CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we ...describe a Staphylococcus aureus Cas9-based repressor (dSaCas9
) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9
efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9
and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9
expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9
. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.
The advancement of underrepresented minority and women PhD students to elite postdoctoral and faculty positions in the STEM fields continues to lag that of majority males, despite decades of efforts ...to mitigate bias and increase opportunities for students from diverse backgrounds. In 2015, the National Science Foundation Alliance for Graduate Education and the Professoriate (NSF AGEP) California Alliance (Berkeley, Caltech, Stanford, UCLA) conducted a wide-ranging survey of graduate students across the mathematical, physical, engineering, and computer sciences in order to identify levers to improve the success of PhD students, and, in time, improve diversity in STEM leadership positions, especially the professoriate. The survey data were interpreted via path analysis, a method that identifies significant relationships, both direct and indirect, among various factors and outcomes of interest. We investigated two important outcomes: publication rates, which largely determine a new PhD student's competitiveness in the academic marketplace, and subjective well-being. Women and minority students who perceived that they were well-prepared for their graduate courses and accepted by their colleagues (faculty and fellow students), and who experienced well-articulated and structured PhD programs, were most likely to publish at rates comparable to their male majority peers. Women PhD students experienced significantly higher levels of distress than their male peers, both majority and minority, while both women and minority student distress levels were mitigated by clearly-articulated expectations, perceiving that they were well-prepared for graduate level courses, and feeling accepted by their colleagues. It is unclear whether higher levels of distress in women students is related directly to their experiences in their STEM PhD programs. The findings suggest that mitigating factors that negatively affect diversity should not, in principle, require the investment of large resources, but rather requires attention to the local culture and structure of individual STEM PhD programs.
We map the extent, infer the life-cycle length and describe spatial and temporal patterns of flowering of sarmentose bamboos (Guadua spp) in upland forests of the southwest Amazon. We first examine ...the spectra and the spectral separation of forests with different bamboo life stages. False-color composites from orbital sensors going back to 1975 are capable of distinguishing life stages. These woody bamboos flower produce massive quantities of seeds and then die. Life stage is synchronized, forming a single cohort within each population. Bamboo dominates at least 161,500 km(2) of forest, coincident with an area of recent or ongoing tectonic uplift, rapid mechanical erosion and poorly drained soils rich in exchangeable cations. Each bamboo population is confined to a single spatially continuous patch or to a core patch with small outliers. Using spatial congruence between pairs of mature-stage maps from different years, we estimate an average life cycle of 27-28 y. It is now possible to predict exactly where and approximately when new bamboo mortality events will occur. We also map 74 bamboo populations that flowered between 2001 and 2008 over the entire domain of bamboo-dominated forest. Population size averaged 330 km(2). Flowering events of these populations are temporally and/or spatially separated, restricting or preventing gene exchange. Nonetheless, adjacent populations flower closer in time than expected by chance, forming flowering waves. This may be a consequence of allochronic divergence from fewer ancestral populations and suggests a long history of widespread bamboo in the southwest Amazon.
The combination of LiDAR and optical remotely sensed data provides unique information about ecosystem structure and function. Here, we describe the development, validation and application of a new ...airborne system that integrates commercial off the shelf LiDAR hyperspectral and thermal components in a compact, lightweight and portable system. Goddard’s LiDAR, Hyperspectral and Thermal (G-LiHT) airborne imager is a unique system that permits simultaneous measurements of vegetation structure, foliar spectra and surface temperatures at very high spatial resolution (~1 m) on a wide range of airborne platforms. The complementary nature of LiDAR, optical and thermal data provide an analytical framework for the development of new algorithms to map plant species composition, plant functional types, biodiversity, biomass and carbon stocks, and plant growth. In addition, G-LiHT data enhance our ability to validate data from existing satellite missions and support NASA Earth Science research. G-LiHT’s data processing and distribution system is designed to give scientists open access to both low- and high-level data products (http://gliht.gsfc.nasa.gov), which will stimulate the community development of synergistic data fusion algorithms. G-LiHT has been used to collect more than 6,500 km2 of data for NASA-sponsored studies across a broad range of ecoregions in the USA and Mexico. In this paper, we document G-LiHT design considerations, physical specifications, instrument performance and calibration and acquisition parameters. In addition, we describe the data processing system and higher-level data products that are freely distributed under NASA’s Data and Information policy.
Microbially Induced Calcite Precipitation (MICP) is a biomediated soil cementation process that offers an environmentally conscious alternative to conventional geotechnical soil improvement ...technologies. This study provides the first comparison of ureolytic bacteria isolated from sand cemented in parallel, meter-scale, MICP experiments using either biostimulation or bioaugmentation approaches, wherein colonies resembling the augmented strain (Sporosarcina pasteurii ATCC 11859) were interrogated. Over the 13 day experiment, 47 of the 57 isolates collected were strains of Sporosarcina and the diversity of these strains was high, with 20 distinct strains belonging to 5 species identified. Although the S. pasteurii inoculant used for augmentation was recovered immediately after introduction in the augmented specimen, the strain was not recovered after 8 days in either augmented or stimulated soils, suggesting that it competes poorly with indigenous bacteria. Past studies on the physiological properties of S. pasteurii ATCC 11859 suggest that close relatives may have selective advantages under the biogeochemical conditions employed during MICP; however, the extent to which these properties apply to isolates of the current study is unknown. Whole cell urease kinetic properties were investigated for representative isolates and suggest up to 100-fold higher rates of carbonate production when compared to other biomediated processes proposed for MICP.
