Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs

Site-specific homing endonucleases are capable of inducing gene conversion via homologous recombination. Reprogramming their cleavage specificities allows the targeting of specific biological sites for gene correction or conversion. We used computational protein design to alter the cleavage specificity of I-MsoI for three contiguous base pair substitutions, resulting in an endonuclease whose activity and specificity for its new site rival that of wild-type I-MsoI for the original site. Concerted design for all simultaneous substitutions was more successful than a modular approach against individual substitutions, highlighting the importance of context-dependent redesign and optimization of protein–DNA interactions. We then used computational design based on the crystal structure of the designed complex, which revealed significant unanticipated shifts in DNA conformation, to create an endonuclease that specifically cleaves a site with four contiguous base pair substitutions. Our results demonstrate that specificity switches for multiple concerted base pair substitutions can be computationally designed, and that iteration between design and structure determination provides a route to large scale reprogramming of specificity

All-Pay Auctions with Weakly Risk-Averse Buyers

We use perturbation analysis to study independent private-value all-pay auctions with weakly risk-averse buyers. We show that under weak risk aversion: 1) Buyers with low values bid lower and buyers with high values bid higher than they would bid in the risk neutral case. 2) Buyers with low values bid lower and buyers with high values bid higher than they would bid in a first-price auction. 3) Buyers' expected utilities in an all-pay auction are lower than in a first-price auction. 4) The seller's expected payoff in an all-pay auction may be either higher or lower than in the risk neutral case. 5) The seller's expected payoff in an all-pay auction may be either higher or lower than in a first-price auction

Inhibition of Vaginal Lactobacilli by a Bacteriocin-Like Inhibitor Produced by Enterococcus faecium 62-6: Potential Significance for Bacterial Vaginosis

Objective: Bacterial vaginosis (BV) is characterized by a shift in vaginal tract ecology, which includes a decrease in the concentration and/or prevalence of facultative lactobacilli. Currently, mechanisms which could account for the disappearance of lactobacilli are not well understood. The objective of this study was to determine whether vaginal streptococci/enterococci can produce bacteriocin-like inhibitors antagonistic to vaginal lactobacilli. Methods: Seventy strains of vaginal streptococci or enterococci were tested for antagonistic activities against vaginal lactobacilli using the deferred antagonism technique. Results: One strain, Enterococcus faecium 62-6, which strongly inhibited growth of lactobacilli was selected for further characterization. The spectrum of inhibitory activity of strain 62-6 included Gram-positive organisms from the vaginal environment, although native lactobacilli from the same host were resistant to inhibitor action. Following growth inMRSbroth the strain 62-6 inhibitor was shown to be heat- (100℃, 30 minutes), cold- (4℃, less than 114 days) and pH- (4–7) stable. The sensitivity of inhibitor-containing supernatants to pepsin and α-chymotrypsin suggested an essential proteinaceous component. The inhibitor was sensitive to lipase but resistant to lysozyme. Dialysis of inhibitor-containing culture supernatants suggested a molecular mass greater than 12 000 Da. All physicochemical properties were consistent with its classification as a bacteriocin-like inhibitor. Kinetic assays demonstrated a sharp onset of inhibitor production coinciding with a concentration of 62-6 of 10(7) cfu/ml, suggesting that production may be regulated by quorum sensing. Conclusions: These results may have clinical significance as a novel mechanism to account for the decline of vaginal Lactobacillus populations and contribute to both the establishment and recurrence of BV

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display

Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions

Land, Environmental Externalities and Tourism Development

In a two sectors dynamic model we analyze the process of tourism development based on the accumulation of capital (building of tourism facilities) and the reallocation of land from traditional activities to the tourism sector. The model incorporates the conflict between occupation of the territory by the tourism facilities, other productive activities and availability of cultural, natural and environmental assets that are valued by residents and visitors. We characterize the process of tourism development in two settings: the socially optimal solution and a situation where the costs of tourism expansion are external to the decision makers, where externalities on residents as well as intraindustry externalities are considered. Regarding the optimal solution, we show that it is optimal to limit tourism expansion before it reaches its maximum capacity even in a context where the economic attractiveness of tourism relative to other productive sectors rise continuously. However, in this context and when all the costs of tourism development are externalities the only limit to tourism quantitative expansion is its maximum capacity determined by the availability of land. Finally, we show that excessive environmental degradation from the future generations' point of view is not a problem of discounting the future but rather a problem of externalities that affects negatively the current and future generations

