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Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here, we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by reduced clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 20 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in the CD46 gene may reduce the burden of BVDV-associated diseases in cattle and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.

Expanded Materials and Methods, Supplemental Figures, and Supplemental Tables Figure S1. Flow cytometric analysis of peripheral blood from SS, SS.BN3, SSIL2Rγ, and SS.BN3IL2Rγ rats. Figure S2.SH2B3 specifically marks CD31+ blood vessels, but not LYVE1+ lymphatic vessels. Table S1. Peripheral blood analysis of SS, SS.BN3, SSIL2Rγ and SS.BN3IL2Rγ rats. Table S2. Genes located in the overlapping portions of the rat chromosome 3 cancer QTLs. Table S3. Variants that cosegregate between tumor resistant (BN and Cop) and tumor susceptible (ACI, F344, and SS) rat strains.

The majority of causative variants in familial breast cancer remain unknown. Of the known risk variants, most are tumor cell autonomous, and little attention has been paid yet to germline variants that may affect the tumor microenvironment. In this study, we developed a system called the Consomic Xenograft Model (CXM) to map germline variants that affect only the tumor microenvironment. In CXM, human breast cancer cells are orthotopically implanted into immunodeficient consomic strains and tumor metrics are quantified (e.g., growth, vasculogenesis, and metastasis). Because the strain backgrounds vary, whereas the malignant tumor cells do not, any observed changes in tumor progression are due to genetic differences in the nonmalignant microenvironment. Using CXM, we defined genetic variants on rat chromosome 3 that reduced relative tumor growth and hematogenous metastasis in the SS.BN3IL2Rγ consomic model compared with the SSIL2Rγ parental strain. Paradoxically, these effects occurred despite an increase in the density of tumor-associated blood vessels. In contrast, lymphatic vasculature and lymphogenous metastasis were unaffected by the SS.BN3IL2Rγ background. Through comparative mapping and whole-genome sequence analysis, we narrowed candidate variants on rat chromosome 3 to six genes with a priority for future analysis. Collectively, our results establish the utility of CXM to localize genetic variants affecting the tumor microenvironment that underlie differences in breast cancer risk. Cancer Res; 74(22); 6419–29. ©2014 AACR.

Introduction: Most male pigs are surgically castrated to avoid puberty-derived boar taint and aggressiveness. However, this surgical intervention represents a welfare concern in swine production. Disrupting porcine KISS1 is hypothesized to delay or abolish puberty by inducing variable hypogonadotropism and thus preventing the need for castration.Methods: To test this hypothesis, we generated the first KISS1-edited large animal using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the sequence encoding a conserved core motif of kisspeptin. Genome editors were intracytoplasmically injected into 684 swine zygotes and transferred to 19 hormonally synchronized surrogate sows. In nine litters, 49 American Yorkshire and 20 Duroc liveborn piglets were naturally farrowed.Results: Thirty-five of these pigs bore KISS1-disruptive alleles ranging in frequency from 5% to 97% and did not phenotypically differ from their wild-type counterparts. In contrast, four KISS1-edited pigs (two boars and two gilts) with disruptive allele frequencies of 96% and 100% demonstrated full hypogonadotropism, infantile reproductive tracts, and failed to reach sexual maturity. Change in body weight during development was unaffected by editing KISS1. Founder pigs partially carrying KISS1-disruptive alleles were bred resulting in a total of 53 KISS1+/+, 60 KISS1+/−, and 34 KISS1−/− F1 liveborn piglets, confirming germline transmission.Discussion: Results demonstrate that a high proportion of KISS1 alleles in pigs must be disrupted before variation in gonadotropin secretion is observed, suggesting that even a small amount of kisspeptin ligand is sufficient to confer proper sexual development and puberty in pigs. Follow-on studies will evaluate fertility restoration in KISS1 KO breeding stock to fully realize the potential of KISS1 gene edits to eliminate the need for surgical castration.

