The Mini‐Organo: A rapid high‐throughput 3D coculture organotypic assay for oncology screening and drug development

Background: The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease. Aim: The development of the 3D coculture collagen contraction and invasion assay, the "organotypic assay," has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development. Methods and results Here, we describe a modified 96-well organotypic assay, the "Mini-Organo," which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions. Conclusions: The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner

Tamanho de amostra para avaliação de caracteres de cenoura em sistemas de cultivo agroecológico.

O correto dimensionamento de experimentos deve ser utilizado para que se possa reduzir o erro experimental e com isso, maximizar a precisão das informações obtidas. O objetivo deste trabalho foi estimar o número mínimo de famílias e de plantas por parcela para avaliação de caracteres de raiz em uma população de cenoura cultivada em sistema agroecológico. Os ensaios foram conduzidos no verão de 2006/2007 em duas propriedades com cultivo agroecológico em Brasília: Associação Mokiti Okada, que segue modelo de Agricultura Natural em Brazlândia-DF e Núcleo Rural Taguatinga, seguindo modelo de Agricultura Orgânica em Taguatinga-DF. Foram avaliadas 100 famílias de meio-irmãos de cenoura dispostas em delineamento de blocos casualizados com duas repetições e parcelas de 1 m2. Foram colhidas 20 plantas competitivas por parcela e avaliadas individualmente para os caracteres comprimento de raiz, diâmetro da raiz, massa da raiz, diâmetro do xilema da raiz, relação diâmetro do xilema/diâmetro da raiz, tipo de ponta da raiz, tipo de ombro da raiz, parâmetro a* para os tecidos xilema e floema. Foi realizada análise de variância conjunta e para cada sistema, com informação entre e dentro de parcelas. Foram realizadas ainda análises de representatividade do número mínimo de famílias e de plantas para representar uma população de cenoura. Foi verificado que uma amostra de 18 plantas competitivas/parcela coletadas em ensaios com 2 repetições, bem como de 74 famílias, são suficientes para garantir uma adequada avaliação de famílias meio-irmãos de cenoura para os caracteres estudados

Genome wide scan for quantitative trait loci affecting tick resistance in cattle (Bos taurus × Bos indicus)

<p><b>Abstract</b></p> <p><b>Background</b></p> <p>In tropical countries, losses caused by bovine tick <it>Rhipicephalus (Boophilus) microplus</it> infestation have a tremendous economic impact on cattle production systems. Genetic variation between <it>Bos taurus</it> and <it>Bos indicus</it> to tick resistance and molecular biology tools might allow for the identification of molecular markers linked to resistance traits that could be used as an auxiliary tool in selection programs. The objective of this work was to identify QTL associated with tick resistance/susceptibility in a bovine F2 population derived from the Gyr (<it>Bos indicus</it>) × Holstein (<it>Bos taurus</it>) cross.</p> <p>Results</p> <p>Through a whole genome scan with microsatellite markers, we were able to map six genomic regions associated with bovine tick resistance. For most QTL, we have found that depending on the tick evaluation season (dry and rainy) different sets of genes could be involved in the resistance mechanism. We identified dry season specific QTL on BTA 2 and 10, rainy season specific QTL on BTA 5, 11 and 27. We also found a highly significant genome wide QTL for both dry and rainy seasons in the central region of BTA 23.</p> <p>Conclusions</p> <p>The experimental F2 population derived from Gyr × Holstein cross successfully allowed the identification of six highly significant QTL associated with tick resistance in cattle. QTL located on BTA 23 might be related with the bovine histocompatibility complex. Further investigation of these QTL will help to isolate candidate genes involved with tick resistance in cattle.</p

Practical computational toolkits for dendrimers and dendrons structure design

Dendrimers and dendrons offer an excellent platform for developing novel drug delivery systems and medicines. The rational design and further development of these repetitively branched systems are restricted by difficulties in scalable synthesis and structural determination, which can be overcome by judicious use of molecular modelling and molecular simulations. A major difficulty to utilise in silico studies to design dendrimers lies in the laborious generation of their structures. Current modelling tools utilise automated assembly of simpler dendrimers or the inefficient manual assembly of monomer precursors to generate more complicated dendrimer structures. Herein we describe two novel graphical user interface (GUI) toolkits written in Python that provide an improved degree of automation for rapid assembly of dendrimers and generation of their 2D and 3D structures. Our first toolkit uses the RDkit library, SMILES nomenclature of monomers and SMARTS reaction nomenclature to generate SMILES and mol files of dendrimers without 3D coordinates. These files are used for simple graphical representations and storing their structures in databases. The second toolkit assembles complex topology dendrimers from monomers to construct 3D dendrimer structures to be used as starting points for simulation using existing and widely available software and force fields. Both tools were validated for ease-of-use to prototype dendrimer structure and the second toolkit was especially relevant for dendrimers of high complexity and size.Peer reviewe