Cortactin Phosphorylated by ERK1/2 Localizes to Sites of Dynamic Actin Regulation and Is Required for Carcinoma Lamellipodia Persistence

Tumor cell motility and invasion is governed by dynamic regulation of the cortical actin cytoskeleton. The actin-binding protein cortactin is commonly upregulated in multiple cancer types and is associated with increased cell migration. Cortactin regulates actin nucleation through the actin related protein (Arp)2/3 complex and stabilizes the cortical actin cytoskeleton. Cortactin is regulated by multiple phosphorylation events, including phosphorylation of S405 and S418 by extracellular regulated kinases (ERK)1/2. ERK1/2 phosphorylation of cortactin has emerged as an important positive regulatory modification, enabling cortactin to bind and activate the Arp2/3 regulator neuronal Wiskott-Aldrich syndrome protein (N-WASp), promoting actin polymerization and enhancing tumor cell movement.In this report we have developed phosphorylation-specific antibodies against phosphorylated cortactin S405 and S418 to analyze the subcellular localization of this cortactin form in tumor cells and patient samples by microscopy. We evaluated the interplay between cortactin S405 and S418 phosphorylation with cortactin tyrosine phosphorylation in regulating cortactin conformational forms by Western blotting. Cortactin is simultaneously phosphorylated at S405/418 and Y421 in tumor cells, and through the use of point mutant constructs we determined that serine and tyrosine phosphorylation events lack any co-dependency. Expression of S405/418 phosphorylation-null constructs impaired carcinoma motility and adhesion, and also inhibited lamellipodia persistence monitored by live cell imaging.Cortactin phosphorylated at S405/418 is localized to sites of dynamic actin assembly in tumor cells. Concurrent phosphorylation of cortactin by ERK1/2 and tyrosine kinases enables cells with the ability to regulate actin dynamics through N-WASp and other effector proteins by synchronizing upstream regulatory pathways, confirming cortactin as an important integration point in actin-based signal transduction. Reduced lamellipodia persistence in cells with S405/418A expression identifies an essential motility-based process reliant on ERK1/2 signaling, providing additional understanding as to how this pathway impacts tumor cell migration

The Genomics of Speciation in Drosophila: Diversity, Divergence, and Introgression Estimated Using Low-Coverage Genome Sequencing

In nature, closely related species may hybridize while still retaining their distinctive identities. Chromosomal regions that experience reduced recombination in hybrids, such as within inversions, have been hypothesized to contribute to the maintenance of species integrity. Here, we examine genomic sequences from closely related fruit fly taxa of the Drosophila pseudoobscura subgroup to reconstruct their evolutionary histories and past patterns of genic exchange. Partial genomic assemblies were generated from two subspecies of Drosophila pseudoobscura (D. ps.) and an outgroup species, D. miranda. These new assemblies were compared to available assemblies of D. ps. pseudoobscura and D. persimilis, two species with overlapping ranges in western North America. Within inverted regions, nucleotide divergence among each pair of the three species is comparable, whereas divergence between D. ps. pseudoobscura and D. persimilis in non-inverted regions is much lower and closer to levels of intraspecific variation. Using molecular markers flanking each of the major chromosomal inversions, we identify strong crossover suppression in F1 hybrids extending over 2 megabase pairs (Mbp) beyond the inversion breakpoints. These regions of crossover suppression also exhibit the high nucleotide divergence associated with inverted regions. Finally, by comparison to a geographically isolated subspecies, D. ps. bogotana, our results suggest that autosomal gene exchange between the North American species, D. ps. pseudoobscura and D. persimilis, occurred since the split of the subspecies, likely within the last 200,000 years. We conclude that chromosomal rearrangements have been vital to the ongoing persistence of these species despite recent hybridization. Our study serves as a proof-of-principle on how whole genome sequencing can be applied to formulate and test hypotheses about species formation in lesser-known non-model systems

Analytical Framework for Identifying and Differentiating Recent Hitchhiking and Severe Bottleneck Effects from Multi-Locus DNA Sequence Data