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 16 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in CD46 may reduce the burden of BVDV-associated diseases in cattle, and is consistent with our stepwise,in vitroandex vivoexperiments with cell lines and matched fetal clones.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium’s plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled—along with validated datasets—into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit—and the knowledge generated by its applications—as a means to accelerate the clinical development of new therapies for a wide range of conditions., This Perspective discusses how the Somatic Cell Genome Editing Consortium aims to accelerate the implementation of safe and effective genome-editing therapies in the clinic.

SummaryThe aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

Changes in the distribution of coastal macrophytes in Greenland, and elsewhere in the Arctic are difficult to quantify as the region remains challenging to access and monitor. Satellite imagery, in particular Sentinel-2 (S2), may enable large-scale monitoring of coastal areas in Greenland but its use is impacted by the optically complex environments and the scarcity of supporting data in the region. Additionally, the canopies of the dominant macrophyte species in Greenland do not extend to the sea surface, limiting the use of indices that exploit the reflection of near-infrared radiation by vegetation due to its absorption by seawater. Three hypotheses are tested: I) 10-m S2 imagery and commonly used detection methods can identify intertidal macrophytes that are exposed at low tide in an optically complex fjord system in Greenland impacted by marine and land terminating glaciers; II) detached and floating macrophytes accumulate in patches that are sufficiently large to be detected by 10-m S2 images; III) iceberg scour and/or turbid meltwater runoff shape the spatial distribution of intertidal macroalgae in fjord systems with marine-terminating glaciers. The NDVI produced the best results in optically complex fjord systems in Greenland. 12 km2 of exposed intertidal macrophytes were identified in the study area at low tide. Floating mats of macrophytes ranged in area from 400 m2 to 326,800 m2 and were most common at the mouth of the fjord. Icebergs and turbidity appear to play a role in structuring the distribution of intertidal macrophytes and the retreat of marine terminating glaciers could allow macrophytes cover to expand. The challenges and solutions presented here apply to most fjords in Greenland and, therefore, the methodology may be extended to produce a Greenland-wide estimate of intertidal macrophytes.

Bovine viral diarrhea virus’s (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46, however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Most mammalian cells possess a single, non-motile primary cilium that plays an important role in mediating cellular signaling pathways, such as Hedgehog (Hh) signaling. Primary cilia are present on testicular somatic cells and demonstrate a temporal expression during development; however, their role in testicular morphogenesis is not well characterized. To investigate the role of primary cilia and Hh signaling in Sertoli cells on morphogenesis, we inhibited assembly of primary cilia through CRISPR Cas9–mediated gene editing of ODF2, a structural component of primary cilia and siRNA-mediated gene silencing of IFT88, a functional component of the intraflagellar transport system. Knockdown of ODF2 and IFT88 resulted in a 50% reduction in the number of cells with primary cilia and significant shortening of the remaining cilia. The expression of GLI1, a downstream target of Hh signaling, was significantly reduced when IFT88 but not ODF2, was downregulated. When morphogenesis was examined using tubule formation in vitro and a novel testicular organoid system, loss of cilia after knockdown of both targets affected cellular assembly and organization. While the Hh pathway was found to be active during morphogenesis in vitro, addition of the Hh antagonist cyclopamine did not affect morphogenesis in either in vitro system. These results indicate that primary cilia are important for morphogenesis in vitro but Hh signaling is not the cilia-mediated pathway responsible for orchestrating morphogenic organization.

Summary Rat embryonic stem cells (rESCs) are capable of contributing to all differentiated tissues, including the germ line in chimeric animals, and represent a unique, authentic alternative to mouse embryonic stem cells for studying stem cell pluripotency and self-renewal. Here, we describe an EGFP reporter transgene that tracks expression of the benchmark naive pluripotency marker gene Rex1 (Zfp42) in the rat. Insertion of the EGFP reporter gene downstream of the Rex1 promoter disrupted Rex1 expression, but REX1-deficient rESCs and rats were viable and apparently normal, validating this targeted knockin transgene as a neutral reporter. The Rex1-EGFP gene responded to self-renewal/differentiation factors and validated the critical role of β-catenin/LEF1 signaling. The stem cell reporter also allowed the identification of functionally distinct sub-populations of cells within rESC cultures, thus demonstrating its utility in discriminating between cell states in rat stem cell cultures, as well as providing a tool for tracking Rex1 expression in the rat., Highlights • Rex1-EGFP transgene is a neutral reporter of pluripotency and self-renewal in the rat • Rex1-EGFP transgene responds appropriately to self-renewal and differentiation signaling • Rex1-EGFP transgene allows the discrimination between rat ESC pluripotent states, Rat embryonic stem cells (ESCs) differentiate into all adult cells, including functional gametes, and uniquely qualify as a comparator with mouse ESCs to study self-renewal and pluripotency. In this article, Burdon and colleagues generated a Rex1-EGFP stem cell reporter as a sensitive tool to monitor and interrogate the regulation of rat ESCs, and contribute to understanding stem cells and fate determination in mammals.

