CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, noncanonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wildtype NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell typespecific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.Published versio

Synorogenic evolution of large-scale drainage patterns: Isotope paleohydrology of sequential Laramide basins

In the past decade, we and others have compiled an extensive dataset of O, C and Sr isotope stratigraphies from sedimentary basins throughout the Paleogene North American Cordillera. In this study, we present new results from the Piceance Creek Basin of northwest Colorado, which record the evolving hydrology of the Eocene Green River Lake system. We then place the new data in the context of the broader Cordilleran dataset and summarize implications for understanding the synorogenic evolution of large-scale drainage patterns. The combined data reflect (1) a period of throughgoing foreland rivers heading in the Sevier fold-and-thrust belt and flowing east, (2) ponding of freshwater lakes in the foredeep as Laramide uplifts blocked drainage, (3) hydrologic closure that led to both intensive evaporation in the terminal sink of the Piceance Creek Basin and integration of catchments over length-scales \u3e1000 km, (4) infilling of basin accommodation by southward migrating magmatism in distal catchments, leading to the freshening and demise of intraforeland lakes that also stepped south over time

Normalization of prostate specific antigen in patients treated with intensity modulated radiotherapy for clinically localized prostate cancer

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to determine the expected time to prostate specific antigen (PSA) normalization with or without neoadjuvant androgen deprivation (NAAD) therapy after treatment with intensity modulated radiotherapy (IMRT) for patients with clinically localized prostate cancer.</p> <p>Methods</p> <p>A retrospective cohort research design was used. A total of 133 patients with clinical stage T1c to T3b prostate cancer (2002 AJCC staging) treated in a community setting between January 2002 and July 2005 were reviewed for time to PSA normalization using 1 ng/mL and 2 ng/mL as criteria. All patients received IMRT as part of their management. Times to PSA normalization were calculated using the Kaplan-Meier method. Significance was assessed at p < 0.05.</p> <p>Results</p> <p>Fifty-six of the 133 patients received NAAD (42.1%). Thirty-one patients (23.8%) received radiation to a limited pelvic field followed by an IMRT boost, while 99 patients received IMRT alone (76.2%). The times to serum PSA normalization < 2 ng/mL when treated with or without NAAD were 298 ± 24 and 302 ± 33 days (mean ± SEM), respectively (p > 0.05), and 303 ± 24 and 405 ± 46 days, respectively, for PSA < 1 ng/mL (p < 0.05). Stage T1 and T2 tumors had significantly increased time to PSA normalization < 1 ng/mL in comparison to Stage T3 tumors. Also, higher Gleason scores were significantly correlated with a faster time to PSA normalization < 1 ng/mL.</p> <p>Conclusions</p> <p>Use of NAAD in conjunction with IMRT leads to a significantly shortened time to normalization of serum PSA < 1 ng/mL in patients with clinically localized prostate cancer.</p

Ecosystem carbon 7 dioxide fluxes after disturbance in forests of North America

Disturbances are important for renewal of North American forests. Here we summarize more than 180 site years of eddy covariance measurements of carbon dioxide flux made at forest chronosequences in North America. The disturbances included stand-replacing fire (Alaska, Arizona, Manitoba, and Saskatchewan) and harvest (British Columbia, Florida, New Brunswick, Oregon, Quebec, Saskatchewan, and Wisconsin) events, insect infestations (gypsy moth, forest tent caterpillar, and mountain pine beetle), Hurricane Wilma, and silvicultural thinning (Arizona, California, and New Brunswick). Net ecosystem production (NEP) showed a carbon loss from all ecosystems following a stand-replacing disturbance, becoming a carbon sink by 20 years for all ecosystems and by 10 years for most. Maximum carbon losses following disturbance (g C m−2y−1) ranged from 1270 in Florida to 200 in boreal ecosystems. Similarly, for forests less than 100 years old, maximum uptake (g C m−2y−1) was 1180 in Florida mangroves and 210 in boreal ecosystems. More temperate forests had intermediate fluxes. Boreal ecosystems were relatively time invariant after 20 years, whereas western ecosystems tended to increase in carbon gain over time. This was driven mostly by gross photosynthetic production (GPP) because total ecosystem respiration (ER) and heterotrophic respiration were relatively invariant with age. GPP/ER was as low as 0.2 immediately following stand-replacing disturbance reaching a constant value of 1.2 after 20 years. NEP following insect defoliations and silvicultural thinning showed lesser changes than stand-replacing events, with decreases in the year of disturbance followed by rapid recovery. NEP decreased in a mangrove ecosystem following Hurricane Wilma because of a decrease in GPP and an increase in ER

