target sites with the specified number of mismatched bases and insertions or deletions when compared with the guide strand. For each site, amplification primers optimal for the chosen application are also given as output. This ranked-list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas induced off-target cleavage in cells.

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains.

Science.gov (United States)

Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; Zhao, Huimin

2018-06-01

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories. © 2018 Wiley Periodicals, Inc.