Estimates of nitrate loading to the Arctic Ocean are limited by the lack of field observations within deltas partly due to logistical constraints. To overcome this limitation, we use a remote sensing ...framework to estimate retention of nitrate in Arctic deltas. We achieve this by coupling hydrological and biogeochemical process models at the network scale for five major Arctic deltas. Binary masks of delta channels were used to simulate flow direction and magnitude through networks. Models were parameterized using historical and seasonal observations. Simulated nitrate retention ranged from 2.9% to 15% of the incoming load. Retention rates were largest during winter but smallest during spring conditions when increased discharges export large nitrate masses to the coast. Under future climate scenarios, retention rates fall by ∼1%–10%. Arctic deltas have an important effect on the magnitude of nitrate entering Arctic seas and the inclusion of processing in deltas can improve flux estimates.
Plain Language Summary
The Arctic Ocean is the smallest and shallowest of the world's oceans but receives the largest riverine input per basin volume. Thus, the flux of nitrate from land to sea through rivers in the circumpolar Arctic can have large impacts on coastal life and global biogeochemical cycles. To assess the impact of terrestrially derived nitrate on Arctic Ocean chemistry under current and future scenarios, accurate accounting of nitrate loading is needed. Deltas act as filters that reduce nitrate loads, but current estimates of flux exclude the effects of Arctic deltas on nitrate export. We use a novel approach based on satellite imagery to estimate the potential impact of deltas on nitrate retention on five Arctic deltas, representing a range of morphologies and sizes. Our analysis shows seasonal and morphological differences in nitrate processing rates in each delta with retention rates ranging from 2.9% to 15% of incoming load and most retention occurring during winter months. Under future climate scenarios, the efficiency of nitrate retention decreases by up to 10%. Our models suggest that Arctic deltas alter the magnitude of nitrate entering the ocean and future predictions of loading or current earth system models could be improved by incorporating their effect.
Key Points
Remotely sensed data provide a useful tool for revealing patterns of nitrate retention in Arctic deltas
Channel networks of Arctic deltas are most efficient at retaining nitrate during spring and winter
Hotspots of nitrate removal cluster according to network structure and discharge on Arctic deltas
As climate changes and populations grow, a deeper understanding of deltaic surface and subsurface processes will help design sustainable management practices of delta resources. Numerical delta ...models are useful tools for understanding the relationship between the surface and subsurface, but quantitatively linking surface dynamics to subsurface structures remains difficult. The challenges stem from uncertainty in the numerical model parameters and the selection of informative post-processing metrics. In this work a Monte Carlo- and metric-based probabilistic framework is proposed for testing how well a numerical delta model captures the link between surface dynamics and subsurface structure. Probabilistic analysis of three graph-theoretic metrics describing morphology, morphodynamics, and subsurface structure shows that, at the laboratory scale, certain delta surface features, including channelization and channel stability, are informative of the spatial organization of sediment in the subsurface. Other surface features, such as sheet flows, are less informative. The surface dynamics metrics are also applied to data from a laboratory-scale physical experiment to show key differences in the numerical and experimental surface dynamics. The experimental morphology is more channelized than the numerical model and also undergoes more dramatic morphologic changes. These differences are likely due to a combination of numerical model resolution limitations, assumptions in the numerical model physics, and differences in flow field extraction in the numerical model and physical experiment.
The CRISPR-Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these ...lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR-Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies.
In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising ...approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610).
Understanding the diversity of repair outcomes after introducing a genomic cut is essential for realizing the therapeutic potential of genomic editing technologies. Targeted PCR amplification ...combined with Next Generation Sequencing (NGS) or enzymatic digestion, while broadly used in the genome editing field, has critical limitations for detecting and quantifying structural variants such as large deletions (greater than approximately 100 base pairs), inversions, and translocations.
To overcome these limitations, we have developed a Uni-Directional Targeted Sequencing methodology, UDiTaS, that is quantitative, removes biases associated with variable-length PCR amplification, and can measure structural changes in addition to small insertion and deletion events (indels), all in a single reaction. We have applied UDiTaS to a variety of samples, including those treated with a clinically relevant pair of S. aureus Cas9 single guide RNAs (sgRNAs) targeting CEP290, and a pair of S. pyogenes Cas9 sgRNAs at T-cell relevant loci. In both cases, we have simultaneously measured small and large edits, including inversions and translocations, exemplifying UDiTaS as a valuable tool for the analysis of genome editing outcomes.
UDiTaS is a robust and streamlined sequencing method useful for measuring small indels as well as structural rearrangements, like translocations, in a single reaction. UDiTaS is especially useful for pre-clinical and clinical application of gene editing to measure on- and off-target editing, large and small.