Microbially Induced Calcite Precipitation (MICP), or bio-cementation, is a promising bio-mediated technology that can improve the engineering properties of soils through the precipitation of calcium ...carbonate. Despite significant advances in the technology, concerns regarding the fate of produced NH
by-products have remained largely unaddressed. In this study, five 3.7-meter long soil columns each containing one of three different soils were improved using ureolytic bio-cementation, and post-treatment NH
by-product removal was investigated during the application of 525 L of a high pH and high ionic strength rinse solution. During rinsing, reductions in aqueous NH
were observed in all columns from initial concentrations between ≈100 mM to 500 mM to final values between ≈0.3 mM and 20 mM with higher NH
concentrations observed at distances furthest from the injection well. In addition, soil V
measurements completed during rinse injections suggested that no significant changes in cementation integrity occurred during NH
removal. After rinsing and a 12 hour stop flow period, all column solutions achieved cumulative NH
removals exceeding 97.9%. Soil samples collected following rinsing, however, contained significant sorbed NH
masses that appeared to have a near linear relationship with surrounding aqueous NH
concentrations. While these results suggest that NH
can be successfully removed from bio-cemented soils, acceptable limits for NH
aqueous concentrations and sorbed NH
masses will likely be governed by site-specific requirements and may require further investigation and refinement of the developed techniques.
Microbially induced calcite precipitation is a biomineralization process with numerous civil engineering and ground improvement applications. In replicate soil columns, the efficacy and microbial ...composition of soil bioaugmented with the ureolytic bacterium Sporosarcina pasteurii were compared to a biostimulation method that enriches native ureolytic soil bacteria in situ under conditions analogous to field implementation. The selective enrichment resulting from sequential stimulation treatments strongly selected for Firmicutes (>97%), with Sporosarcina and Lysinibacillus comprising 60 to 94% of high-throughput 16S rDNA sequences in each suspended community sample. Seven species of the former and two of the latter were present in greater than 10% abundance at different times, demonstrating unexpected within-genus diversity and robustness in the suspended phase of this highly selective environment. Based on longer 16S sequences, it was inferred that augmented S. pasteurii competed poorly with natural bacteria, decreasing to below detection after nine treatments, while the native microbial community was enriched to approximately that present in the stimulated columns. These analyses were corroborated by the observed convergence in bulk ureolytic rates and calcite contents between techniques. However, a 10-fold discrepancy between the observed cell density and an activity-based estimate indicates the attached community, uncharacterized despite efforts, substantially contributes to bulk behavior.
Microbially Induced Calcite Precipitation (MICP) is a bio-mediated cementation process that can improve the engineering properties of granular soils through the precipitation of calcite. The process ...is made possible by soil microorganisms containing urease enzymes, which hydrolyze urea and enable carbonate ions to become available for precipitation. While most researchers have injected non-native ureolytic bacteria to complete bio-cementation, enrichment of native ureolytic microorganisms may enable reductions in process treatment costs and environmental impacts. In this study, a large-scale bio-cementation experiment involving two 1.7-meter diameter tanks and a complementary soil column experiment were performed to investigate biogeochemical differences between bio-cementation mediated by either native or augmented (Sporosarcina pasteurii) ureolytic microorganisms. Although post-treatment distributions of calcite and engineering properties were similar between approaches, the results of this study suggest that significant differences in ureolysis rates and related precipitation rates between native and augmented microbial communities may influence the temporal progression and spatial distribution of bio-cementation, solution biogeochemical changes, and precipitate microstructure. The role of urea hydrolysis in enabling calcite precipitation through sustained super-saturation following treatment injections is explored.
Objective
Zero echo time (ZTE) and ultrashort echo time (UTE) pulse sequences for MRI offer unique advantages of being able to detect signal from rapidly decaying short-T2 tissue components. In this ...paper, we applied 3D ZTE and UTE pulse sequences at 7T to assess differences between these methods.
Materials and methods
We matched the ZTE and UTE pulse sequences closely in terms of readout trajectories and image contrast. Our ZTE used the water- and fat-suppressed solid-state proton projection imaging method to fill the center of k-space. Images from healthy volunteers obtained at 7T were compared qualitatively, as well as with SNR and CNR measurements for various ultrashort, short, and long-T2 tissues.
Results
We measured nearly identical contrast-to-noise and signal-to-noise ratios (CNR/SNR) in similar scan times between the two approaches for ultrashort, short, and long-T2 components in the brain, knee and ankle. In our protocol, we observed gradient fidelity artifacts in UTE, and our chosen flip angle and readout also resulted in shading artifacts in ZTE due to inadvertent spatial selectivity. These can be corrected by advanced reconstruction methods or with different chosen protocol parameters.
Conclusion
The applied ZTE and UTE pulse sequences achieved similar contrast and SNR efficiency for volumetric imaging of ultrashort-T2 components. Key differences include that ZTE is limited to volumetric imaging, but has substantially reduced acoustic noise levels during the scan. Meanwhile, UTE has higher acoustic noise levels and greater sensitivity to gradient fidelity, but offers more flexibility in image contrast and volume selection.
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