We present an analysis of ocean surface dispersion characteristics, on 1–100-m scales, obtained by optically tracking a release of bamboo plates for 2 h in the northern Gulf of Mexico. Under sustained 5–6 m s−1 winds, energetic Langmuir cells are clearly delineated in the spatially dense plate observations. Within 10 min of release, the plates collect in windrows with 15-m spacing aligned with the wind. Windrow spacing grows, through windrow merger, to 40 m after 20 min and then expands at a slower rate to 50 m. The presence of Langmuir cells produces strong horizontal anisotropy and scale dependence in all surface dispersion statistics computed from the plate observations. Relative dispersion in the crosswind direction initially dominates but eventually saturates, while downwind dispersion exhibits continual growth consistent with contributions from both turbulent fluctuations and organized mean shear. Longitudinal velocity differences in the crosswind direction indicate mean convergence at scales below the Langmuir cell diameter and mean divergence at larger scales. Although the second-order structure function measured by contemporaneous GPS-tracked surface drifters drogued at ~0.5 m shows persistent r2/3 power law scaling down to 100–200-m separation scales, the second-order structure function for the very near surface plates observations has considerably higher energy and significantly shallower slope at scales below 100 m. This is consistent with contemporaneous data from undrogued surface drifters and previously published model results indicating shallowing spectra in the presence of direct wind-wave forcing mechanisms.

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

In this paper, a high-frequency (HF) coastal radar network is described, which is established and maintained by the Institute of Marine Sciences (ISMAR) of the National Research Council of Italy (CNR) for the measurement of surface current velocities in the Gulf of Manfredonia, located in the semienclosed Adriatic Sea (Mediterranean Sea), during 2013–2015. The network consisted of four HF radars (HFRs) that provided hourly sea surface velocity data in real-time mode in a netCDF format compliant to the Climate and Forecast Metadata Conventions CF-1.6 and the INSPIRE directive. The hourly netCDF files are disseminated via a Thematic Real-time Environmental Distributed Data Services catalog supporting OGC compliant distributions and protocols for data visualization, metadata interrogation, and data download. HFR velocity data were compared with in situ velocity measurements by Global Positioning System tracked surface drifters deployed within the radar footprint. The results show a good agreement, with the root mean square (rms) of the difference between radial velocities from HFR and drifters ranging between $20\%$ and $50\%$ of the drifter velocity rms. The HFR data have also been compared with subsurface velocity profiles from an upward looking acoustic Doppler current profiler (ADCP) during winter 2015, to gain information on the correlation between surface and water column velocities. This information is especially relevant for fishery and coastal management applications, where transport of larvae, sediments, and pollutants in the water column are considered. Results show that, at least in the considered period, the velocity in the water column is well correlated, and there is a good agreement between surface HFR and ADCP data (correlations between 0.95 and 0.75). The Gulf of Manfredonia network has been instrumental to the set up of a core of quality control practices and interoperable data and metadata formats that have been subsequently adopted within the Italian RITMARE network and that are presently discussed and refined at the European level through the projects Jerico-NEXT and INCREASE.

