Non-viral in vivo cytidine base editing in hepatocytes using focused ultrasound targeted microbubbles.

CRISPR-Cas9-based genome editing technologies, such as base editing, have the potential for clinical translation, but delivering nucleic acids into target cells in vivo is a major obstacle. Viral vectors are widely used but come with safety concerns, while current non-viral methods are limited by low transfection efficiency. Here we describe a new method to deliver CRISPR-Cas9 base editing vectors to the mouse liver using focused ultrasound targeted microbubble destruction (FUTMD). We demonstrate, using the example of cytosine base editing of the Pde3b gene, that FUTMD-mediated delivery of cytosine base editing vectors can introduce stop codons (up to ∼2.5% on-target editing) in mouse liver cells in vivo. However, base editing specificity is less than one might hope with these DNA constructs. Our findings suggest that FUTMD-based gene editing tools can be rapidly and transiently deployed to specific organs and sites, providing a powerful platform for the development of non-viral genome editing therapies. Non-viral delivery also reveals greater off-target base exchange in vivo than in vitro.

Large interannual variability in supraglacial lakes around East Antarctica.

Antarctic supraglacial lakes (SGLs) have been linked to ice shelf collapse and the subsequent acceleration of inland ice flow, but observations of SGLs remain relatively scarce and their interannual variability is largely unknown. This makes it difficult to assess whether some ice shelves are close to thresholds of stability under climate warming. Here, we present the first observations of SGLs across the entire East Antarctic Ice Sheet over multiple melt seasons (2014-2020). Interannual variability in SGL volume is >200% on some ice shelves, but patterns are highly asynchronous. More extensive, deeper SGLs correlate with higher summer (December-January-February) air temperatures, but comparisons with modelled melt and runoff are complex. However, we find that modelled January melt and the ratio of November firn air content to summer melt are important predictors of SGL volume on some potentially vulnerable ice shelves, suggesting large increases in SGLs should be expected under future atmospheric warming.

Dissolution and transport of insensitive munitions formulations IMX-101 and IMX-104 in saturated soil columns.

Military training exercises can result in deposition of energetic residues on range soils, which ultimately can contaminate groundwater with munitions constituents. Column experiments followed by HYDRUS-1D modeling were conducted to evaluate dissolution and transport of energetic constituents from the new insensitive munitions (IM) formulations IMX-101, a mixture of 3-nitro-1,2,4-triazol-5-one (NTO), nitroguanidine (NQ), and 2, 4-dinitroanisole (DNAN), and IMX-104, a mixture of NTO, 1,3,5-hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and DNAN. NTO and DNAN are emerging contaminants associated with the development of insensitive munitions as replacements for traditional munitions. Flow interruption experiments were performed to investigate dissolution kinetics and sorption non-equilibrium between soil and solution phases. The results indicated that insensitive munitions compounds dissolved in order of their aqueous solubility, consistent with prior dissolution studies conducted in the absence of soil. Initial elution of the high concentration pulse of highly soluble NTO and NQ was followed by lower concentrations, while DNAN had generally lower and more constant concentrations in leachate. The sorption of NTO and NQ was low, while RDX, 1,3,5,7-octahydro-1,3,5,7-tetranitrotetrazocine (HMX, an impurity in technical grade RDX), and DNAN all exhibited appreciable sorption. DNAN transformation was observed, with formation of amino-reduction products 2-ANAN (2-amino-4-nitroanisole) and 4-ANAN (4-amino-2-nitroanisole). HYDRUS-1D model, incorporating one-dimensional advective-dispersive transport with particle dissolution and first-order solute transformation was used to simulate the measured breakthrough curves. Optimized dissolution parameters varied widely but were correlated between compounds in the same formulation. Determined adsorption coefficients generally agreed with values determined from batch and column studies conducted with pure NTO and DNAN, while mass-loss rate coefficients were in better agreement with ones from batch than column studies possibly due to suppression of microbial transformation during elution of high concentrations of explosives. Even in the low organic matter soils selected in this study DNAN experienced significant retardation and transformation, indicating potential for its natural attenuation.

Batch soil adsorption and column transport studies of 2,4-dinitroanisole (DNAN) in soils.