Assessing the Effectiveness of Tradable Landuse Rights for Biodiversity Conservation: An Application to Canada's Boreal Mixedwood Forest

Ecological reserve networks are an important strategy for conserving biodiversity. One approach to selecting reserves is to use optimization algorithms that maximize an ecological objective function subject to a total reserve area constraint. Under this approach, economic factors such as potential land values and tenure arrangements are often ignored. Tradable landuse rights are proposed as an alternative economic mechanism for selecting reserves. Under this approach economic considerations determine the spatial distribution of development and reserves are allocated to sites with the lowest development value, minimizing the cost of the reserve network. The configuration of the reserve network as well as the biodiversity outcome is determined as a residual. However cost savings can be used to increase the total amount of area in reserve and improve biodiversity outcomes. The appropriateness of this approach for regional planning is discussed in light of key uncertainties associated with biodiversity protection. A comparison of biodiversity outcomes and costs under ecological versus economic approaches is undertaken for the Boreal Forest Natural Region of Alberta, Canada. We find a significant increase in total area protected and an increase in species representation under the TLR approach

Empirical Analysis of National Income and So2 Emissions in Selected European Countries

Data on GDP per capita and sulfur emissions for twelve European countries were analyzed to determine the relationship between emissions and income in these countries. As a whole, the relationship between sulfur emissions and per capita income is a fourth order polynomial and not a quadratic one as found in most studies. When countries were examined individually, seven out of the twelve countries depicted the same relationship. Looking closely at the regulations restricting sulfur emissions in the UK, the impact of all regulations supported the inverted U-shaped Kuznets curve. Individually, however, it is found that only two regulations have statistically significant impacts: Smoke Abatement Act in 1926 (reduced the amount of sulfur associated with a given level of GDP); and Clean Air Act in 1956 (increased the amount of sulfur emissions associated with a given level of GDP)

First results of the CAST-RADES haloscope search for axions at 34.67 μeV

We present results of the Relic Axion Dark-Matter Exploratory Setup (RADES), a detector which is part of the CERN Axion Solar Telescope (CAST), searching for axion dark matter in the 34.67μeV mass range. A radio frequency cavity consisting of 5 sub-cavities coupled by inductive irises took physics data inside the CAST dipole magnet for the first time using this filter-like haloscope geometry. An exclusion limit with a 95% credibility level on the axion-photon coupling constant of gaγ & 4 × 10−13 GeV−1 over a mass range of 34.6738μeV < ma < 34.6771μeV is set. This constitutes a significant improvement over the current strongest limit set by CAST at this mass and is at the same time one of the most sensitive direct searches for an axion dark matter candidate above the mass of 25μeV. The results also demonstrate the feasibility of exploring a wider mass range around the value probed by CAST-RADES in this work using similar coherent resonant cavitiesWe wish to thank our colleagues at CERN, in particular Marc Thiebert from the coating lab, as well as the whole team of the CERN Central Cryogenic Laboratory for their support and advice in speci c aspects of the project. We thank Arefe Abghari for her contributions as the project's summer student during 2018. This work has been funded by the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) under project FPA-2016-76978-C3-2-P and PID2019-108122GB-C33, and was supported by the CERN Doctoral Studentship programme. The research leading to these results has received funding from the European Research Council and BD, JG and SAC acknowledge support through the European Research Council under grant ERC-2018-StG-802836 (AxScale project). BD also acknowledges fruitful discussions at MIAPP supported by DFG under EXC-2094 { 390783311. IGI acknowledges also support from the European Research Council (ERC) under grant ERC-2017-AdG-788781 (IAXO+ project). JR has been supported by the Ramon y Cajal Fellowship 2012-10597, the grant PGC2018-095328-B-I00(FEDER/Agencia estatal de investigaci on) and FSE-GA2017-2019-E12/7R (Gobierno de Aragón/FEDER) (MINECO/FEDER), the EU through the ITN \Elusives" H2020-MSCA-ITN-2015/674896 and the Deutsche Forschungsgemeinschaft under grant SFB-1258 as a Mercator Fellow. CPG was supported by PROMETEO II/2014/050 of Generalitat Valenciana, FPA2014-57816-P of MINECO and by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreements 690575 and 674896. AM is supported by the European Research Council under Grant No. 742104. Part of this work was performed under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344