A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination

BACKGROUND: Cre-loxP recombination refers to the process of site-specific recombination mediated by two loxP sequences and the Cre recombinase protein. Transgenic experiments exploit integrative recombination, where a donor plasmid carrying a loxP site and DNA of interest integrate into a recipient loxP site in a target genome. Unfortunately, integrative recombination is highly inefficient because the insert is flanked by two loxP sites, which themselves become targets for Cre and lead to subsequent excision of the insert. A small number of mutations have been discovered in parts of the loxP sequence, specifically the spacer and inverted repeat segments, that increase the efficiency of integrative recombination. In this study we introduce a high-throughput in vitro assay to rapidly detect novel loxP spacer mutants and describe the sequence characteristics of successful recombinants. RESULTS: We created synthetic loxP oligonucleotides that contained a combination of inverted repeat mutations (the lox66 and lox71 mutations) and mutant spacer sequences, degenerate at 6 of the 8 positions. After in vitro Cre recombination, 3,124 recombinant clones were identified by sequencing. Included in this set were 31 unique, novel, self-recombining sequences. Using network visualization tools, we recognized 12 spacer sets with restricted promiscuity. We observed that increased guanine content at all spacer positions save for position 8 resulted in increased recombination. Interestingly, recombination between identical spacers was not preferred over non-identical spacers. We also identified a set of 16 pairs of loxP spacers that reacted at least twice with another spacer, but not themselves. Further, neither the wild-type P1 phage loxP sequence nor any of the known loxP spacer mutants appeared to be kinetically favoured by Cre recombinase. CONCLUSION: This study approached loxP spacer mutant screening in an unbiased manner, assuming nothing about candidate loxP sites save for the conserved 4 and 5 spacer positions. Candidate sites were free to recombine with any other sequence in the pool of all possible sites. The subset of loxP sites identified here are candidates for in vivo serial recombination as they have already demonstrated limited promiscuity with other loxP spacer and stability in the presence of Cre

Analise das diferenças e similaridades no conteúdo dos regimentos internos dos comitês de autoria das empresas de capital aberto

O estudo objetivou analisar o conteúdo dos regulamentos internos dos comitês de auditoria estatutários das empresas brasileiras de capital aberto listadas na B3. Foi utilizada uma pesquisa descritiva, documental e qualitativa com base em uma amostra de 111 empresas que divulgaram os Regimentos Internos dos Comitês de Auditoria Estatutários, no site da Comissão de Valores Mobiliários, no período de 01/01/2018 a 26/11/2021. Por fim, o objetivo do trabalho consiste em analisar o conteúdo disseminado pelas organizações nos respectivos relatórios, expondo quais conteúdos as empresas abordam ou deixam de abordar e ainda se seguem as normas e instruções das agências e instituições reguladoras. Em linhas gerais, pode-se concluir que as empresas nem sempre formalizam no Regimento Interno do Comitê de Auditoria o que é exigido pelas normas, ou seja, não mencionam as competências do comitê, a composição do comitê e nem tampouco deveres dos membros do Comitê. Além disso, nota-se que as empresas não seguem fidedignamente o conteúdo que deve mencionar em concordância com o que é pedido pelos órgãos regulamentadores

From citizen science to jellyfish dispersion models and molecular studies : tracking the progress of jellyfish science in Malta (Central Mediterranean)

Following participation within the 1980”s FAO-mediated monitoring exercises of Pelagia noctiluca blooms within Maltese waters, little scientific effort was invested in studying the dynamics of jellyfish blooms within same waters and at developing management and public information strategies concerning the same blooms. A renewed scientific effort at studying such aspects within Maltese waters was registered from 2010 onwards, with the launch of the Spot the Jellyfish citizen science campaign (www.ioikids.net/jellyfish) which provided a user-friendly, multivalent and web-based through which maritime stakehold- ers and the public at large could submit their jellyfish records for Maltese waters. The web- based portal was also supported by other promotional initiatives in the field, such as the installation of seaside boards on beaches. Through this initiative, several previously-un- documented species of gelatinous plankton were recorded for the first time from the same waters, including Rhopilema nomadica, Aequorea forskalea, Porpita porpita, Discomedua lobata, Geryonia proboscidalis, Neotima lucullana, Physophora hydrostatica, Chrysaora hysoscella and Oceania armata. The maintenance of an updated jellyfish record database has been made possible through the conduction of such a citizen science initiative.peer-reviewe

Perseus and The Gorgon Medusa

18 cm; 52 ha

Sons and Daughters of the Cherokee Strip Collection

Photograph of the Enid O.T. Square, east side, showing Hockaday Hardware

"A Morbid Longing for the Picturesque" : The Pursuit of Beauty in Donna Tartt's The Secret History

