An Allosteric Inhibitor of KRas Identified Using a Barcoded Rapid Assay Microchip Platform

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC_(50)) of ∼24 μM

An Allosteric Inhibitor of KRas Identified Using a Barcoded Rapid Assay Microchip Platform

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC_(50)) of ∼24 μM

The chipá and the feminine stamp: memories of paraguayan women who are part of the cultural heritage of Paraguay.

Ko jehesa‟ÿijópe oñeñe‟ë peteï arandukuaa mba‟e porä ñemomba‟ére, peteï mba‟e ojekuaa ha ojejapo meméva Paraguáipe: pe chipa (mbujape ojejapóva aramirö mandi‟ógui, avati aramirö, kure ñandy, ryguasu rupi‟a, kesu, kamby ha ambue mba‟e oñemoï joparáva), kuña rembiapóicha ojehechaukáva ñandéve oñekuave‟ëhápe ko oñemomba‟eva‟ekue Tetä rembi‟úicha (arandukuaa mba‟e tee) Paraguáipe. Ko chipáre ñeñemu, maymave ojehecha kuñanguéra pópe, umi ojekuaáva opárupi chipéraicha. Upéicha avei, ko tembiapo rupive ojehechaukase ha oñeikumby poräseve umi kuña paraguái rembiapo katupyry, yvypóra omoañetéva gueteri ohóvo pe ñande reko ymágui oúva, ñane retäygua mandu‟áicha ha mba‟e porä oikovéva chipáre oñeñemuhápe. Upéicha avei, ko ñe‟ë haipýpe ojehechauka umi tekove rapykuere, hembiapopekuéra ha mba‟apohára kuñáicha upe chipa ñemoñemúme; mba‟éichagua tekoha ha mba‟éichapa ojereko hikuái pe imba‟apohápe, mba‟eichahárupi oñemoï ha oje‟u pe chipa, ha mba‟éicha ojereko pe mba‟e tee rehegua mandu‟a, upe teko arandu guasu kuña mba‟évape. Ko jehesa‟ÿijo ohechase pe mba‟e apytu‟ü oipurukuévo Marandeko Jurugua Ñe‟ërä.O assunto em questão lida com o capital de um bem cultural, produto característico do Paraguai; o chipá ou a chipa (pão feito principalmente com amido de mandioca, farinha de milho, banha de porco, ovos, queijo, leite, entre outros ingredientes que se combinam de forma variada), à luz do preponderante trabalho feminino na comercialização deste bem, declarado alimento nacional (patrimônio cultural) do Paraguai. Este trabalho, de venda do chipá, em sua grande maioria, ocorre nas mãos de mulheres popularmente conhecidas como chiperas. Assim, o principal objetivo da pesquisa é tornar visível e comprender o papel das mulheres paraguaias como pessoas que representam e preservam uma tradição cultural, uma memória coletiva e um patrimônio vivo, através da comercialização do chipá. Assim também, a dissertação apresenta as trajetorias de vida, experiências laborais e pessoais das mulheres em relação à chipá; a descrição das condições de trabalho em que as mesmas se encontram; os vários usos e formas de comensalidade e sociabilidade associados ao chipá e a análise dos conceitos de memória e patrimônio, principalmente, envolvidos em um universo cultural essencialmente feminino. A pesquisa é qualitativa, na qual foi aplicada a metodologia de História Oral Temática.The subject in question deals with the capital of a cultural asset, a characteristic product of Paraguay; the chipá or chipa (type of bread made mainly with cassava starch, corn flour, pork fat, egg, cheese, milk, among other ingredients that are combined in a variety of ways), through the preponderant female labor in the commercialization of this cultural good, declared as a national food (cultural heritage) of Paraguay. This work, to sell chipá, is developed, mostly, in the hands of women popularly known as chiperas. Thus, the main objective of the research is to make visible and understand the protagonism of paraguayan women as people who represent and preserve a cultural tradition, a collective memory and a living heritage, through the commercialization of the chipá. Also, the present dissertation presents the life and labor trajectories and personal experiences of women towards the commercialization of the chipá; the description of the working conditions in which they are found, the various uses and forms of commensality and sociability associated with the chipá and the analysis of the concepts of memory and cultural heritage, mainly, involved in an essentially feminine cultural universe. The research is qualitative, in which was applied the Thematic Oral History methodology.La materia en cuestión trata sobre el capital de un bien cultural, producto característico del Paraguay; el chipá o la chipa (panecillo elaborado principalmente con almidón de mandioca, harina de maíz, grasa de cerdo, huevo, queso, leche, entre otros ingredientes que se combinan de manera variada), a la luz de la preponderante labor femenina en el oficio de la comercialización de este bien, declarado alimento nacional (patrimonio cultural) del Paraguay. Esta labor, de vender chipá, en su gran mayoría, se da en manos de mujeres popularmente conocidas como las chiperas. Así pues, el objetivo principal de la investigación es visibilizar y comprender el protagonismo de mujeres paraguayas como personas que representan y preservan una tradición cultural, una memoria colectiva y un patrimonio vivo, a través de la comercialización del chipá. Así también, la presente disertación expone las trayectorias de vida, experiencias laborales y personales de mujeres en relación a la comercialización del chipá; la descripción de las condiciones laborales en las que las mismas se encuentran, los varios usos y formas de comensalía y sociabilidad asociadas al chipá y el análisis de los conceptos de memoria y patrimonio, principalmente, envueltos en un universo cultural esencialmente femenino. La investigación es de carácter cualitativa, en la cual se aplicó la metodología de Historia Oral Temática