A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation

BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12860-014-0041-5) contains supplementary material, which is available to authorized users

Hilbert-Post completeness for the state and the exception effects

In this paper, we present a novel framework for studying the syntactic completeness of computational effects and we apply it to the exception effect. When applied to the states effect, our framework can be seen as a generalization of Pretnar's work on this subject. We first introduce a relative notion of Hilbert-Post completeness, well-suited to the composition of effects. Then we prove that the exception effect is relatively Hilbert-Post complete, as well as the "core" language which may be used for implementing it; these proofs have been formalized and checked with the proof assistant Coq.Comment: Siegfried Rump (Hamburg University of Technology), Chee Yap (Courant Institute, NYU). Sixth International Conference on Mathematical Aspects of Computer and Information Sciences , Nov 2015, Berlin, Germany. 2015, LNC

Soil indigenous microbiome and plant genotypes cooperatively modify soybean rhizosphere microbiome assembly

Background Plants have evolved intimate interactions with soil microbes for a range of beneficial functions including nutrient acquisition, pathogen resistance and stress tolerance. Further understanding of this system is a promising way to advance sustainable agriculture by exploiting the versatile benefits offered by the plant microbiome. The rhizosphere is the interface between plant and soil, and functions as the first step of plant defense and root microbiome recruitment. It features a specialized microbial community, intensive microbe-plant and microbe-microbe interactions, and complex signal communication. To decipher the rhizosphere microbiome assembly of soybean (Glycine max), we comprehensively characterized the soybean rhizosphere microbial community using 16S rRNA gene sequencing and evaluated the structuring influence from both host genotype and soil source. Results Comparison of the soybean rhizosphere to bulk soil revealed significantly different microbiome composition, microbe-microbe interactions and metabolic capacity. Soil type and soybean genotype cooperatively modulated microbiome assembly with soil type predominantly shaping rhizosphere microbiome assembly while host genotype slightly tuned this recruitment process. The undomesticated progenitor species, Glycine soja, had higher rhizosphere diversity in both soil types tested in comparison to the domesticated soybean genotypes. Rhizobium, Novosphingobium, Phenylobacterium, Streptomyces, Nocardioides,etc. were robustly enriched in soybean rhizosphere irrespective of the soil tested. Co-occurrence network analysis revealed dominant soil type effects and genotype specific preferences for key microbe-microbe interactions. Functional prediction results demonstrated converged metabolic capacity in the soybean rhizosphere between soil types and among genotypes, with pathways related to xenobiotic degradation, plant-microbe interactions and nutrient transport being greatly enriched in the rhizosphere. Conclusion This comprehensive comparison of the soybean microbiome between soil types and genotypes expands our understanding of rhizosphere microbe assembly in general and provides foundational information for soybean as a legume crop for this assembly process. The cooperative modulating role of the soil type and host genotype emphasizes the importance of integrated consideration of soil condition and plant genetic variability for future development and application of synthetic microbiomes. Additionally, the detection of the tuning role by soybean genotype in rhizosphere microbiome assembly provides a promising way for future breeding programs to integrate host traits participating in beneficial microbiota assembly

Measurement of Sub-23 nm Particulate Emissions from GDI Engines: A Comparison of Processing Methods

Engine research has increasingly focused on emission of sub 23 nm particulates in recent years. Likewise, current legislative efforts are being made for particulate number (PN) emission limits to include this previously omitted size range. In Europe, PN measurement equipment and procedures for regulatory purposes are defined by the particle measurement programme (PMP). Latest regulation drafts for sub 23 nm measurements specify counting efficiencies with a 65% cutoff size at 10 nm (d65) and a minimum of 90% above 15 nm (d90). Even though alternative instruments, such as differential mobility spectrometers (DMS), are widely used in laboratory environments, the interpretation of their sub 23 nm measurements has not yet been widely discussed. For this study, particulate emissions of a 1.0L gasoline direct injection (GDI) engine have been measured with a DMS system for low to medium speeds with two load steps. While the particle size distribution (PSD) at the higher load conditions exhibited a bimodal shape, the PSD for the other conditions was unimodal with a peak position below 30 nm. Lognormal fitting of nucleation and accumulation modes previously yielded results comparable to the established PMP, with d50 and d90 of 23 nm and 41 nm, respectively. However, this approach was found not suitable for sub 23 nm PN measurements due to incorrect assignment of the nucleation and accumulation modes. Recent literature suggests digital filtering of the PSD from DMS. Here, a modified filtering equation is proposed based on the latest legislative proposals. Subsequently, the new filter was compared with filters for both PMP equivalent and sub 23 nm processing of DMS data. Compared to the latter, results with the new filter showed up to 17% higher PN emissions and up to 13.6 nm lower geometric mean diameter (GMD) of the PSD

Domain perturbation for parabolic equations

Doctor of PhilosophyWe study the effect of domain perturbation on the behaviour of parabolic equations. The first aspect considered in this thesis is the behaviour of solutions under changes of the domain. We show how solutions of linear and semilinear parabolic equations behave as a sequence of domains $\Omega_n$ converges to an open set $\Omega$ in a certain sense. In particular, we are interested in singular domain perturbations so that a change of variables is not possible on these domains. For autonomous linear equations, it is known that convergence of solutions under domain perturbation is closely related to the corresponding elliptic equations via a standard semigroup theory. We show that there is also a relation between domain perturbation for non-autonomous linear parabolic equations and domain perturbation for elliptic equations. The key result for this is the equivalence of Mosco convergences between various closed and convex subsets of Banach spaces. An important consequence is that the same conditions for a sequence of domains imply convergence of solutions under domain perturbation for both parabolic and elliptic equations. By applying variational methods, we obtain the convergence of solutions of initial value problems under Dirichlet or Neumann boundary conditions. A similar technique can be applied to obtain the convergence of weak solutions of parabolic variational inequalities when the underlying convex set is perturbed. Using the linear theory, we then study domain perturbation for initial boundary value problems of semilinear type. We are also interested in the behaviour of bounded entire solutions of parabolic equations defined on the whole real line. We establish a convergence result for bounded entire solutions of linear parabolic equations under $L^2$ and $L^p$-norms. For the $L^p$-theory, we also prove H\"{o}lder regularity of bounded entire solutions with respect to time. In addition, the persistence of some classes of bounded entire solutions is given for semilinear equations using the Leray-Schauder degree theory. The second aspect is to study the dynamics of parabolic equations under domain perturbation. In this part, we consider parabolic equation as a dynamical system in an $L^2$ space and study the stability of invariant manifolds near a stationary solution. In particular, we prove the continuity (upper and lower semicontinuity) of both, the local stable invariant manifolds and the local unstable invariant manifolds under domain perturbation

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PMCID: PMC379391

Loved Ones Near and Far: Feinberg's Personal Significance Theory

This paper examines Todd Feinberg's theory of the misidentification syndromes