Comparison between the Comet Assay and Fast Micromethod® for Measuring DNA Damage in HeLa Cells

The sensitivity and precision of the single cell gel electrophoresis (Comet) assay and Fast Micromethod® for DNA damage determinations in human HeLa cell line were compared. The first assay allows analysis of DNA breaks in individual cells while the second is a rapid and convenient procedure for DNA breaks determination in cell suspensions on single microplates. Both assays detect DNA strand breaks, alkali-labile sites and transient breaks occurring at sites of ongoing repair and might be applied for the assessment of surface water genotoxic potential as well as for clinical use. DNA damage in HeLa cells was induced by different doses of γ-rays generated by Cs137 (8 to 500 cGy), UV-C light (10 to 1000 J m-2) and by different concentrations of 4-nitroquinoline-V-oxide (0.026-2.6 μmol dm-3). Gamma rays induced a dose-depended response with the average Comet tail moment values from 7 mm for the negative control to 291 mm for 200 cGy, from 6.1 to 192 mm for 500 J m-2 of UV-C light and from 7.1 to 238 mm for 1.0 μmol dm-3 of 4-nitro-quinoline-N-oxide. The Fast Micromethod® strand scission factor varied from 0.010 for negative control to 0.701 for 500 cGy, from 0.019 to 1.196 for 1000 J m-2 and from 0.003 to 0.810 for 0.5 μmol dm-3 of 4-nitroquinoline-IV-oxide. Sensitivity was the same for both methods and in the case of 4-nitroquinoline-IV-oxide even better precision (lower variation coefficient) was achieved with the Fast Micromethod®. Since the time required for multiple analysis by the Fast Micromethod® is short (2 hours or less), its use in measuring DNA breakage in cells can be recommended for environmental genotoxicity monitoring

Comparison between the Comet Assay and Fast Micromethod® for Measuring DNA Damage in HeLa Cells

The sensitivity and precision of the single cell gel electrophoresis (Comet) assay and Fast Micromethod® for DNA damage determinations in human HeLa cell line were compared. The first assay allows analysis of DNA breaks in individual cells while the second is a rapid and convenient procedure for DNA breaks determination in cell suspensions on single microplates. Both assays detect DNA strand breaks, alkali-labile sites and transient breaks occurring at sites of ongoing repair and might be applied for the assessment of surface water genotoxic potential as well as for clinical use. DNA damage in HeLa cells was induced by different doses of γ-rays generated by Cs137 (8 to 500 cGy), UV-C light (10 to 1000 J m-2) and by different concentrations of 4-nitroquinoline-V-oxide (0.026-2.6 μmol dm-3). Gamma rays induced a dose-depended response with the average Comet tail moment values from 7 mm for the negative control to 291 mm for 200 cGy, from 6.1 to 192 mm for 500 J m-2 of UV-C light and from 7.1 to 238 mm for 1.0 μmol dm-3 of 4-nitro-quinoline-N-oxide. The Fast Micromethod® strand scission factor varied from 0.010 for negative control to 0.701 for 500 cGy, from 0.019 to 1.196 for 1000 J m-2 and from 0.003 to 0.810 for 0.5 μmol dm-3 of 4-nitroquinoline-IV-oxide. Sensitivity was the same for both methods and in the case of 4-nitroquinoline-IV-oxide even better precision (lower variation coefficient) was achieved with the Fast Micromethod®. Since the time required for multiple analysis by the Fast Micromethod® is short (2 hours or less), its use in measuring DNA breakage in cells can be recommended for environmental genotoxicity monitoring

Orion Capsule Handling Qualities for Atmospheric Entry

Two piloted simulations were conducted at NASA's Johnson Space Center using the Cooper-Harper scale to study the handling qualities of the Orion Command Module capsule during atmospheric entry flight. The simulations were conducted using high fidelity 6-DOF simulators for Lunar Return Skip Entry and International Space Station Return Direct Entry flight using bank angle steering commands generated by either the Primary (PredGuid) or Backup (PLM) guidance algorithms. For both evaluations, manual control of bank angle began after descending through Entry Interface into the atmosphere until drogue chutes deployment. Pilots were able to use defined bank management and reversal criteria to accurately track the bank angle commands, and stay within flight performance metrics of landing accuracy, g-loads, and propellant consumption, suggesting that the pilotability of Orion under manual control is both achievable and provides adequate trajectory performance with acceptable levels of pilot effort. Another significant result of these analyses is the applicability of flying a complex entry task under high speed entry flight conditions relevant to the next generation Multi Purpose Crew Vehicle return from Mars and Near Earth Objects

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

Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.

Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology

Combining series elastic actuation and magneto-rheological damping for the control of agile locomotion

All-terrain robot locomotion is an active topic of research. Search and rescue maneuvers and exploratory missions could benefit from robots with the abilities of real animals. However, technological barriers exist to ultimately achieving the actuation system, which is able to meet the exigent requirements of these robots. This paper describes the locomotioncontrol of a leg prototype, designed and developed to make a quadruped walk dynamically while exhibiting compliant interaction with the environment. The actuation system of the leg is based on the hybrid use of series elasticity and magneto-rheological dampers, which provide variable compliance for natural-looking motion and improved interaction with the ground. The locomotioncontrol architecture has been proposed to exploit natural leg dynamics in order to improve energy efficiency. Results show that the controller achieves a significant reduction in energy consumption during the leg swing phase thanks to the exploitation of inherent leg dynamics. Added to this, experiments with the real leg prototype show that the combined use of series elasticity and magneto-rheologicaldamping at the knee provide a 20 % reduction in the energy wasted in braking the knee during its extension in the leg stance phase

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

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

Social Participation and Disaster Risk Reduction Behaviors in Tsunami Prone Areas

This paper examines the relationships between social participation and disaster risk reduction actions. A survey of 557 households in tsunami prone areas in Phang Nga, Thailand was conducted following the 2012 Indian Ocean earthquakes. We use a multivariate probit model to jointly estimate the likelihood of undertaking three responses to earthquake and tsunami hazards (namely, (1) following disaster-related news closely, (2) preparing emergency kits and/or having a family emergency plan, and (3) having an intention to migrate) and community participation.We find that those who experienced losses from the 2004 tsunami are more likely to participate in community activities and respond to earthquake hazards. Compared to men, women are more likely to prepare emergency kits and/or have an emergency plan and have a greater intention to migrate. Living in a community with a higher proportion of women with tertiary education increases the probability of engaging in community activities and carrying out disaster risk reduction measures. Individuals who participate in village-based activities are 5.2% more likely to undertake all three risk reduction actions compared to those not engaging in community activities. This implies that encouraging participation in community activities can have positive externalities in disaster mitigation

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