To reach the mammalian gut, enteric bacteria must pass through the stomach. Many such organisms survive exposure to the harsh gastric environment (pH 1.5-4) by mounting extreme acid-resistance ...responses, one of which, the arginine-dependent system of Escherichia coli, has been studied at levels of cellular physiology, molecular genetics and protein biochemistry. This multiprotein system keeps the cytoplasm above pH 5 during acid challenge by continually pumping protons out of the cell using the free energy of arginine decarboxylation. At the heart of the process is a 'virtual proton pump' in the inner membrane, called AdiC, that imports l-arginine from the gastric juice and exports its decarboxylation product agmatine. AdiC belongs to the APC superfamily of membrane proteins, which transports amino acids, polyamines and organic cations in a multitude of biological roles, including delivery of arginine for nitric oxide synthesis, facilitation of insulin release from pancreatic -cells, and, when inappropriately overexpressed, provisioning of certain fast-growing neoplastic cells with amino acids. High-resolution structures and detailed transport mechanisms of APC transporters are currently unknown. Here we describe a crystal structure of AdiC at 3.2 Å resolution. The protein is captured in an outward-open, substrate-free conformation with transmembrane architecture remarkably similar to that seen in four other families of apparently unrelated transport proteins.
The CLC family of Cl⁻-transporting proteins includes both Cl⁻ channels and Cl⁻/H⁺ exchange transporters. CLC-ec1, a structurally known bacterial homolog of the transporter subclass, exchanges two Cl⁻ ...ions per proton with strict, obligatory stoichiometry. Point mutations at two residues, Glu¹⁴⁸ and Tyr⁴⁴⁵, are known to impair H⁺ movement while preserving Cl⁻ transport. In the x-ray crystal structure of CLC-ec1, these residues form putative "gates" flanking an ion-binding region. In mutants with both of the gate-forming side chains reduced in size, H⁺ transport is abolished, and unitary Cl⁻ transport rates are greatly increased, well above values expected for transporter mechanisms. Cl⁻ transport rates increase as side-chain volume at these positions is decreased. The crystal structure of a doubly ungated mutant shows a narrow conduit traversing the entire protein transmembrane width. These characteristics suggest that Cl⁻ flux through uncoupled, ungated CLC-ec1 occurs via a channel-like electrodiffusion mechanism rather than an alternating-exposure conformational cycle that has been rendered proton-independent by the gate mutations.
The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ...ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure–activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 μM, BRET IC50 = 0.41 μM, BRD4(1) IC50 = 13 μM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.
Much of modern science takes place in a computational environment, and, increasingly, that environment is programmed using R, Python, or Julia. Furthermore, most scientific data now live on the ...cloud, so the first step in many workflows is to query a cloud database and load the response into a computational environment for further analysis. Thus, tools that facilitate programmatic data retrieval represent a critical component in reproducible scientific workflows. Earth science is no different in this regard. To fulfill that basic need, we developed R, Python, and Julia packages providing programmatic access to the U.S. Geological Survey’s National Water Information System database and the multi-agency Water Quality Portal. Together, these packages create a common interface for retrieving hydrologic data in the Jupyter ecosystem, which is widely used in water research, operations, and teaching. Source code, documentation, and tutorials for the packages are available on GitHub. Users can go there to learn, raise issues, or contribute improvements within a single platform, which helps foster better engagement and collaboration between data providers and their users.
RNA-guided endonucleases such as Cas9 provide efficient on-target genome editing in cells but may also cleave at off-target loci throughout the genome. Engineered variants of Streptococcus pyogenes ...Cas9 (SpCas9) have been developed to globally reduce off-target activity, but individual off-targets may remain, or on-target activity may be compromised. In order to evolve against activity at specific off-targets while maintaining strong on-target editing, we developed a novel M13 bacteriophage-mediated selection method. Using this method, sequential rounds of positive and negative selection are used to identify mutations to Cas9 that enhance or diminish editing activity at particular genomic sequences. We also introduce scanning mutagenesis of oligo-directed targets (SMOOT), a comprehensive mutagenesis method to create highly diverse libraries of Cas9 variants that can be challenged with phage-based selection. Our platform identifies novel SpCas9 mutants which mitigate cleavage against off-targets both in biochemical assays and in T-cells while maintaining higher on-target activity than previously described variants. We describe an evolved variant, S. pyogenes Adapted to Reduce Target Ambiguity Cas9 (SpartaCas), composed of the most enriched mutations, each of unknown function. This evolved Cas9 mutant reduces off-target cleavage while preserving efficient editing at multiple therapeutically relevant targets. Directed evolution of Cas9 using our system demonstrates an improved structure-independent methodology to effectively engineer nuclease activity.
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