A Rapid and Simple Procedure for the Establishment of Human Normal and Cancer Renal Primary Cell Cultures from Surgical Specimens

The kidney is a target organ for the toxicity of several xenobiotics and is also highly susceptible to the development of malignant tumors. In both cases, in vitro studies provide insight to cellular damage, and represent adequate models to study either the mechanisms underlying the toxic effects of several nephrotoxicants or therapeutic approaches in renal cancer. The development of efficient methods for the establishment of human normal and tumor renal cell models is hence crucial. In this study, a technically simple and rapid protocol for the isolation and culture of human proximal tubular epithelial cells and human renal tumor cells from surgical specimens is presented. Tumor and normal tissues were processed by using the same methodology, based on mechanical disaggregation of tissue followed by enzymatic digestion and cell purification by sequential sieving. The overall procedure takes roughly one hour. The resulting cell preparations have excellent viabilities and yield. Establishment of primary cultures from all specimens was achieved successfully. The origin of primary cultured cells was established through morphological evaluation. Normal cells purity was confirmed by immunofluorescent staining and reverse transcription-polymerase chain reaction analysis for expression of specific markers

Effects of Ploidy and Recombination on Evolution of Robustness in a Model of the Segment Polarity Network

Many genetic networks are astonishingly robust to quantitative variation, allowing these networks to continue functioning in the face of mutation and environmental perturbation. However, the evolution of such robustness remains poorly understood for real genetic networks. Here we explore whether and how ploidy and recombination affect the evolution of robustness in a detailed computational model of the segment polarity network. We introduce a novel computational method that predicts the quantitative values of biochemical parameters from bit sequences representing genotype, allowing our model to bridge genotype to phenotype. Using this, we simulate 2,000 generations of evolution in a population of individuals under stabilizing and truncation selection, selecting for individuals that could sharpen the initial pattern of engrailed and wingless expression. Robustness was measured by simulating a mutation in the network and measuring the effect on the engrailed and wingless patterns; higher robustness corresponded to insensitivity of this pattern to perturbation. We compared robustness in diploid and haploid populations, with either asexual or sexual reproduction. In all cases, robustness increased, and the greatest increase was in diploid sexual populations; diploidy and sex synergized to evolve greater robustness than either acting alone. Diploidy conferred increased robustness by allowing most deleterious mutations to be rescued by a working allele. Sex (recombination) conferred a robustness advantage through “survival of the compatible”: those alleles that can work with a wide variety of genetically diverse partners persist, and this selects for robust alleles

A Dynamic Landscape for Antibody Binding Modulates Antibody-Mediated Neutralization of West Nile Virus

Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this “multiple-hit” perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus “breathing” in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

The movement tracker: A flexible system for automated movement analysis in invertebrate model organisms

10.1002/cpns.17Current Protocols in Neuroscience201618.37.1-8.37.2