In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

Wall expansion assessment of an intracranial aneurysm model by a 3D digital image correlation system

Intracranial aneurysm is a local dilatation of an intracranial artery with high risk of rupture and death. Although it is generally accepted that the weakening of the arterial wall is the main cause for the rupture of an aneurysm, it still no consensus about the reasons for its creation, expansion and rupture. In particular, what is the role played by the blood flow in these phenomena. In this way, the aim of this work is the in vitro mechanical assessment of the wall expansion, namely the displacements, deformations and strains occurring in a saccular intracranial aneurysm model, when subjected to different flow rates. To obtain new insights into the mechanisms involved in the aneurysm rupture, a 3D-VicTM Digital Image Correlation System was used and validated with a finite element analysis. The wall expansion results have revealed that the displacements, deformations and principal strains are directly related to the internal pressure caused by the fluid on the wall of the aneurism. These findings were especially observed in the weakened areas of the aneurysm model, where the wall was thinner. Furthermore, the technique used in this study has shown to be a potential method to validate numerical simulations of aneurysms, allowing the future performance of more complex and realistic haemodynamic studies.The authors acknowledge the financial support provided by PTDC/SAU-ENB/116929/2010, EXPL/EMSSIS/2215/2013 and UID/EQU/50020/2013 from FCT (Fundação para a Ciência e a Tecnologia), COMPETE, QREN and European Union (FEDER). R.O. Rodrigues, D. Pinho and D. Bento acknowledge, respectively, the PhD scholarships SFRH/BD/97658/2013, SFRH/BD/89077/2012 and SFRH/BD/91192/2012 granted by FCT. Also the authors acknowledge the Vic-3DTM DIC System that was kindly borrowed by the University of Coimbra to the experimental execution of this work.info:eu-repo/semantics/publishedVersio

Scaling Reliably: Improving the Scalability of the Erlang Distributed Actor Platform

Distributed actor languages are an effective means of constructing scalable reliable systems, and the Erlang programming language has a well-established and influential model. While the Erlang model conceptually provides reliable scalability, it has some inherent scalability limits and these force developers to depart from the model at scale. This article establishes the scalability limits of Erlang systems and reports the work of the EU RELEASE project to improve the scalability and understandability of the Erlang reliable distributed actor model. We systematically study the scalability limits of Erlang and then address the issues at the virtual machine, language, and tool levels. More specifically: (1) We have evolved the Erlang virtual machine so that it can work effectively in large-scale single-host multicore and NUMA architectures. We have made important changes and architectural improvements to the widely used Erlang/OTP release. (2) We have designed and implemented Scalable Distributed (SD) Erlang libraries to address language-level scalability issues and provided and validated a set of semantics for the new language constructs. (3) To make large Erlang systems easier to deploy, monitor, and debug, we have developed and made open source releases of five complementary tools, some specific to SD Erlang. Throughout the article we use two case studies to investigate the capabilities of our new technologies and tools: a distributed hash table based Orbit calculation and Ant Colony Optimisation (ACO). Chaos Monkey experiments show that two versions of ACO survive random process failure and hence that SD Erlang preserves the Erlang reliability model. While we report measurements on a range of NUMA and cluster architectures, the key scalability experiments are conducted on the Athos cluster with 256 hosts (6,144 cores). Even for programs with no global recovery data to maintain, SD Erlang partitions the network to reduce network traffic and hence improves performance of the Orbit and ACO benchmarks above 80 hosts. ACO measurements show that maintaining global recovery data dramatically limits scalability; however, scalability is recovered by partitioning the recovery data. We exceed the established scalability limits of distributed Erlang, and do not reach the limits of SD Erlang for these benchmarks at this scal

Fully adaptive multiresolution schemes for strongly degenerate parabolic equations with discontinuous flux

A fully adaptive finite volume multiresolution scheme for one-dimensional strongly degenerate parabolic equations with discontinuous flux is presented. The numerical scheme is based on a finite volume discretization using the Engquist--Osher approximation for the flux and explicit time--stepping. An adaptivemultiresolution scheme with cell averages is then used to speed up CPU time and meet memory requirements. A particular feature of our scheme is the storage of the multiresolution representation of the solution in a dynamic graded tree, for the sake of data compression and to facilitate navigation. Applications to traffic flow with driver reaction and a clarifier--thickener model illustrate the efficiency of this method

Revising mtDNA haplotypes of the ancient Hungarian conquerors with next generation sequencing

As part of the effort to create a high resolution representative sequence database of the medieval Hungarian conquerors we have resequenced the entire mtDNA genome of 24 published ancient samples with Next Generation Sequencing, whose haplotypes had been previously determined with traditional PCR based methods. We show that PCR based methods are prone to erroneous haplotype or haplogroup determination due to ambiguous sequence reads, and many of the resequenced samples had been classified inaccurately. The SNaPshot method applied with published ancient DNA authenticity criteria is the most straightforward and cheapest PCR based approach for testing a large number of coding region SNP-s, which greatly facilitates correct haplogroup determination

Insight from the draft genome of Dietzia cinnamea P4 reveals mechanisms of survival in complex tropical soil habitats and biotechnology potential

The draft genome of Dietzia cinnamea strain P4 was determined using pyrosequencing. In total, 428 supercontigs were obtained and analyzed. We here describe and interpret the main features of the draft genome. The genome contained a total of 3,555,295 bp, arranged in a single replicon with an average G+C percentage of 70.9%. It revealed the presence of complete pathways for basically all central metabolic routes. Also present were complete sets of genes for the glyoxalate and reductive carboxylate cycles. Autotrophic growth was suggested to occur by the presence of genes for aerobic CO oxidation, formate/formaldehyde oxidation, the reverse tricarboxylic acid cycle and the 3-hydropropionate cycle for CO2 fixation. Secondary metabolism was evidenced by the presence of genes for the biosynthesis of terpene compounds, frenolicin, nanaomycin and avilamycin A antibiotics. Furthermore, a probable role in azinomycin B synthesis, an important product with antitumor activity, was indicated. The complete alk operon for the degradation of n-alkanes was found to be present, as were clusters of genes for biphenyl ring dihydroxylation. This study brings new insights in the genetics and physiology of D. cinnamea P4, which is useful in biotechnology and bioremediation