The explosive 2,4,6-trinitrotoluene (TNT) is currently a main ingredient in munitions; however the compound has failed to meet the new sensitivity requirements. The replacement compound being tested is 2,4-dinitroanisole (DNAN). DNAN is less sensitive to shock, high temperatures, and has good detonation characteristics. However, DNAN is more soluble than TNT, which can influence transport and fate behavior and thus bioavailability and human exposure potential. The objective of this study was to investigate the environmental fate and transport of DNAN in soil, with specific focus on sorption processes. Batch and column experiments were conducted using soils collected from military installations located across the United States. The soils were characterized for pH, electrical conductivity, specific surface area, cation exchange capacity, and organic carbon content. In the batch rate studies, change in DNAN concentration with time was evaluated using the first order equation, while adsorption isotherms were fitted using linear and Freundlich equations. Solution mass-loss rate coefficients ranged between 0.0002h-1 and 0.0068h-1. DNAN was strongly adsorbed by soils with linear adsorption coefficients ranging between 0.6 and 6.3Lg-1, and Freundlich coefficients between 1.3 and 34mg1-nLnkg-1. Both linear and Freundlich adsorption coefficients were positively correlated with the amount of organic carbon and cation exchange capacity of the soil, indicating that similar to TNT, organic matter and clay minerals may influence adsorption of DNAN. The results of the miscible-displacement column experiments confirmed the impact of sorption on retardation of DNAN during transport. It was also shown that under flow conditions DNAN transforms readily with formation of amino transformation products, 2-ANAN and 4-ANAN. The magnitudes of retardation and transformation observed in this study result in significant attenuation potential for DNAN, which would be anticipated to contribute to a reduced risk for contamination of ground water from soil residues.

Day Traders in South Australia: Similarities and Differences with Traditional Gamblers.

The present study investigated the relationship between engaging in day trading and engaging in traditional forms of gambling in South Australia. Consistent with a previous study on this issue, it would appear that most individuals who engage in day trading are heavily involved traditional gamblers who include day trading in their repertoire of activities. They differ somewhat from most gamblers in their strong preference for skill-based types of gambling, their higher overall involvement in gambling, and their higher rates of problem gambling. They also have some demographic differences, in particular, being older and having higher incomes. The present findings provide further evidence that not only do certain types of financial speculation bear some conceptual similarity to gambling, they also appear to be empirically related.

Column transport studies of 3-nitro-1,2,4-triazol-5-one (NTO) in soils.

Development of the new, insensitive, energetic compound, NTO (3-nitro-1,2,4-triazol-5-one), creates need for the data on NTO's fate and transport to predict its behavior in the environment and potential for groundwater contamination. To measure the transport of NTO in soils, we conducted miscible-displacement experiments under steady state and interrupted flow conditions using eight soils having varying physical and geochemical properties. The breakthrough curve (BTC) data were analyzed using temporal moment analysis and simulated using HYDRUS-1D to determine transport parameters and better understand the mechanisms of sorption and transformation. Parameters determined from the miscible-displacement study were compared to results obtained from batch experiments conducted for the same soils, and examined in relation to soil properties. Column NTO linear adsorption coefficients (Kd) were low and correlated well (P = 0.000049) with measurements from the batch studies. NTO transformation rate constants increased and NTO recovery decreased with increase in soil organic carbon (OC) content. Autoclaved soils had slower transformation rates and greater NTO recoveries indicating that microorganisms play a role in NTO transformation. In addition, the transformation rate increased with time in soils with higher OC. Monod-type kinetics was implemented in HYDRUS-1D to simulate the observed increase in transformation rate with time. We think this phenomenon is due to bacterial growth. Results indicate very low adsorption of NTO in a range of soils, but natural attenuation through transformation that, depending on soil OC content and hydraulic residence time, could result in complete removal of NTO.

The conceptual and empirical relationship between gambling, investing, and speculation.

Background and aims To review the conceptual and empirical relationship between gambling, investing, and speculation. Methods An analysis of the attributes differentiating these constructs as well as identification of all articles speaking to their empirical relationship. Results Gambling differs from investment on many different attributes and should be seen as conceptually distinct. On the other hand, speculation is conceptually intermediate between gambling and investment, with a few of its attributes being investment-like, some of its attributes being gambling-like, and several of its attributes being neither clearly gambling or investment-like. Empirically, gamblers, investors, and speculators have similar cognitive, motivational, and personality attributes, with this relationship being particularly strong for gambling and speculation. Population levels of gambling activity also tend to be correlated with population level of financial speculation. At an individual level, speculation has a particularly strong empirical relationship to gambling, as speculators appear to be heavily involved in traditional forms of gambling and problematic speculation is strongly correlated with problematic gambling. Discussion and conclusions Investment is distinct from gambling, but speculation and gambling have conceptual overlap and a strong empirical relationship. It is recommended that financial speculation be routinely included when assessing gambling involvement, and there needs to be greater recognition and study of financial speculation as both a contributor to problem gambling as well as an additional form of behavioral addiction in its own right.

Adsorption and attenuation behavior of 3-nitro-1,2,4-triazol-5-one (NTO) in eleven soils.

