Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line

This project was carried out during CD’s BBSRC Eastbio PIPS placement at Marine Scotland. The authors are grateful to Dr Milena Monte (University of Aberdeen) for help with the FACS analysis. The authors wish to thank Dr Filippo Del Bene (Neuronal Circuit Development, Institut Curie) and Dr Wenbiao Chen (School of Medicine, Vanderbilt University) for the Addgene plasmids, #61051 and #47929, respectively, and Prof. Nancy C. Reich Marshall (Department of Molecular Genetics and Microbiology, Stony Brooks University) for the plasmid pmEGFP-N1.

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line

This project was carried out during CD’s BBSRC Eastbio PIPS placement at Marine Scotland. The authors are grateful to Dr Milena Monte (University of Aberdeen) for help with the FACS analysis. The authors wish to thank Dr Filippo Del Bene (Neuronal Circuit Development, Institut Curie) and Dr Wenbiao Chen (School of Medicine, Vanderbilt University) for the Addgene plasmids, #61051 and #47929, respectively, and Prof. Nancy C. Reich Marshall (Department of Molecular Genetics and Microbiology, Stony Brooks University) for the plasmid pmEGFP-N1.

CRISPR/Cas9-Mediated Gene Editing of ARG1 in Cell Lines and Primary Cells

Arginase 1 deficiency, a urea cycle disorder resulting from an inability of the body to convert arginine into urea, results in hyperargininemia and sporadic episodes of hyperammonemia. Arginase 1 deficiency can lead to a range of developmental disorders and progressive spastic diplegia in children, and current therapeutic options are limited. Clustered regularly interspaced short palindromic repeat (CRISPR) /CRISPR associated protein (Cas) 9 gene editing systems serve as a novel means of treating genetic disorders such as Arginase 1 (ARG1) deficiency, and must be thoroughly examined to determine their curative capabilities. In these experiments numerous guide RNAs and CRISPR/Cas9 systems targeting the ARG1 gene were designed and observed by heteroduplex assay for their targeting capabilities and cleavage efficiencies in multiple cell lines. The CRISPR/Cas9 system utilized in these experiments, along with a panel of guide RNAs targeting various locations in the arginase 1 gene, successfully produced targeted cleavage in HEK293, MCF7, A549, K562, HeLa, and HepG2 cells; however, targeted cleavage in human dermal fibroblasts, blood outgrowth endothelial cells, and induced pluripotent stem cells was not observed. Additionally, a CRISPR/Cas system involving partially inactivated Cas9 was capable of producing targeted DNA cleavage in intron 1 of ARG1, while a Cas protein termed Cpf1 was incapable of producing targeted cleavage. These results indicate a complex set of variables determining the CRISPR/Cas9 systems’ capabilities in the cell lines and primary cells tested. By examining epigenetic factors and alternative CRISPR/Cas9 gene targeting systems, the CRISPR/Cas9 system can be more thoroughly considered in its ability to act as a means towards editing the genome of arginase 1-deficient individuals.

Un-Editing Alfred: Rethinking Modern Editions of Pre-modern Texts from a Post-modern Sensibility

Thesis (Ph.D.)--University of Washington, 2016-06

megaTAL-mediated Gene Editing at the CCR5 locus

Thesis (Ph.D.)--University of Washington, 2016-08

Page Composition using PPML as a Link-editing Script

The advantages of a COG (Component Object Graphic) approach to the composition of PDF pages have been set out in a previous paper [1]. However, if pages are to be composed in this way then the individual graphic objects must have known bounding boxes and must be correctly placed on the page in a process that resembles the link editing of a multi-module computer program. Ideally the linker should be able to utilize all declared resource information attached to each COG. We have investigated the use of an XML application called Personalized Print Markup Language (PPML) to control the link editing process for PDF COGs. Our experiments, though successful, have shown up the shortcomings of PPML's resource handling capabilities which are currently active at the document and page levels but which cannot be elegantly applied to individual graphic objects at a sub-page level. Proposals are put forward for modifications to PPML that would make easier any COG-based approach to page composition.

Engineering the zinc finger recombinase for use in targeted genomic editing

No abstract available.

