Fixed-target serial crystallography at the Structural Biology Center

Serial synchrotron crystallography enables the study of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D-printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin Mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed using a high-performance computing pipeline. Here, the system was applied to two β-lactamases: a class D serine β-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-β-lactamase from Stenotrophomonas maltophilia K279a

Cobalt, manganese, and iron near the Hawaiian Islands : a potential concentrating mechanism for cobalt within a cyclonic eddy and implications for the hybrid-type trace metals

Author Posting. © Elsevier B.V., 2008. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Deep Sea Research Part II: Topical Studies in Oceanography 55 (2008): 1473-1490, doi:10.1016/j.dsr2.2008.02.010.The vertical distributions of cobalt, iron, and manganese in the water column were studied during the E-Flux Program (E-Flux II and III), which focused on the biogeochemistry of cold-core cyclonic eddies that form in the lee of the Hawaiian Islands. During E-Flux II (January 2005) and E-Flux III (March 2005), 17 stations were sampled for cobalt (n =147), all of which demonstrated nutrient-like depletion in surface waters. During E-Flux III, two depth profiles collected from within a mesoscale coldcore eddy, Cyclone Opal, revealed small distinct maxima in cobalt at ~100m depth and a larger inventory of cobalt within the eddy. We hypothesize that this was due to a cobalt concentrating effect within the eddy, where upwelled cobalt was subsequently associated with sinking particulate organic carbon (POC) via biological activity and was released at a depth coincident with nearly complete POC remineralization (Benitez-Nelson et al. 2007). There is also evidence for the formation of a correlation between cobalt and soluble reactive phosphorus during E-Flux III relative to the E-Flux II cruise that we suggest is due to increased productivity, implying a minimum threshold of primary production below which cobalt-phosphate coupling does not occur. Dissolved iron was measured in E-Flux II and found in somewhat elevated concentrations (~0.5nM) in surface waters relative to the iron depleted waters of the surrounding Pacific (Fitzwater et al. 1996), possibly due to island effects associated with the iron-rich volcanic soil from the Hawaiian Islands and/or anthropogenic inputs. Distinct depth maxima in total dissolved cobalt were observed at 400 to 600m depth, suggestive of the release of metals from the shelf area of comparable depth that surrounds these islands.This research was supported by NSF Grants OCE-0327225, OCE-0452883, OPP-0440840, the Office of Naval Research, the Center for Environmental Bioinorganic Chemistry at Princeton, and the Center for Microbial Oceanography and Education

Above–belowground herbivore interactions in mixed plant communities are influenced by altered precipitation patterns

Root- and shoot-feeding herbivores have the capacity to influence one another by modifying the chemistry of the shared host plant. This can alter rates of nutrient mineralization and uptake by neighboring plants and influence plant–plant competition, particularly in mixtures combining grasses and legumes. Root herbivory-induced exudation of nitrogen (N) from legume roots, for example, may increase N acquisition by co-occurring grasses, with knock-on effects on grassland community composition. Little is known about how climate change may affect these interactions, but an important and timely question is how will grass–legume mixtures respond in a future with an increasing reliance on legume N mineralization in terrestrial ecosystems. Using a model grass–legume mixture, this study investigated how simultaneous attack on lucerne (Medicago sativa) by belowground weevils (Sitona discoideus) and aboveground aphids (Acyrthosiphon pisum) affected a neighboring grass (Phalaris aquatica) when subjected to drought, ambient, and elevated precipitation. Feeding on rhizobial nodules by weevil larvae enhanced soil water retention under ambient and elevated precipitation, but only when aphids were absent. While drought decreased nodulation and root N content in lucerne, grass root and shoot chemistry were unaffected by changes in precipitation. However, plant communities containing weevils but not aphids showed increased grass height and N concentrations, most likely associated with the transfer of N from weevil-attacked lucerne plants containing more nodules and higher root N concentrations compared with insect-free plants. Drought decreased aphid abundance by 54% but increased total and some specific amino acid concentrations (glycine, lysine, methionine, tyrosine, cysteine, histidine, arginine, aspartate, and glutamate), suggesting that aphid declines were being driven by other facets of drought (e.g., reduced phloem hydraulics). The presence of weevil larvae belowground decreased aphid numbers by 30%, likely associated with a significant reduction in proline in weevil-treated lucerne plants. This study demonstrates how predicted changes to precipitation patterns and indirect interactions between herbivores can alter the outcome of competition between N-fixing legumes and non-N-fixing grasses, with important implications for plant community structure and productivity

