Monitoring Replication Protein A (RPA) Dynamics in Homologous Recombination Through Site-specific Incorporation of Non-canonical Amino Acids

An essential coordinator of all DNA metabolic processes is Replication Protein A (RPA). RPA orchestrates these processes by binding to single-stranded DNA (ssDNA) and interacting with several other DNA binding proteins. Determining the real-time kinetics of single players such as RPA in the presence of multiple DNA processors to better understand the associated mechanistic events is technically challenging. To overcome this hurdle, we utilized non-canonical amino acids and bio-orthogonal chemistry to site-specifically incorporate a chemical fluorophore onto a single subunit of heterotrimeric RPA. Upon binding to ssDNA, this fluorescent RPA (RPAf) generates a quantifiable change in fluorescence, thus serving as a reporter of its dynamics on DNA in the presence of multiple other DNA binding proteins. Using RPAf, we describe the kinetics of facilitated self-exchange and exchange by Rad51 and mediator proteins during various stages in homologous recombination. RPAf is widely applicable to investigate its mechanism of action in processes such as DNA replication, repair and telomere maintenance

Novel Weapons Testing: Are Invasive Plants More Chemically Defended than Native Plants?

Background: Exotic species have been hypothesized to successfully invade new habitats by virtue of possessing novel biochemistry that repels native enemies. Despite the pivotal long-term consequences of invasion for native food-webs, to date there are no experimental studies examining directly whether exotic plants are any more or less biochemically deterrent than native plants to native herbivores. Methodology/Principal Findings: In a direct test of this hypothesis using herbivore feeding assays with chemical extracts from 19 invasive plants and 21 co-occurring native plants, we show that invasive plant biochemistry is no more deterrent (on average) to a native generalist herbivore than extracts from native plants. There was no relationship between extract deterrence and length of time since introduction, suggesting that time has not mitigated putative biochemical novelty. Moreover, the least deterrent plant extracts were from the most abundant species in the field, a pattern that held for both native and exotic plants. Analysis of chemical deterrence in context with morphological defenses and growth-related traits showed that native and exotic plants had similar trade-offs among traits. Conclusions/Significance: Overall, our results suggest that particular invasive species may possess deterrent secondary chemistry, but it does not appear to be a general pattern resulting from evolutionary mismatches between exotic plants and native herbivores. Thus, fundamentally similar processes may promote the ecological success of both native and exotic species

Guiding the design of well-powered Hi-C experiments to detect differential loops

Motivation: Three-dimensional chromatin structure plays an important role in gene regulation by connecting regulatory regions and gene promoters. The ability to detect the formation and loss of these loops in various cell types and conditions provides valuable information on the mechanisms driving these cell states and is critical for understanding long-range gene regulation. Hi-C is a powerful technique for characterizing 3D chromatin structure; however, Hi-C can quickly become costly and labor-intensive, and proper planning is required to ensure efficient use of time and resources while maintaining experimental rigor and well-powered results. Results: To facilitate better planning and interpretation of human Hi-C experiments, we conducted a detailed evaluation of statistical power using publicly available Hi-C datasets, paying particular attention to the impact of loop size on Hi-C contacts and fold change compression. In addition, we have developed Hi-C Poweraid, a publicly hosted web application to investigate these findings. For experiments involving well-replicated cell lines, we recommend a total sequencing depth of at least 6 billion contacts per condition, split between at least two replicates to achieve the power to detect differences in the majority of loops. For experiments with higher variation, more replicates and deeper sequencing depths are required. Values for specific cases can be determined by using Hi-C Poweraid. This tool simplifies Hi-C power calculations, allowing for more efficient use of time and resources and more accurate interpretation of experimental results. Availability and implementation: Hi-C Poweraid is available as an R Shiny application deployed at http://phanstiel-lab.med.unc.edu/poweraid/, with code available at https://github.com/sarmapar/poweraid

The Nature and Frequency of the Gas Outbursts in Comet 67P/Churyumov-Gerasimenko observed by the Alice Far-ultraviolet Spectrograph on Rosetta