Bovine viral diarrhea virus (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genesPTPN12,GRID2, andRABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss ofPTPN12,GRID2, orRABGAP1Lgene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Background There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. Methods The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. Results Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. Conclusions This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype. The male germline stem cell or spermatogonial stem cell (SSC) is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Here we report that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing in porcine spermatogonia that include SSCs is significantly more efficient than previously reported editing with TALENs and allows precise gene editing by homology directed repair (HDR). We also established homology-mediated end joining (HMEJ) as a second approach to targeted gene editing to enable introduction of larger transgenes and/or humanizing parts of the pig genome for disease modeling or regenerative medicine. In summary, the approaches established in the current study result in efficient targeted genome editing in porcine germ cells for precise replication of human disease alleles.

Vertical mixing of upper water masses in Arctic fjords is important for circulation and transport of nutrients and heat. However, the distribution of turbulent mixing is poorly known. Here we present hydrographic and microscale turbulence measurements from six fjords in Northwestern (69°N–75°N) Greenland. Water mass distributions showed the presence of warm bottom water of Atlantic origin in all fjord transects and a significant modification of upper water masses in fjords with tidewater outlet glaciers. Spatial and temporal distribution of turbulence in the fjordsshowed, in general, low rates of turbulent kinetic energy, ε (∼10 −9 W kg−1), and strong stratification implies that vertical exchange in the upper ∼200 m was relatively weak. However, measurements within 2 km of the terminus of a tidewater outlet glaciershowed values of ε and inferred turbulent diffusion coefficients >10 −4 m2 s−1 that were about two orders of magnitude larger than elsewhere in the deep fjords. This elevated mixing corresponded to comparatively large heat and salt fluxes. These observations suggest that vertical mixing near tidewater outlet glaciers results in localized mixing hot spots, likely due to subglacial discharge and near-glacial current shear, that contribute significantly to vertical exchange in the deep fjords.

Icebergs account for approximately half of the freshwater flux from the Greenland Ice Sheet and they can impact marine ecosystems by releasing nutrients and sediments into the ocean as they drift and melt. Parameterizing iceberg fluxes of nutrients and sediments to fjord and ocean waters remains a difficult task due to the complexity of ice-ocean interactions and is complicated by a lack of observations. Acquiring iceberg samples can be difficult and dangerous, as icebergs can break apart and roll without warning. Here we present open source design files for a small, lightweight ice coring drill that can be reproduced using modern computer numerical control (CNC) machining and 3D printing technology. This ice core drill can rapidly acquire small ice samples from icebergs and bergy bits using a standard commercial, off-the-shelf battery-operated hand drill. Design files and a recent field expedition to Northwest Greenland are described. Ice core collection required only 30 s, thereby minimizing risks to scientists.

Background: Heart rate variability (HRV) and pulse rate variability are indices of autonomic cardiac modulation. Increased pericardial fat is associated with worse cardiovascular outcomes. We hypothesized that progressive increases in pericardial fat volume and inflammation prospectively dampen HRV in hypercholesterolemic pigs. Methods: WT (wild type) or PCSK9 (proprotein convertase subtilisin-like/kexin type-9) gain-of-function Ossabaw mini-pigs were studied in vivo before and after 3 and 6 months of a normal diet (WT-normal diet, n=4; PCSK9-normal diet, n=6) or high-fat diet (HFD; WT-HFD, n=3; PCSK9-HFD, n=6). The arterial pulse waveform was obtained from an arterial telemetry transmitter to analyze HRV indices, including SD (SD of all pulse-to-pulse intervals over a single 5-minute period), root mean square of successive differences, proportion >50 ms of normal-to-normal R-R intervals, and the calculated ratio of low-to-high frequency distributions (low-frequency power/high-frequency power). Pericardial fat volumes were evaluated using multidetector computed tomography and its inflammation by gene expression of TNF (tumor necrosis factor)-α. Plasma lipid panel and norepinephrine level were also measured. Results: At diet completion, hypercholesterolemic PCSK9-HFD had significantly ( P 50 ms, high-frequency power, low-frequency power), and both HFD groups had higher sympathovagal balance (SD of all pulse-to-pulse intervals over a single 5-minute period/root mean square of successive differences, low-frequency power/high-frequency power) compared with normal diet. Pericardial fat volumes and LDL (low-density lipoprotein) cholesterol concentrations correlated inversely with HRV and directly with sympathovagal balance, while sympathovagal balance correlated directly with plasma norepinephrine. Pericardial fat TNF-α expression was upregulated in PCSK9-HFD, colocalized with nerve fibers, and correlated inversely with root mean square of successive differences and proportion >50 ms. Conclusions: Progressive pericardial fat expansion and inflammation are associated with a fall in HRV in Ossabaw mini-pigs, implying aggravated autonomic imbalance. Hence, pericardial fat accumulation is associated with alterations in HRV and the autonomic nervous system. Visual Overview: A visual overview is available for this article.

