Optimization of a 96-Well Electroporation Assay for Postnatal Rat CNS Neurons Suitable for Cost–Effective Medium-Throughput Screening of Genes that Promote Neurite Outgrowth

Following an injury, central nervous system (CNS) neurons show a very limited regenerative response which results in their failure to successfully form functional connections with their original target. This is due in part to the reduced intrinsic growth state of CNS neurons, which is characterized by their failure to express key regeneration-associated genes (RAGs) and by the presence of growth inhibitory molecules in CNS environment that form a molecular and physical barrier to regeneration. Here we have optimized a 96-well electroporation and neurite outgrowth assay for postnatal rat cerebellar granule neurons (CGNs) cultured upon an inhibitory cellular substrate expressing myelin-associated glycoprotein or a mixture of growth inhibitory chondroitin sulfate proteoglycans. Optimal electroporation parameters resulted in 28% transfection efficiency and 51% viability for postnatal rat CGNs. The neurite outgrowth of transduced neurons was quantitatively measured using a semi-automated image capture and analysis system. The neurite outgrowth was significantly reduced by the inhibitory substrates which we demonstrated could be partially reversed using a Rho Kinase inhibitor. We are now using this assay to screen large sets of RAGs for their ability to increase neurite outgrowth on a variety of growth inhibitory and permissive substrates

categoryCompare, an analytical tool based on feature annotations

Assessment of high-throughput—omics data initially focuses on relative or raw levels of a particular feature, such as an expression value for a transcript, protein, or metabolite. At a second level, analyses of annotations including known or predicted functions and associations of each individual feature, attempt to distill biological context. Most currently available comparative- and meta-analyses methods are dependent on the availability of identical features across data sets, and concentrate on determining features that are differentially expressed across experiments, some of which may be considered “biomarkers.” The heterogeneity of measurement platforms and inherent variability of biological systems confounds the search for robust biomarkers indicative of a particular condition. In many instances, however, multiple data sets show involvement of common biological processes or signaling pathways, even though individual features are not commonly measured or differentially expressed between them. We developed a methodology, categoryCompare, for cross-platform and cross-sample comparison of high-throughput data at the annotation level. We assessed the utility of the approach using hypothetical data, as well as determining similarities and differences in the set of processes in two instances: (1) denervated skin vs. denervated muscle, and (2) colon from Crohn's disease vs. colon from ulcerative colitis (UC). The hypothetical data showed that in many cases comparing annotations gave superior results to comparing only at the gene level. Improved analytical results depended as well on the number of genes included in the annotation term, the amount of noise in relation to the number of genes expressing in unenriched annotation categories, and the specific method in which samples are combined. In the skin vs. muscle denervation comparison, the tissues demonstrated markedly different responses. The Crohn's vs. UC comparison showed gross similarities in inflammatory response in the two diseases, with particular processes specific to each disease

Heparan sulfate expression in the neural crest is essential for mouse cardiogenesis

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1(-/-) embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect

Impacts of shifting phenology on boundary layer dynamics in North America in the CESM

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Reflections: OD or Not OD that is the Question! A Constructivist's Thoughts on the Changing Nature of Change

The landscape of organization development (OD) has changed significantly over the last several decades. This article provides a broad commentary on these changes. In particular, it offers a critique of 'current OD' in terms of the marginalization of materiality in discourse-based OD techniques and the neglect of problem-centred, diagnostic approaches in favour of solution-driven, emergent approaches. The future of OD is also explored in relation to the scope for meaningful 'bottom-up OD' (i.e. employee-instigated change) and 'outside-in OD' (i.e. involving a range of non-organizational stakeholders). © 2013 Taylor & Francis

The Cell Cycle Time of CD8+ T Cells Responding In Vivo Is Controlled by the Type of Antigenic Stimulus

A hallmark of cells comprising the mammalian adaptive immune system is the requirement for these rare naïve T (and B) lymphocytes directed to a specific microorganism to undergo proliferative expansion upon first encounter with this antigen. In the case of naïve CD8+ T cells the ability of these rare quiescent lymphocytes to rapidly activate and expand into effector T cells in numbers sufficient to control viral and certain bacterial infections can be essential for survival. In this report we examined the activation, cell cycle time and initial proliferative response of naïve murine CD8+ T cells responding in vivo to Influenza and Vaccinia virus infection or vaccination with viral antigens. Remarkably, we observed that CD8+ T cells could divide and proliferate with an initial cell division time of as short as 2 hours. The initial cell cycle time of responding CD8+ T cells is not fixed but is controlled by the antigenic stimulus provided by the APC in vivo. Initial cell cycle time influences the rate of T cell expansion and the numbers of effector T cells subsequently accumulating at the site of infection. The T cell cycle time varies with duration of the G1 phase of the cell cycle. The duration of G1 is inversely correlated with the phosphorylation state of the retinoblastoma (Rb) protein in the responding T cells. The implication of these findings for the development of adaptive immune responses and the regulation of cell cycle in higher eukaryotic cells is discussed

Taking It to the Extreme:The Effect of Coalition Cabinets on Foreign Policy

Institutional constraints have been offered by some scholars as an explanation for why multiparty coalitions should be more peaceful than single-party cabinets. Yet others see the same institutional setting as a prescription for more aggressive behavior. Recent research has investigated these conflicting expectations, but with mixed results. We examine the theoretical bases for these alternative expectations about the effects of coalition politics on foreign policy. We find that previous research is limited theoretically by confounding institutional effects with policy positions, and empirically by analyzing only international conflict data. We address these limitations by examining cases of foreign policy behavior using the World Event/Interaction Survey (WEIS) dataset. Consistent with our observation that institutional constraints have been confounded with policy positions, we find that coalitions are neither more aggressive nor more peaceful, but do engage in more extreme foreign policy behaviors. These findings are discussed with regard to various perspectives on the role of institutions in shaping foreign policy behavior.</p

Superconducting properties and Fermi-surface topology of the quasi-two-dimensional organic superconductor λ\lambda-(BETS)2_{2}GaCl4_{4}

The Fermi surface topology of the organic superconductor \lbets has been determined using the Shubnikov-de Haas and magnetic breakdown effects and angle-dependent magnetoresistance oscillations. The former experiments were carried out in pulsed fields of up to 60 T, whereas the latter employed quasistatic fields of up to 30 T. All of these data show that the Fermi-surface topology of \lbets is very similar to that of the most heavily-studied organic superconductor, \cuscn, except in one important respect; the interplane transfer integral in \lbets is a factor $\sim 10$ larger than that in \cuscn . The increased three-dimensionality of \lbets is manifested in radiofrequency penetration-depth measurements, which show a clear dimensional crossover in the behaviour of $H_{c2}(T)$. The radiofrequency measurements have also been used to extract the Labusch parameter determining the fluxoid interactions as a function of temperature, and to map the flux-lattice melting curve.Comment: 24 pages 10 figure