Hitchhiking and severe bottleneck effects have impact on the dynamics of genetic diversity of a population by inducing homogenization at a single locus and at the genome-wide scale, respectively. As a result, identification and differentiation of the signatures of such events from DNA sequence data at a single locus is challenging. This paper develops an analytical framework for identifying and differentiating recent homogenization events at multiple neutral loci in low recombination regions. The dynamics of genetic diversity at a locus after a recent homogenization event is modeled according to the infinite-sites mutation model and the Wright-Fisher model of reproduction with constant population size. In this setting, I derive analytical expressions for the distribution, mean, and variance of the number of polymorphic sites in a random sample of DNA sequences from a locus affected by a recent homogenization event. Based on this framework, three likelihood-ratio based tests are presented for identifying and differentiating recent homogenization events at multiple loci. Lastly, I apply the framework to two data sets. First, I consider human DNA sequences from four non-coding loci on different chromosomes for inferring evolutionary history of modern human populations. The results suggest, in particular, that recent homogenization events at the loci are identifiable when the effective human population size is 50000 or greater in contrast to 10000, and the estimates of the recent homogenization events are agree with the “Out of Africa” hypothesis. Second, I use HIV DNA sequences from HIV-1-infected patients to infer the times of HIV seroconversions. The estimates are contrasted with other estimates derived as the mid-time point between the last HIV-negative and first HIV-positive screening tests. The results show that significant discrepancies can exist between the estimates

An application of the Rasch model to reading comprehension measurement

An effective reading comprehension measurement demands robust psychometric tools that allow teachers and researchers to evaluate the educational practices and track changes in students’ performance. In this study, we illustrate how Rasch model can be used to attend such demands and improve reading comprehension measurement. We discuss the construction of two reading comprehension tests: TRC-n, with narrative texts, and TRC-e, with expository texts. Three vertically scaled forms were generated for each test (TRC-n-2, TRC-n-3, TRC-n-4; TRC-e-2, TRC-e-3 and TRC-e-4), each meant to assess Portuguese students in second, third and fourth grade of elementary school. The tests were constructed according to a nonequivalent groups with anchor test design and data were analyzed using the Rasch model. The results provided evidence for good psychometric qualities for each test form, including unidimensionality and local independence and adequate reliability. A critical view of this study and future researches are discussed.CIEC – Research Centre on Child Studies, IE, UMinho (FCT R&D unit 317), PortugalThis research was supported by Grant FCOMP-01-0124-FEDER-010733 from Fundação para a Ciência e Tecnologia (FCT) and the European Regional Development Fund (FEDER) through the European program COMPETE (Operational Program for Competitiveness Factors) under the National Strategic Reference Framework (QREN).info:eu-repo/semantics/publishedVersio

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis

Epstein-Barr Virus-Encoded LMP1 Interacts with FGD4 to Activate Cdc42 and Thereby Promote Migration of Nasopharyngeal Carcinoma Cells

Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a human malignancy notorious for its highly metastatic nature. Among EBV-encoded genes, latent membrane protein 1 (LMP1) is expressed in most NPC tissues and exerts oncogenicity by engaging multiple signaling pathways in a ligand-independent manner. LMP1 expression also results in actin cytoskeleton reorganization, which modulates cell morphology and cell motility— cellular process regulated by RhoGTPases, such as Cdc42. Despite the prominent association of Cdc42 activation with tumorigenesis, the molecular basis of Cdc42 activation by LMP1 in NPC cells remains to be elucidated. Here using GST-CBD (active Cdc42-binding domain) as bait in GST pull-down assays to precipitate active Cdc42 from cell lysates, we demonstrated that LMP1 acts through its transmembrane domains to preferentially induce Cdc42 activation in various types of epithelial cells, including NPC cells. Using RNA interference combined with re-introduction experiments, we identified FGD4 (FYVE, RhoGEF and PH domain containing 4) as the GEF (guanine nucleotide exchange factor) responsible for the activation of Cdc42 by LMP1. Serial deletion experiments and co-immunoprecipitation assays further revealed that ectopically expressed FGD4 modulated LMP1-mediated Cdc42 activation by interacting with LMP1. Moreover, LMP1, through its transmembrane domains, directly bound FGD4 and enhanced FGD4 activity toward Cdc42, leading to actin cytoskeleton rearrangement and increased motility of NPC cells. Depletion of FGD4 or Cdc42 significantly reduced (∼50%) the LMP1-stimulated cell motility, an effect that was partially reversed by expression of a constitutively active mutant of Cdc42. Finally, quantitative RT-PCR and immunohistochemistry analyses showed that FGD4 and LMP1 were expressed in NPC tissues, supporting the potential physiologically relevance of this mechanism in NPC. Collectively, our results not only uncover a novel mechanism underlying LMP1-mediated Cdc42 activation, namely LMP1 interaction with FGD4, but also functionally link FGD4 to NPC tumorigenesis