Evidence for the linked biogeochemical cycling of zinc, cobalt, and phosphorus in the western North Atlantic Ocean

Author Posting. © American Geophysical Union, 2008. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Global Biogeochemical Cycles 22 (2008): GB4012, doi:10.1029/2007GB003119.Many trace metals such as iron, copper, and manganese have lower concentrations in the surface waters of the North Pacific Ocean than in North Atlantic surface waters. However, cobalt and zinc concentrations in North Atlantic surface waters are often as low as those reported in the North Pacific. We studied the relationship between the distribution of cobalt, zinc, and phosphorus in surface waters of the western North Atlantic Ocean. Both metals show strong depletion in the southern Sargasso Sea, a region characterized by exceedingly low dissolved inorganic phosphorus (generally <4 nmol L−1) and measurable alkaline phosphatase activity. Alkaline phosphatase is a metalloenzyme (typically containing zinc) that cleaves phosphate monoesters and is a diagnostic indicator of phosphorus stress in phytoplankton. In contrast to the North Pacific Ocean, cobalt and zinc appear to be drawn down to their lowest values only when inorganic phosphorus is below 10 nmol L−1 in the North Atlantic Ocean. Lower levels of phosphorus in the Atlantic may contribute to these differences, possibly through an increased biological demand for zinc and cobalt associated with dissolved organic phosphorus acquisition. This hypothesis is consistent with results of a culture study where alkaline phosphatase activity decreased in the model coccolithophore Emiliania huxleyi upon zinc and cobalt limitation.This research was supported by NSF grant OCE- 0136835 to J.W.M. and S.D. R.W.J. was supported by an EPA STAR Fellowship

Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844–848

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000–3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons—Leu844, Cys845, Ala846, Leu847, and Gly848—located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844–848 exists and will be valuable in the management and genetic counseling of a significant number of individuals

Transcriptome response of high- and low-light-adapted Prochlorococcus strains to changing iron availability

Prochlorococcus contributes significantly to ocean primary productivity. The link between primary productivity and iron in specific ocean regions is well established and iron-limitation of Prochlorococcus cell division rates in these regions has been demonstrated. However, the extent of ecotypic variation in iron metabolism among Prochlorococcus and the molecular basis for differences is not understood. Here, we examine the growth and transcriptional response of Prochlorococcus strains, MED4 and MIT9313, to changing iron concentrations. During steady-state, MIT9313 sustains growth at an order-of-magnitude lower iron concentration than MED4. To explore this difference, we measured the whole-genome transcriptional response of each strain to abrupt iron starvation and rescue. Only four of the 1159 orthologs of MED4 and MIT9313 were differentially-expressed in response to iron in both strains. However, in each strain, the expression of over a hundred additional genes changed, many of which are in labile genomic regions, suggesting a role for lateral gene transfer in establishing diversity of iron metabolism among Prochlorococcus. Furthermore, we found that MED4 lacks three genes near the iron-deficiency induced gene (idiA) that are present and induced by iron stress in MIT9313. These genes are interesting targets for studying the adaptation of natural Prochlorococcus assemblages to local iron conditions as they show more diversity than other genomic regions in environmental metagenomic databases.Gordon and Betty Moore FoundationNational Science Foundation (U.S.) (Biological Oceanography)United States. Office of Naval Research (ONR Young Investigator Award)National Science Foundation (U.S.) (Chemical Oceanography)National Science Foundation (U.S.) (Environmental Genomics grants