Formed in the aftermath of the Deepwater Horizon event, the largest accidental marine oil spill, the Consortium for Advanced Research on Transport of Hydrocarbons in the Environment (CARTHE) focused on understanding the physical processes controlling the transport of material from a deep blowout all the way to the coast. Even though CARTHE was initially a modeling-oriented team, it progressively became more focused on observations in order to collect the data needed for model evaluation. A number of new technological advances needed to be made to collect the necessary data. This article reviews most of these, with special focus on surface sampling, where much of the oil is located during oil spills, as well as the measurement of near-field droplet size distribution.

Neurofibromatosis Type 1 (NF1) is a genetic disease caused by mutations in Neurofibromin 1 (NF1). NF1 patients present with a variety of clinical manifestations and are predisposed to cancer development. Many NF1 animal models have been developed, yet none display the spectrum of disease seen in patients and the translational impact of these models has been limited. We describe a minipig model that exhibits clinical hallmarks of NF1, including café au lait macules, neurofibromas, and optic pathway glioma. Spontaneous loss of heterozygosity is observed in this model, a phenomenon also described in NF1 patients. Oral administration of a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor suppresses Ras signaling. To our knowledge, this model provides an unprecedented opportunity to study the complex biology and natural history of NF1 and could prove indispensable for development of imaging methods, biomarkers, and evaluation of safety and efficacy of NF1-targeted therapies., Isakson et al. report a genetically engineered minipig model of Neurofibromatosis Type 1 (NF1) that exhibits clinical hallmarks of the disease, including neurofibromas and optic pathway glioma. This model may expedite the development of imaging methods, biomarkers, and therapies for NF1 patients.

Two major episodes of seagrass mortality have occurred in Florida Bay in the past 30 years: The first occurred between 1987 and 1991 and the second began in 2015. In both episodes, dense beds of turtle grass (Thalassia testudinum) were decimated. Elevated water temperature and hypersalinity have been implicated as contributing factors in both mortality events. During both die-off events seagrass beds on shallow (Z≈0.3 m) mudbanks in western Florida Bay were disproportionately affected when compared to neighboring seagrass in deeper basins (Z≈1.5 m). A network of in situ monitoring stations has measured sea surface temperature and salinity at hourly intervals in 16 basins in Florida Bay from August 2009 to the present day and these data aided in diagnosing the 2015–2016 die-off event. However, very few in situ observations of sea surface temperature have been collected on Florida Bay's shallow mudbanks as most are inaccessible by boat. As a result, our understanding of the role of elevated SST in shaping the spatial patterns of seagrass mortality is hindered by the spatial distribution of the in situ monitoring data and its record length. Therefore, we turn to remotely sensed SST data to expand our spatial coverage to include the entire Florida Bay ecosystem and to extend the record length to include the 1987–1991 die-off event. 1 km MODIS SST shows that shallow mudbanks were consistently warmer (by up to 6°C) than nearby deeper basins. While water depth is likely the primary driver of spatial variability in SST, Landsat-8 surface reflectance data suggest that shallow seagrass beds could have suffered from the added influence of low surface reflectance, which might have further contributed to their thermal stress. Daily 0.25° AVHRR SST shows that the August maximum SST has increased by 1°C from 1981 to 2016, which is a cause for concern for the future of seagrass in Florida Bay. Correlation of monthly AVHRR SST anomalies with the multivariate ENSO index shows that ENSO can only partially explain the anomalous temperatures. When viewed together, the in situ and remotely sensed SST suggests that both extended exposure to anomalously warm temperatures and large, rapid changes in temperature could have contributed to seagrass mortality during both events.