This essay analyzes the theme of the pursuit of beauty in The Secret History. It analyzes the main characters’ concept of beauty, their manner of seeking beauty, as well as the result of this search. For this analysis, I use Friedrich Nietzsche’s theories of the Apollonian and the Dionysian as outlined in The Birth of Tragedy and in scholarly texts that analyze TBT— which describe the Apollonian/Dionysian dichotomy as the opposed worlds of order and madness— to define the main characters’ concept of beauty. The narrator of the novel once says that “beauty is terror” (Tartt 45), a statement which paints beauty as harsh and shocking, and potentially destructive. Likewise, in this essay I argue that for these characters beauty is created through the interplay between the Apollonian and the Dionysian, and that its pursuit leads to destruction. I analyze this through the characters of Richard Papen, Henry Winter, and Bunny Corcoran. Richard and Henry pursue beauty in that the actions they take are aimed at embodying an aesthetic ideal. In Richard’s case, it is his longing for beauty which leads him to imitate and join the classicists— particularly by mimicking their socio-economic class— and which eventually places him in a disordered Dionysian world of madness and murder. Henry, on the other hand, is the embodiment of Apollonian order, and it is his search for beauty through a bacchanal which leads him to commit murder twice and, eventually, to take his own life. Lastly, Bunny is different in that he is neither beautiful nor interested in beauty as his peers define it. It is because of this that he is excluded from the others’ pursuit of beauty, that he is murdered, and that his murder is justifiable in the eyes of his murderers. This study finds that, in The Secret History, where beauty is defined as the dance between Apollonian order and Dionysian madness, the Dionysian ends up as the victorious half of the dichotomy, causing the loss of reason and the triumph of destruction and disaster. This portrayal of beauty as destruction and vice versa, rather than serving as the vehicle for a moral indictment, is instead the very purpose of the novel

Sons and Daughters of the Cherokee Strip Collection

Photograph of a very early image of downtown Enid, Oklahoma. Depicted is the Crescent Hotel, Club Saloon, Buttrey Groceries. There is a large puddle in middle of street and shadows of photography crew

“People, Corrupted”: Monstruösa förvandlingar i “The Whistlers” och “Whitefall”

This essay explores monstrosity in two contemporary horror stories: “The Whistlers” by Amity Argot, and “Whitefall” by C.K. Walker, focusing on how the humans in these texts are monstrously transformed. The monsters and monstrosity present in the texts are read against some of the cultural anxieties of postmodernity, and against various monstrous frameworks such as that of the zombie, the terrorist, and the monstrous space and nature. Both texts present monstrous spaces intent on perverting humans by eroding them physically until they reach a state of bare life that mimics zombification and may allegorize socioeconomic inequality, displacement, and the effects of capitalism; as well as by enticing them to commit atrocities against each other and transgress the very moral boundaries that defined them as human, up to and including cannibalism. In this way, these monsters reveal humans as their own annihilators, laying bare an innate human monstrosity that emerges from the traumatic conditions of postmodernity

“People, Corrupted”: Monstruösa förvandlingar i “The Whistlers” och “Whitefall”

This essay explores monstrosity in two contemporary horror stories: “The Whistlers” by Amity Argot, and “Whitefall” by C.K. Walker, focusing on how the humans in these texts are monstrously transformed. The monsters and monstrosity present in the texts are read against some of the cultural anxieties of postmodernity, and against various monstrous frameworks such as that of the zombie, the terrorist, and the monstrous space and nature. Both texts present monstrous spaces intent on perverting humans by eroding them physically until they reach a state of bare life that mimics zombification and may allegorize socioeconomic inequality, displacement, and the effects of capitalism; as well as by enticing them to commit atrocities against each other and transgress the very moral boundaries that defined them as human, up to and including cannibalism. In this way, these monsters reveal humans as their own annihilators, laying bare an innate human monstrosity that emerges from the traumatic conditions of postmodernity

CIS-features mediating CAG/CTG repeat instability the Satellog database, and candidate repeat prioritization in schizophrenia

Polyglutamine repeat expansions in the coding regions of unrelated genes have been implicated in the neurodegenerative phenotype of nine separate diseases. However, little is known about the role of flanking c/'s-sequences in mediating this repeat instability. Brock et al. identified an association between flanking GC content and CAG/CTG repeat instability at many of these disease loci by using a relative measure of repeat instability called 'expandability'. Using this measure, we have extended the analysis of Brock and colleagues and utilized the expandability metric to associate other features theorized to contribute to CAG/CTG repeat instability such as repeat length and purity, proximity to CCCTC-binding factor (CTCF) binding sites, and the nucleosome formation potential of the surrounding DNA. Our results confirmed earlier relationships regarding flanking G C content and CAG/CTG repeat instability and also suggest a novel one involving flanking CTCF binding sites. Conversely, no relationships between expandability and repeat length, purity, and nucleosome formation were detected. Anticipation refers to the progressive worsening of a disease phenotype and earlier age of onset in successive generations. Anticipation has been reported in a number of diseases in which repeat expansion may have a role in etiology. We developed Satellog, a database that catalogs all pure 1-16 repeat unit repeats in the human genome along with supplementary data of use for the prioritization of repeats in disease association studies. For each pure repeat we calculate the percentile rank of its length relative to other repeats of the same class in the genome, its polymorphism within UniGene clusters, its location either within or adjacent to EnsEMBL-defined genes, and its expression profile in normal tissues according to the GeneNote database. By examining the global repeat polymorphism profile, we found that highly polymorphic coding repeats were mostly restricted to trinucleotide repeats, whereas a wider range of repeat unit lengths were tolerated in untranslated sequence. We also found that 3'-UTR sequence tolerates more repeat polymorphisms than 5'-UTR or exonic sequence. Lastly, we use Satellog to prioritize repeats for disease-association studies in schizophrenia. Satellog is available as a freely downloadable MySQL and web-based database.Medicine, Faculty ofMedical Genetics, Department ofGraduat