Metodologia di editing immagini per analisi dati morfometrici dei primi stadi di sviluppo di Engraulis encrasicolus (Linnaeus, 1758)

Tra le più importanti risorse alieutiche di molte regioni del Mar Mediterraneo vi sonole acciughe (Engraulisencrasicolus, Linnaeus, 1758), piccoli pesci pelagici appartenenti alle famiglie degli Engraulidae. Dati IREPA del 2009, hanno di fatto reso noto che in Italia, la pesca di E. encrasicolusha rappresentano in media il 26% circa del pescato totale. Questa specie viene continuamente monitorata e grazie a tali programmi è stato evidenziato che vi sono delle fluttuazioni inter-annuali molto pronunciate (Cergoleet al., 2002; Cingolani, 2004), le cui cause possono essere molteplici, da fattori antropicicome l’elevato sforzo di pesca a fattori naturali (Borjia et al., 1996). Va però posta molta attenzione sulle dinamiche biologiche ed ambientali che influiscono sulla sopravvivenza dei primi stadi di vita di questa specie, che ricadendo sul successivo reclutamento, possono essere una delle cause fondamentali delle contrazioni e degli incrementi annuali dello stock adulto (Thikonova et al., 2000; James et al., 2003; Cuttitta et al., 2003, 2006).Lo studio delle fasi ittioplanctoniche e delle sue relazioni con l’ambiente e gli altri organismi, risulta quindi di primaria importanza nell’ambito delle conoscenze necessarie per il corretto sfruttamento delle risorse alieutiche.

En/titled: The space where writing, editing and teaching intersect

Editing a literary magazine offers us a cultural space where our ideas and aesthetics can be expressed collectively and therefore be heard more effectively. This informs and frames our own writing by increasing our confidence in our own unusual voices. The sense of belonging Brand creates further breaks down the isolation of the writing life. The internationalism of Brand reinforces our own cultural identities as non-English writers. However, acting as a facilitator of others’ creativity can sometimes dissipate or even deplete creative energy. Editing and teaching can take over your writing to the point of annihilation. Further, in terms of external perceptions, you run the risk of disappearing as a writer. We shall look at how this can happen and explore ways that we can prevent it e.g. keeping the boundaries firm and clear.

Convergent bioenergetic defects in Coenzyme Q10 depleted cells by pharmacological inhibition of coq2 enzyme (p-hydroxybenzoate polyprenyl transferase) and by genome editing technology targeting the encoding gene (COQ2)

Primary CoQ10 deficiency diseases encompass a heterogeneous spectrum of clinical phenotypes. Among these, defect or mutation on COQ2 gene, encoding a para-hydroxybenzoate polyprenyl transferase, have been associated with different diseases. Understanding the functional and metabolic impact of COQ2 mutation and the consequent CoQ10 deficiency is still a matter of debate. To date the aetiology of the neurological phenotypes correlated to CoQ10 deficiency does not present a clear genotype-phenotype association. In addition to the metabolic alterations due to Coenzyme Q depletion, the impairment of mitochondrial function, associated with the reduced CoQ level, could play a significant role in the metabolic flexibility of cancer. This study aimed to characterize the effect of varying degrees of CoQ10 deficiency and investigate the multifaceted aspect of CoQ10 depletion and its impact on cell metabolism. To induced CoQ10 depletion, different cell models were used, employing a chemical and genome editing approach. In T67 and MCF-7 CoQ10 depletion was achieved by a competitive inhibitor of the enzyme, 4-nitrobenzoate (4-NB), whereas in SH-SY5Y the COQ2 gene was edited via CRISPR-Cas9 cutting edge technology.