Spermatogonia represent a diploid germ cell population that includes spermatogonial stem cells. In this report, we describe new methods for isolation of highly enriched porcine spermatogonia based on light scatter properties, and for targeted mutagenesis in porcine spermatogonia using nucleofection and TALENs. We optimized a nucleofection protocol to deliver TALENs specifically targeting the DMD locus in porcine spermatogonia. We also validated specific sorting of porcine spermatogonia based on light scatter properties. We were able to obtain a highly enriched germ cell population with over 90% of cells being UCH-L1 positive undifferentiated spermatogonia. After gene targeting in porcine spermatogonia, indel (insertion or deletion) mutations as a result of non-homologous end joining (NHEJ) were detected in up to 18% of transfected cells. Our report demonstrates for the first time an approach to obtain a live cell population highly enriched in undifferentiated spermatogonia from immature porcine testes, and that gene targeting can be achieved in porcine spermatogonia which will enable germ line modification.

Hypercholesterolemia is a major risk factor for atherosclerosis. Remaining challenges in the management of atherosclerosis necessitate development of animal models that mimic human pathophysiology. We characterized a novel mutant pig model with DNA transposition of D374Y gain-of-function (GOF) cDNA of chimp proprotein convertase subtilisin/kexin type-9 (PCSK9), and tested the hypothesis that it would develop peripheral vascular remodeling and target organ injury in the kidney. Wild-type or PCSK9-GOF Ossabaw miniature pigs fed a standard or atherogenic diet (AD) (n = 7 each) were studied in vivo after 3 and 6 months of diet. Single-kidney hemodynamics and function were studied using multidetector computed tomography and kidney oxygenation by blood oxygen level-dependent magnetic resonance imaging. The renal artery was evaluated by intravascular ultrasound, aortic stiffness by multidetector computed tomography, and kidney stiffness by magnetic resonance elastography. Subsequent ex vivo studies included the renal artery endothelial function and morphology of abdominal aorta, renal, and femoral arteries by histology. Compared with wild type, PCSK9-GOF pigs had elevated cholesterol, triglyceride, and blood pressure levels at 3 and 6 months. Kidney stiffness increased in GOF groups, but aortic stiffness only in GOF-AD. Hypoxia, intrarenal fat deposition, oxidative stress, and fibrosis were observed in both GOF groups, whereas kidney function remained unchanged. Peripheral arteries in GOF groups showed medial thickening and development of atheromatous plaques. Renal endothelial function was impaired only in GOF-AD. Therefore, the PCSK9-GOF mutation induces rapid development of atherosclerosis in peripheral vessels of Ossabaw pigs, which is exacerbated by a high-cholesterol diet. This model may be useful for preclinical studies of atherosclerosis.

Drone autopilots are naturally suited for real-time iceberg tracking as they measure position and orientation (pitch, roll, and heading) and they transmit these data to a ground station. We powered an ArduPilot Mega (APM) 2.6 with a 5V 11 Ah lithium ion battery (a smartphone power bank), placed the APM and battery in a waterproof sportsman’s box, and tossed the box and its contents by hand onto an 80 m-long iceberg from an 8 m boat. The data stream could be viewed on a laptop, which greatly enhanced safety while collecting conductivity/temperature/depth (CTD) profiles from the small boat in the iceberg’s vicinity. The 10 s position data allowed us to compute the distance of each CTD profile to the iceberg, which is necessary to determine if a given CTD profile was collected within the iceberg’s meltwater plume. The APM position data greatly reduced position uncertainty when compared to 5 min position data obtained from a Spot Trace unit. The APM functioned for over 10 h without depleting the battery. We describe the specific hardware used and the software settings necessary to use the APM as a real-time iceberg tracker. Furthermore, the methods described here apply to all Ardupilot-compatible autopilots. Given the low cost ($90) and ease of use, drone autopilots like the APM should be included as another tool for studying iceberg motion and for enhancing safety of marine operations. • Commercial off-the-shelf iceberg trackers are typically configured to record positions over relatively long intervals (months to years) and are not well-suited for short-term (hours to few days), high-frequency monitoring • Drone autopilots are cheap and provide high-frequency (>1 Hz) and real-time information about iceberg drift and orientation • Drone autopilots and ground control software can be easily adapted to studies of iceberg-ocean interactions and operational iceberg management Method name: Low-cost, Real-time iceberg drift and orientation observations, Keywords: Iceberg tracking, Iceberg drift and orientation, Operational iceberg management, Drone autopilot, Iceberg-ocean interaction