Delivering Sustained, Coordinated, and Integrated Observations of the Southern Ocean for Global Impact

The Southern Ocean is disproportionately important in its effect on the Earth system, impacting climatic, biogeochemical and ecological systems, which makes recent observed changes to this system cause for global concern. The enhanced understanding and improvements in predictive skill needed for understanding and projecting future states of the Southern Ocean require sustained observations. Over the last decade, the Southern Ocean Observing System (SOOS) has established networks for enhancing regional coordination and research community groups to advance development of observing system capabilities. These networks support delivery of the SOOS 20-year vision, which is to develop a circumpolar system that ensures time series of key variables, and deliver the greatest impact from data to all key end-users. Although the Southern Ocean remains one of the least-observed ocean regions, enhanced international coordination and advances in autonomous platforms have resulted in progress towards addressing the need for sustained observations of this region. Since 2009, the Southern Ocean community has deployed over 5700 observational platforms south of 40°S. Large-scale, multi-year or sustained, multidisciplinary efforts have been supported and are now delivering observations of essential variables at space and time scales that enable assessment of changes being observed in Southern Ocean systems. The improved observational coverage, however, is predominantly for the open ocean, encompasses the summer, consists of primarily physical oceanographic variables and covers surface to 2000 m. Significant gaps remain in observations of the ice-impacted ocean, the sea ice, depths more than 2000 m, the air-sea-ice interface, biogeochemical and biological variables, and for seasons other than summer. Addressing these data gaps in a sustained way requires parallel advances in coordination networks, cyberinfrastructure and data management tools, observational platform and sensor technology, platform interrogation and data-transmission technologies, modeling frameworks, and internationally agreed sampling requirements of key variables. This paper presents a community statement on the major scientific and observational progress of the last decade, and importantly, an assessment of key priorities for the coming decade, towards achieving the SOOS vision and delivering essential data to all end users

Volcanogenic fluxes of iron from the seafloor in the Amundsen Sea, West Antarctica

The Amundsen Sea in the Pacific sector of West Antarctica receives meltwater from the fastest retreating Antarctic glaciers, and its coastal polynyas host the highest primary productivity per unit area observed on the Antarctic continental shelf. Polynya productivity provides the base for a robust, diverse ecosystem and is controlled primarily by light and the availability of the micronutrient iron (Fe). While the sources of Fe in the region are not yet certain, Fe could be transported within modified Circumpolar Deep Water (mCDW) that intrudes onto the retrograde shelf and into ice shelf cavities, where it gains buoyancy through the addition of glacial meltwater and is injected into the upper water column when it exits the cavity. Thus, fluxes of dissolved Fe from the seafloor into in-flowing mCDW may ultimately be a source of Fe to the euphotic zone in the Amundsen Sea. To investigate the surface sediment biogeochemistry and the potential for a significant benthic flux of Fe to the waters on the Amundsen Sea shelf, sediment cores were collected at two sites close to the calving fronts of the Pine Island and Thwaites Glacier ice shelves. Pore water was analyzed for trace element content, and sediment was analyzed for physical and chemical properties including organic carbon and trace elements. Using a novel approach based on hypothesized Fe speciation and colloidal particle radius, theoretical Fe fluxes were calculated from pore water gradients and porosity. The fluxes reveal a spatially variable Fe input to the lower water column that could ultimately fertilize primary productivity. Supported by geochemical and physical evidence, we conclude that submarine weathering of volcanic glass grains observed and quantified in seabed sediments at the Pine Island site drives nonreductive Fe fluxes that are 100-fold higher than at the Thwaites site. This study highlights the need for further investigations of benthic-pelagic coupling in the Amundsen Sea region, which will likely be impacted in coming decades by accelerating glacial melting