A genome editing approach to the study of Parvovirus B19

Parvovirus B19 (B19V) is a ssDNA virus, with a 5596 nt long genome encapsidated within an icosahedral capsid with a diameter of 22 nm. Viral proteins are subdivided into structural and non-structural: the main non-structural one is the NS1, while the 2 structural proteins VP1 and VP2 assemble originating the capsid shell. B19V tropism is mainly limited to erythroid progenitor cells (EPCs), however, virus can be detected in several districts persisting in tissues possibly lifelong. The virus can induce anemia and erythroid aplasia. Therapeutic strategies are only symptomatic, so the search for antivirals is strongly active, with screenings showing the activity in vitro of different compounds like hydroxyurea, cidofovir and brincidofovir. In the first project, a functional minigenome of B19V was developed, able to express only the NS1 protein. This minigenome proved able to replicate and express the NS1 at levels comparable to unmodified clones. Furthermore, the ability of this minigenome to complement the function of NS1-deficient genomes was demonstrated, thus providing a proof-of-concept of B19V genome editing possibility and, at the same time, a useful tool to study the NS1 protein also as an antiviral target. In the second project I addressed the interplay between B19V and the cellular restriction factor APOBEC3B (A3B), a cytidine deaminase acting on ssDNA, whose footprint on B19V genome was proved by a bioinformatic sequence analysis performed by the hosting lab. To understand whether A3B still exerts activity and a potential antiviral effect on B19V, the UT7/EpoS1 cells were transduced with lentiviral vectors to silence A3B expression, then used as a model to study viral behavior. No significant role of A3B on B19V was demonstrated, in agreement with the hypothesis of viral adaptation to this cellular restriction factor; anyway, virus ability to alter A3B expression would deserve further investigations.

Arqueologia das práticas editoriais: 15 anos de um impresso em Movimento

The paper studies the archeology of editorial practices of the journal Movimento regarding the processes of editing, diffusion, circulation and adaptation to the index agencies. It reveals the phases of the printed material emphasizing the choices of the editors with reference to the graphical project of the periodic that puts in circulation different devices of regulation of the appropriations, at the same time when it aims at creating an identity that qualifies the periodic as an authorized gadget able to show the readers what Physical Education is in Brazil.

A synchronous multimedia annotation system for secure collaboratories

In this paper, we describe the Vannotea system - an application designed to enable collaborating groups to discuss and annotate collections of high quality images, video, audio or 3D objects. The system has been designed specifically to capture and share scholarly discourse and annotations about multimedia research data by teams of trusted colleagues within a research or academic environment. As such, it provides: authenticated access to a web browser search interface for discovering and retrieving media objects; a media replay window that can incorporate a variety of embedded plug-ins to render different scientific media formats; an annotation authoring, editing, searching and browsing tool; and session logging and replay capabilities. Annotations are personal remarks, interpretations, questions or references that can be attached to whole files, segments or regions. Vannotea enables annotations to be attached either synchronously (using jabber message passing and audio/video conferencing) or asynchronously and stand-alone. The annotations are stored on an Annotea server, extended for multimedia content. Their access, retrieval and re-use is controlled via Shibboleth identity management and XACML access policies.

Incremental parsing in language-based editors: user needs and how to meet them

Incremental parsing has long been recognized as a technique of great utility in the construction of language-based editors, and correspondingly, the area currently enjoys a mature theory. Unfortunately, many practical considerations have been largely overlooked in previously published algorithms. Many user requirements for an editing system necessarily impact on the design of its incremental parser, but most approaches focus only on one: response time. This paper details an incremental parser based on LR parsing techniques and designed for use in a modeless syntax recognition editor. The nature of this editor places significant demands on the structure and quality of the document representation it uses, and hence, on the parser. The strategy presented here is novel in that both the parser and the representation it constructs are tolerant of the inevitable and frequent syntax errors that arise during editing. This is achieved by a method that differs from conventional error repair techniques, and that is more appropriate for use in an interactive context. Furthermore, the parser aims to minimize disturbance to this representation, not only to ensure other system components can operate incrementally, but also to avoid unfortunate consequences for certain user-oriented services. The algorithm is augmented with a limited form of predictive tree-building, and a technique is presented for the determination of valid symbols for menu-based insertion. Copyright (C) 2001 John Wiley & Sons, Ltd.

Managing Organisational Writing to Enhance Corporate Credibility

Many employees neither trained as career writers nor defining themselves as writers spend a major part of their time writing because, undeniably, writing is a central activity in organizations. To produce the high quality required to create and maintain credibility, organizations need to have professionals who can efficiently produce documents with substance, structure and style. This paper discusses issues relating to the management of corporate writing and editing, and presents practices and processes that managers can implement in their organizations to produce flawless documents, thereby avoiding the credibility crises that occur when writing is not taken seriously.