The accelerated melt of the Greenland Ice Sheet has been linked to a sudden increase in the presence of warm subsurface coastal water in west Greenland. Yet pathways of warm coastal water along the entire west Greenland coast have remained largely unstudied. Here we present the first, near‐synoptic hydrographic observations at both the continental slope and fjord entrances of the west Greenland coastal system from Cape Farewell (59°N) to Melville Bay (75°N) in summer 2016. We observed a distinct north‐south division in the water mass distribution in west Greenland, approximately partitioned by the northern part of Davis Strait, and a division between the continental slope and fjord entrances. Waters from the regional southern freshwater source with origin in the East Greenland Current that rounds Cape Farewell are not observed to enter Baffin Bay. The regional heat source transported by the West Greenland Current is blocked by Southwest Greenland Coastal Water in the south but the deep connections in the north allow warm deep Subpolar Mode Water to enter fjords. Furthermore, we observed cold and relative saline Baffin Bay Polar Water over the inner part of the banks, periodically reaching as far south as 64°N, suggesting the presence of an undescribed southward current at the Southwest Greenland continental shelf.

The Vaigat Iceberg-Microbial Oil Degradation and Archaeological Heritage Investigation (VIMOA) project records the results of archaeological survey of five sites in Greenland that are threatened by extreme weather conditions related to climate change. The project demonstrates the advantages of collaboration between archaeologists and natural scientists, and provides a repository of data to help preserve the archaeological record.

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

Ribonucleoprotein (RNP) granules are biomolecular condensates—liquid–liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation. Dysregulation of ribonucleoprotein complex granules, previously implicated in neuromuscular disease, can drive pathogenesis in a genetic form of dilated cardiomyopathy, as shown in gene-edited pigs and patient-derived cardiomyocytes.

Prediction of the distribution of buoyant oil, plastics, flotsam and marine organisms near the ocean surface is a long-standing fundamental problem of practical importance. Here, progress in this area during the time of the Gulf of Mexico Research Initiative (GoMRI) (2012-2019) is synthesized, emphasizing the well-known accumulation of floating material into highly concentrated streaks on horizontal scales of meters to 10’s of kilometers. In the pre-GoMRI period, two new paradigms were developed: the importance of submesoscale frontal dynamics on the larger scales and of surface-wave-driven Langmuir turbulence on the smaller scales, with a broad transition occurring near 100 m. Rapid progress resulted from the combination of high resolution numerical modeling tools, mostly developed before GoMRI, and new observational techniques developed during GoMRI. New designs for inexpensive and biodegradable satellite tracked surface drifters and new techniques for aerial tracking of surface drift cards enabled the deployment of thousands of such drifters to measure surface currents and to act as calibrated surrogates for oil. Progress in using radar and optical remote sensing of surface waves enabled mapping of surface currents from ships and aircraft. These new tools, combined with traditional oceanographic tools, enabled a set of coordinated measurement programs which supported and expanded the new paradigms. Floating material was found to both accumulate at submesoscale fronts and to disperse by following these fronts as they moved, leading to both higher concentrations and increased dispersion. Analyses confirmed the distinct submesoscale dynamics of this process and complexity of the resulting fields. Existing tools could be developed into predictive models of submesoscale statistics; prediction of individual submesoscale features is limited by data. Away from fronts, measured rates of accumulation of material in and beneath surface windrows was found to be consistent with Langmuir turbulence, but highly dependent on the rise rate of the material and thus, for oil, on the droplet size. Models of this process were developed and tested and could be further developed into predictive tools. Both the submesoscale and Langmuir processes are sensitive to coupling with surface waves and air-sea flux processes; this is a promising area for future studies.