Kinetics of the hydrogen abstraction ·C2H5 + alkane → C2H6 + alkyl reaction class: an application of the reaction class transition state theory

This paper presents an application of the reaction class transition state theory (RC-TST) to predict thermal rate constants for hydrogen abstraction reactions at alkane by the C2H5 radical on-the-fly. The linear energy relationship (LER), developed for acyclic alkanes, was also proven to hold for cyclic alkanes. We have derived all RCTST parameters from rate constants of 19 representative reactions, coupling with LER and the barrier height grouping (BHG) approach. Both the RC-TST/LER, where only reaction energy is needed, and the RC-TST/BHG, where no other information is needed, can predict rate constants for any reaction in this reaction class with satisfactory accuracy for combustion modeling. Our analysis indicates that less than 50% systematic errors on the average exist in the predicted rate constants using either the RC-TST/LER or RC-TST/BHG method, while in comparison with explicit rate calculations, the differences are within a factor of 2 on the average. The results also show that the RC-TST method is not sensitive to the choice of density functional theory used

Calculation of molecular thermochemical data and their availability in databases

Thermodynamic properties of molecules can be obtained by experiment, by statistical mechanics in conjunction with electronic structure theory and by empirical rules like group additivity. The latter two methods are briefly re-viewed in this chapter. The overview of electronic structure methods is intended for readers less experienced in electronic structure theory and focuses on concepts without going into mathematical details. This is followed by a brief description of group additivity schemes; finally, an overview of databases listing reliable thermochemical data is given

The Global Nutrition Report 2014: Actions and Accountability to Accelerate the World’s Progress on Nutrition

In 2013, the Nutrition for Growth Summit called for a Global Nutrition Report (GNR) to strengthen accountability in nutrition so that progress in reducing malnutrition could be accelerated. This article summarizes the results of the first GNR. By focusing on undernutrition and overweight, the GNR puts malnutrition in a new light. Nearly every country in the world is affected by malnutrition, and multiple malnutrition burdens are the ‘‘new normal.’’ Unfortunately, the world is off track to meet the 2025 World Health Assembly (WHA) targets for nutrition. Many countries are, however, making good progress on WHA indicators, providing inspiration and guidance for others. Beyond the WHA goals, nutrition needs to be more strongly represented in the Sustainable Development Goal (SDG) framework. At present, it is only explicitly mentioned in 1 of 169 SDG targets despite the many contributions improved nutritional status will make to their attainment. To achieve improvements in nutrition status, it is vital to scale up nutrition programs. We identify bottlenecks in the scale-up of nutrition-specific and nutrition-sensitive approaches and highlight actions to accelerate coverage and reach. Holding stakeholders to account for delivery on nutrition actions requires a well-functioning accountability infrastructure, which is lacking in nutrition. New accountability mechanisms need piloting and evaluation, financial resource flows to nutrition need to be made explicit, nutrition spending targets should be established, and some key data gaps need to be filled. For example, many UN member states cannot report on their WHA progress and those that can often rely on data >5 y old. The world can accelerate malnutrition reduction substantially, but this will require stronger accountability mechanisms to hold all stakeholders to account. J Nutr doi: 10.3945/jn.114.206078