Dark matter halo concentrations in the Wilkinson Microwave Anisotropy Probe year 5 cosmology

We use a combination of three large N-body simulations to investigate the dependence of dark matter halo concentrations on halo mass and redshift in the WMAP year 5 cosmology. The median relation between concentration and mass is adequately described by a power-law for halo masses in the range 10^11 - 10^15 Msol/h and redshifts z < 2, regardless of whether the halo density profiles are fit using NFW or Einasto profiles. Compared with recent analyses of the Millennium Simulation, which uses a value of sigma_8 that is higher than allowed by WMAP5, z = 0 halo concentrations are reduced by factors ranging from 23 per cent at 10^11 Msol/h to 16 per cent at 10^14 Msol/h. The predicted concentrations are much lower than inferred from X-ray observations of groups and clusters.Comment: 6 pages, 4 figures, Accepted by MNRAS letters. Version 4: Typo fixe

Rapid Heterotrophic Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-Expressing MSCs

Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving the viability and functionality of PEG-encapsulated BMP2-transduced MSCs

Ice-lens formation and geometrical supercooling in soils and other colloidal materials

We present a new, physically-intuitive model of ice-lens formation and growth during the freezing of soils and other dense, particulate suspensions. Motivated by experimental evidence, we consider the growth of an ice-filled crack in a freezing soil. At low temperatures, ice in the crack exerts large pressures on the crack walls that will eventually cause the crack to split open. We show that the crack will then propagate across the soil to form a new lens. The process is controlled by two factors: the cohesion of the soil, and the geometrical supercooling of the water in the soil; a new concept introduced to measure the energy available to form a new ice lens. When the supercooling exceeds a critical amount (proportional to the cohesive strength of the soil) a new ice lens forms. This condition for ice-lens formation and growth does not appeal to any ad hoc, empirical assumptions, and explains how periodic ice lenses can form with or without the presence of a frozen fringe. The proposed mechanism is in good agreement with experiments, in particular explaining ice-lens pattern formation, and surges in heave rate associated with the growth of new lenses. Importantly for systems with no frozen fringe, ice-lens formation and frost heave can be predicted given only the unfrozen properties of the soil. We use our theory to estimate ice-lens growth temperatures obtaining quantitative agreement with the limited experimental data that is currently available. Finally we suggest experiments that might be performed in order to verify this theory in more detail. The theory is generalizable to complex natural-soil scenarios, and should therefore be useful in the prediction of macroscopic frost heave rates.Comment: Submitted to PR

From Dose to Response: In Vivo Nanoparticle Processing and Potential Toxicity

Adverse human health impacts due to occupational and environmental exposures to manufactured nanoparticles are of concern and pose a potential threat to the continued industrial use and integration of nanomaterials into commercial products. This chapter addresses the inter-relationship between dose and response and will elucidate on how the dynamic chemical and physical transformation and breakdown of the nanoparticles at the cellular and subcellular levels can lead to the in vivo formation of new reaction products. The dose-response relationship is complicated by the continuous physicochemical transformations in the nanoparticles induced by the dynamics of the biological system, where dose, bio-processing, and response are related in a non-linear manner. Nanoscale alterations are monitored using high-resolution imaging combined with in situ elemental analysis and emphasis is placed on the importance of the precision of characterization. The result is an in-depth understanding of the starting particles, the particle transformation in a biological environment, and the physiological response