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL’s absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis—after organogenesis has begun—in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer.

Background & Aims Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology. Methods Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663–L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs). Results Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver. Conclusions We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.

The ecological impacts of meltwater produced by icebergs and sea ice in the waters around Greenland are poorly understood, due in part to limited observations. Current field sampling methods are resource and labor-intensive, and not without significant risk. We developed a small, unmanned, and robotic platform to retrieve ice samples, while simultaneously eliminating risks to scientists and their support infrastructure. The IceDrone consists of a commercial hexcopter that is modified to retrieve shallow ice cores. We describe the design requirements, construction, and testing of the IceDrone. IceDrone’s capabilities were validated in the laboratory and during a field test in January 2019 near Nuuk (southwest Greenland). IceDrone retrieved shallow ice cores in hard and dry ice in harsh winter conditions. The field test led to modifications in the drilling head design and drilling process that enable it to retrieve samples in thin sea ice. All design files and software are provided in an attempt to rapidly enhance our collective understanding of ice-ocean interactions while improving the safety and productivity of field sampling campaigns.

Freshwater discharge from glaciers is increasing across the Arctic in response to anthropogenic climate change, which raises questions about the potential downstream effects in the marine environment. Whilst a combination of long-term monitoring programmes and intensive Arctic field campaigns have improved our knowledge of glacier–ocean interactions in recent years, especially with respect to fjord/ocean circulation, there are extensive knowledge gaps concerning how glaciers affect marine biogeochemistry and productivity. Following two cross-cutting disciplinary International Arctic Science Committee (IASC) workshops addressing the importance of glaciers for the marine ecosystem, here we review the state of the art concerning how freshwater discharge affects the marine environment with a specific focus on marine biogeochemistry and biological productivity. Using a series of Arctic case studies (Nuup Kangerlua/Godthåbsfjord, Kongsfjorden, Kangerluarsuup Sermia/Bowdoin Fjord, Young Sound and Sermilik Fjord), the interconnected effects of freshwater discharge on fjord–shelf exchange, nutrient availability, the carbonate system, the carbon cycle and the microbial food web are investigated. Key findings are that whether the effect of glacier discharge on marine primary production is positive or negative is highly dependent on a combination of factors. These include glacier type (marine- or land-terminating), fjord–glacier geometry and the limiting resource(s) for phytoplankton growth in a specific spatio-temporal region (light, macronutrients or micronutrients). Arctic glacier fjords therefore often exhibit distinct discharge–productivity relationships, and multiple case-studies must be considered in order to understand the net effects of glacier discharge on Arctic marine ecosystems.

Bathymetry measurements are needed in shallow, near-shore environments to estimate the potential distribution of macroalgae and seagrass, which contribute to oceanic carbon sinks in Greenland and other Arctic locations. Additionally, ocean current observations near icebergs and marine-terminating glaciers are necessary to estimate their melt rates. Rocky coastlines, icebergs and glaciers, however, present a significant safety risk to manned vessels. We developed an affordable and portable autonomous surface vehicle (ASV) that can safely obtain bathymetry and ocean current measurements in dangerous environments. The Arctic Research Centre Autonomous Boat (ARCAB) is a student-built ASV that can be transported on a euro-pallet and operated from a small boat and from shore. ARCAB has a minimum battery life of 2 h and accommodates a SonTek M9 RiverSurveyor system for bathymetry and ocean current measurements. ARCAB is controlled by a National Instruments myRIO-1900 and features autonomous waypoint navigation, obstacle avoidance, and return-to-home functionality. We demonstrate the use of ARCAB in Greenland in January 2019. Keywords: Autonomous surface vehicle, Marine robotics, Bathymetry, Ocean currents, Coastal monitoring, Greenland

Background The human genetic disorder Microvillus Inclusion Disease (MVID) results from inactivating mutations in Myosin Vb (MYO5B). MVID is characterized by severe, unremitting diarrhea that typic...