Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

Artificial endonucleases consisting of a Fokl cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off- target sites. Here we describe a structure- based approach to reducing off- target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology

Nd isotope composition of seep carbonates: Towards a new approach for constraining subseafloor fluid circulation at hydrocarbon seeps

The carbonates forming at deep-sea hydrocarbon seeps provide an archive for the source and migration pathways of the seeping fluids. Interpretation of the archived isotopic and elemental signatures is, however, not straightforward because of mixing between the signals of fluids and ambient seawater, limited understanding of subseafloor fluid circulation, and effects of diagenetic alteration. The Nd isotope system is among the most widely used tracers of circulation patterns in modern and past oceans, but to date Nd isotopes have been rarely used in investigations of fluid migration pathways at hydrocarbon seeps. To test the sensitivity of Nd isotope signals of seep carbonates to record past interactions between the seeping fluids and Nd-143-enriched, volcanic-derived mineral components, we performed Nd, Sr and stable isotope analyses of the Lower Cretaceous (Barremian) seep carbonates found in Bagka (Czech Carpathians). These seep deposits are directly underlain by a complex of mafic igneous rocks, which the methane-charged fluids must have passed through on their way to the seafloor. The epsilon(Nd)(t) values measured in the seep carbonates are significantly more radiogenic than the inferred signal of contemporaneous local seawater, and shifted towards the composition typical of the underlying basaltoids. This is in agreement with the Sr-87/Sr-86 ratios, which are markedly lower that the coeval seawater signature. Consistent co-variance trends are observed among the measured epsilon(Nd)(t), Sr-87/Sr-86 and delta C-13 values, attesting increasing role of the volcanic-derived fluid component with increasing contribution of methane-derived carbon. The results of the present approach reveal the potential of Nd isotopes to fingerprint exotic fluid end-members present in plumbing systems of both fossil and modern methane discharges. The method can be of particular use in studies of seeps associated with plate margins, where Nd-143-enriched igneous materials are commonly encountered in the basement or in the sediment column.Web of Science503514

The late Cenozoic evolution of the Humboldt Current System in coastal Peru: Insights from neodymium isotopes

The Humboldt Current System along the Pacific coast of South America creates one of the most productive ecosystems on Earth. To trace the origin of the water masses in this area, we measured neodymium isotope compositions (ԑNd) in tooth enameloid of two genera of coastal sharks from latest Oligocene to early Pleistocene strata in the Pisco and Sacaco basins in southern Peru. Most ԑNd values range from −4 to −1, with a strong negative excursion in the late Miocene (∼8–7 million years ago [Ma]) with values as low as −9.2. The overall trend of the ԑNd values resembles that of equatorial Pacific deep waters, though with an offset of about +2 ԑNd units until about 6 Ma. With a major input of hinterland weathering considered unlikely, we interpret this pattern as reflecting a modern-type upwelling regime, though with a lower contribution of Antarctic waters than today. Starting about 6 Ma, the contribution of Antarctic waters to the upwelling waters increased approximately to present-day levels, coincident with, and possibly driven by, increased Antarctic glaciation and the Andes reaching their present-day elevation, both of which likely enhanced the counter-clockwise circulation in the South Pacific Ocean. The negative excursion of ԑNd values in the Pisco/Sacaco basins ∼8–7 Ma coincides with a late Miocene biogenic bloom in the Pacific Ocean and elsewhere, and with a strongly increased northward bottom current observed on the Nazca Drift System just offshore our sampling area. Thus, the negative excursion of ԑNd values in the Pisco/Sacaco basins likely resulted from a southern sourced input of nutrient-rich, unradiogenic water, which could have been an important contributor to the biogenic bloom.Financial support was provided by the Bolin Centre for Climate Research (Stockholm, Sweden) to SK, and by Prociencia/Concytec research grants (E38-2019-02-Fondecyt-BM, 104-2018-Fondecyt, and 149-2018-Fondecyt-BM-IADT-AV) to RS-G.</p