Alice is a far-ultraviolet imaging spectrograph onboard Rosetta that, amongst multiple objectives, is designed to observe emissions from various atomic and molecular species from within the coma of comet 67P/Churyumov-Gerasimenko. The initial observations, made following orbit insertion in August 2014, showed emissions of atomic hydrogen and oxygen spatially localized close to the nucleus and attributed to photoelectron impact dissociation of H2O vapor. Weaker emissions from atomic carbon were subsequently detected and also attributed to electron impact dissociation, of CO2, the relative H I and C I line intensities reflecting the variation of CO2 to H2O column abundance along the line-of-sight through the coma. Beginning in mid-April 2015, Alice sporadically observed a number of outbursts above the sunward limb characterized by sudden increases in the atomic emissions, particularly the semi-forbidden O I 1356 multiplet, over a period of 10-30 minutes, without a corresponding enhancement in long wavelength solar reflected light characteristic of dust production. A large increase in the brightness ratio O I 1356/O I 1304 suggests O2 as the principal source of the additional gas. These outbursts do not correlate with any of the visible images of outbursts taken with either OSIRIS or the navigation camera. Beginning in June 2015 the nature of the Alice spectrum changed considerably with CO Fourth Positive band emission observed continuously, varying with pointing but otherwise fairly constant in time. However, CO does not appear to be a major driver of any of the observed outbursts.Comment: 6 pages, 4 figures, accepted for publication in the Astrophysical Journal Letter

Tc-99 Ion Exchange Resin Testing

Pacific Northwest National Laboratory was contracted by CHPRC to evaluate the release of 99Tc from spent resin used to treat water from well 299-W15-765 and stored for several years. The key questions to be answered are: 1) does 99Tc readily release from the spent ion exchange resin after being in storage for several years; 2) if hot water stripping is used to remove the co-contaminant carbon tetrachloride, will 99Tc that has been sequestered by the resin be released; and 3) can spent resin be encapsulated into a cementitious waste form; if so, how much 99Tc would be released from the weathering of the monolith waste form? The results from the long term stability leach test results confirm that the resin is not releasing a significant amount of the sequestered 99Tc, evident by the less than 0.02% of the total 99Tc loaded being identified in the solution. Furthermore, it is possible that the measured 99Tc concentration is the result of 99Tc contained in the pore spaces of the resin. In addition to these results, analyses conducted to examine the impact of hot water on the release of 99Tc suggest that only a small percentage of the total is being released. This suggest that hot water stripping to remove carbon tetrachloride will not have a significant affect on the resin’s ability to hold-on to sequestered 99Tc. Finally, encapsulation of spent resin in a cementitious material may be a viable disposal option, but additional tests are needed to examine the extent of physical degradation caused by moisture loss and the effect this degradation process can have on the release of 99Tc

Dimensionless Measures of Turbulent Magnetohydrodynamic Dissipation Rates

The magnetic Reynolds number R_M, is defined as the product of a characteristic scale and associated flow speed divided by the microphysical magnetic diffusivity. For laminar flows, R_M also approximates the ratio of advective to dissipative terms in the total magnetic energy equation, but for turbulent flows this latter ratio depends on the energy spectra and approaches unity in a steady state. To generalize for flows of arbitrary spectra we define an effective magnetic dissipation number, R_{M,e}, as the ratio of the advection to microphysical dissipation terms in the total magnetic energy equation, incorporating the full spectrum of scales, arbitrary magnetic Prandtl numbers, and distinct pairs of inner and outer scales for magnetic and kinetic spectra. As expected, for a substantial parameter range R_{M,e}\sim {O}(1) << R_M. We also distinguish R_{M,e} from {\tilde R}_{M,e} where the latter is an effective magnetic Reynolds number for the mean magnetic field equation when a turbulent diffusivity is explicitly imposed as a closure. That R_{M,e} and {\tilde R}_{M,e} approach unity even if R_M>>1 highlights that, just as in hydrodynamic turbulence,energy dissipation of large scale structures in turbulent flows via a cascade can be much faster than the dissipation of large scale structures in laminar flows. This illustrates that the rate of energy dissipation by magnetic reconnection is much faster in turbulent flows, and much less sensitive to microphysical reconnection rates compared to laminar flows.Comment: 14 pages (including 2 figs), accepted by MNRA