126 research outputs found

    Sequential and Differential Interaction of Assembly Factors During Nitrogenase MoFe Protein Maturation

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    Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states

    Three-dimensional microCT imaging of mouse development from early post-implantation to early postnatal stages

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    AbstractIn this work, we report the use of iodine-contrast microCT to perform high-throughput 3D morphological analysis of mouse embryos and neonates between embryonic day 8.5 to postnatal day 3, with high spatial resolution up to 3µm/voxel. We show that mouse embryos at early stages can be imaged either within extra embryonic tissues such as the yolk sac or the decidua without physically disturbing the embryos. This method enables a full, undisturbed analysis of embryo turning, allantois development, vitelline vessels remodeling, yolk sac and early placenta development, which provides increased insights into early embryonic lethality in mutant lines. Moreover, these methods are inexpensive, simple to learn and do not require substantial processing time, making them ideal for high throughput analysis of mouse mutants with embryonic and early postnatal lethality

    Instability in clinical risk stratification models using deep learning

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    While it has been well known in the ML community that deep learning models suffer from instability, the consequences for healthcare deployments are under characterised. We study the stability of different model architectures trained on electronic health records, using a set of outpatient prediction tasks as a case study. We show that repeated training runs of the same deep learning model on the same training data can result in significantly different outcomes at a patient level even though global performance metrics remain stable. We propose two stability metrics for measuring the effect of randomness of model training, as well as mitigation strategies for improving model stability.Comment: Accepted for publication in Machine Learning for Health (ML4H) 202

    Forum: Critical Decision Dates for Drought Management in Centraland Northern Great Plains Rangeland

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    Ranchers and other land managers of central and northern Great Plains rangelands face recurrent droughts that negatively influence economic returns and environmental resources for ranching enterprises. Accurately estimating annual forage production and initiating drought decision-making actions proactively early in the growing season are both critical to minimize financial losses and degradation to rangeland soil and plant resources. Long-term forage production data sets from Alberta, Kansas, Montana, Nebraska, North Dakota, South Dakota, and Wyoming demonstrated that precipitation in April, May, and June (or some combination of these months) robustly predict annual forage production. Growth curves from clipping experiments and ecological site descriptions (ESDs) indicate that maximum monthly forage growth rates occur 1 mo after the best spring month (April to June) precipitation prediction variable. Key for rangeland managers is that the probability of receiving sufficient precipitation after 1 July to compensate for earlier spring precipitation deficits is extremely low. The complexity of human dimensions of drought decision-making necessitates that forage prediction tools account for uncertainty in matching animal demand to forage availability, and that continued advancements in remote sensing applications address both spatial and temporal relationships in forage production to inform critical decision dates for drought management in these rangeland ecosystems

    Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging

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    The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice

    Crossbow Volume 1

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    Student Integrated ProjectIncludes supplementary materialDistributing naval combat power into many small ships and unmanned air vehicles that capitalize on emerging technology offers a transformational way to think about naval combat in the littorals in the 2020 time frame. Project CROSSBOW is an engineered systems of systems that proposes to use such distributed forces to provide forward presence to gain and maiantain access, to provide sea control, and to project combat power in the littoral regions of the world. Project CROSSBOW is the result of a yearlong, campus-wide, integrated research systems engineering effort involving 40 student researchers and 15 supervising faculty members. This report (Volume I) summarizes the CROSSBOW project. It catalogs the major features of each of the components, and includes by reference a separate volume for each of the major systems (ships, aircraft, and logistics). It also prresents the results of the mission and campaign analysis that informed the trade-offs between these components. It describes certain functions of CROSSBOW in detail through specialized supporting studies. The student work presented here is technologically feasible, integrated and imaginative. The student project cannot by itself provide definitive designs or analyses covering such a broad topic. It does strongly suggest that the underlying concepts have merit and deserve further serious study by the Navy as it transforms itself

    Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer

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    Increased tetrahydrobiopterin (BH4) generated in injured sensory neurons contributes to increased pain sensitivity and its persistence. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme in the de novo BH4 synthetic pathway, and human single-nucleotide polymorphism studies, together with mouse genetic modeling, have demonstrated that decreased GCH1 leads to both reduced BH4 and pain. However, little is known about the regulation of Gch1 expression upon nerve injury and whether this could be modulated as an analgesic therapeutic intervention. We performed a phenotypic screen using about 1000 bioactive compounds, many of which are target-annotated FDA-approved drugs, for their effect on regulating Gch1 expression in rodent injured dorsal root ganglion neurons. From this approach, we uncovered relevant pathways that regulate Gch1 expression in sensory neurons. We report that EGFR/KRAS signaling triggers increased Gch1 expression and contributes to neuropathic pain; conversely, inhibiting EGFR suppressed GCH1 and BH4 and exerted analgesic effects, suggesting a molecular link between EGFR/KRAS and pain perception. We also show that GCH1/BH4 acts downstream of KRAS to drive lung cancer, identifying a potentially druggable pathway. Our screen shows that pharmacologic modulation of GCH1 expression and BH4 could be used to develop pharmacological treatments to alleviate pain and identified a critical role for EGFR-regulated GCH1/BH4 expression in neuropathic pain and cancer in rodents

    The effects of dilution and mixed layer depth on deliberate ocean iron fertilization : 1-D simulations of the southern ocean iron experiment (SOFeX)

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    Author Posting. © Elsevier B.V., 2007. 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 Journal of Marine Systems 71 (2008): 112-130, doi:10.1016/j.jmarsys.2007.07.002.To better understand the role of iron in driving marine ecosystems, the Southern Ocean Iron Experiment (SOFeX) fertilized two surface water patches with iron north and south of the Antarctic Polar Front Zone (APFZ). Using 1-D coupled biological-physical simulations, we examine the biogeochemical dynamics that occurred both inside and outside of the fertilized patches during and shortly after the SOFeX field campaign. We focus, in particular, on three main issues governing the biological response to deliberate iron fertilization: the interaction among phytoplankton, light, macro-nutrient and iron limitation; dilution and lateral mixing between the fertilized patch and external, unfertilized waters; and the effect of varying mixed layer depth on the light field. At the patch south of the APFZ, sensitivity simulations with no dilution results in the maximum bloom magnitude, whereas dilution with external water extends the development of the north patch bloom by relieving silicon limitation. In model sensitivity studies for both sites, maximum chlorophyll concentration and dissolved inorganic carbon depletion inside the fertilized patches are inversely related to mixed layer depth, similar to the patterns observed across a number of iron fertilization field experiments. Our results suggest that Southern Ocean phytoplankton blooms resulting from natural or deliberate iron fertilization will tend to become iron-light co-limited unless the mixed layer depth is quite shallow.This work was supported by NASA grant NAG5-12520 from the NASA Ocean Biogeochemistry Program
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