NTO (3-nitro-1,2,4-triazol-5-one) is one of the new explosive compounds used in insensitive munitions (IM) developed to replace traditional explosives, TNT and RDX. Data on NTO fate and transport is needed to determine its environmental behavior and potential for groundwater contamination. We conducted a series of kinetic and equilibrium batch experiments to characterize the fate of NTO in soils and the effect of soil geochemical properties on NTO-soil interactions. A set of experiments was also conducted using sterilized soils to evaluate the contribution of biodegradation to NTO attenuation. Measured pH values for NTO solutions decreased from 5.98 ± 0.13 to 3.50 ± 0.06 with increase in NTO concentration from 0.78 to 100 mg L(-1). Conversely, the pH of soil suspensions was not significantly affected by NTO in this concentration range. NTO experienced minimal adsorption, with measured adsorption coefficients being less than 1 cm(3) g(-1) for all studied soils. There was a highly significant inverse relationship between the measured NTO adsorption coefficients and soil pH (P = 0.00011), indicating the role of NTO and soil charge in adsorption processes. In kinetic experiments, 1st order transformation rate constant estimates ranged between 0.0004 h(-1) and 0.0142 h(-1) (equivalent to half-lives of 72 and 2 d, respectively), and correlated positively with organic carbon in the soil. Total attenuation of NTO was higher in untreated versus sterilized samples, suggesting that NTO was being biodegraded. The information presented herein can be used to help evaluate NTO potential for natural attenuation in soils.

Salted and preserved duck eggs: a consumer market segmentation analysis.

The combination of increasing ethnic diversity in North America and growing consumer support for local food products may present opportunities for local producers and processors in the ethnic foods product category. Our study examined the ethnic Chinese (pop. 402,000) market for salted and preserved duck eggs in Vancouver, British Columbia (BC), Canada. The objective of the study was to develop a segmentation model using survey data to categorize consumer groups based on their attitudes and the importance they placed on product attributes. We further used post-segmentation acculturation score, demographics and buyer behaviors to define these groups. Data were gathered via a survey of randomly selected Vancouver households with Chinese surnames (n = 410), targeting the adult responsible for grocery shopping. Results from principal component analysis and a 2-step cluster analysis suggest the existence of 4 market segments, described as Enthusiasts, Potentialists, Pragmatists, Health Skeptics (salted duck eggs), and Neutralists (preserved duck eggs). Kruskal Wallis tests and post hoc Mann-Whitney tests found significant differences between segments in terms of attitudes and the importance placed on product characteristics. Health Skeptics, preserved egg Potentialists, and Pragmatists of both egg products were significantly biased against Chinese imports compared to others. Except for Enthusiasts, segments disagreed that eggs are 'Healthy Products'. Preserved egg Enthusiasts had a significantly lower acculturation score (AS) compared to all others, while salted egg Enthusiasts had a lower AS compared to Health Skeptics. All segments rated "produced in BC, not mainland China" products in the "neutral to very likely" range for increasing their satisfaction with the eggs. Results also indicate that buyers of each egg type are willing to pay an average premium of at least 10% more for BC produced products versus imports, with all other characteristics equal. Overall results indicate that opportunities exist for local producers and processors: Chinese Canadians with lower AS form a core part of the potential market.

Survival times for canine intranasal sarcomas treated with radiation therapy: 86 cases (1996-2011).

Sarcomas comprise approximately one-third of canine intranasal tumors, however few veterinary studies have described survival times of dogs with histologic subtypes of sarcomas separately from other intranasal tumors. One objective of this study was to describe median survival times for dogs treated with radiation therapy for intranasal sarcomas. A second objective was to compare survival times for dogs treated with three radiation therapy protocols: daily-fractionated radiation therapy; Monday, Wednesday, and Friday fractionated radiation therapy; and palliative radiation therapy. Medical records were retrospectively reviewed for dogs that had been treated with radiation therapy for confirmed intranasal sarcoma. A total of 86 dogs met inclusion criteria. Overall median survival time for included dogs was 444 days. Median survival time for dogs with chondrosarcoma (n = 42) was 463 days, fibrosarcoma (n = 12) 379 days, osteosarcoma (n = 6) 624 days, and undifferentiated sarcoma (n = 22) 344 days. Dogs treated with daily-fractionated radiation therapy protocols; Monday, Wednesday and Friday fractionated radiation therapy protocols; and palliative radiation therapy protocols had median survival times of 641, 347, and 305 days, respectively. A significant difference in survival time was found for dogs receiving curative intent radiation therapy vs. palliative radiation therapy (P = 0.032). A significant difference in survival time was also found for dogs receiving daily-fractionated radiation therapy vs. Monday, Wednesday and Friday fractionated radiation therapy (P = 0.0134). Findings from this study support the use of curative intent radiation therapy for dogs with intranasal sarcoma. Future prospective, randomized trials are needed for confirmation of treatment benefits.

Characterization of normal tissue complications in 51 dogs undergoing definitive pelvic region irradiation.

Our objective was to further characterize the late normal tissue complications developing after definitive irradiation of pelvic region tumors in dogs, and to search for prognostic factors. The medical records of dogs receiving definitive irradiation of the pelvic region between 1987 and 2005 were reviewed. The following criteria were established for inclusion: total dose > or =45 Gy, a portion of colon in the primary field, and a minimum of 6 months follow-up. Fifty-one dogs were identified. Prognostic factors evaluated included multiple descriptors of the patient, tumor and radiation treatment. One or more late complications were documented in 20 of 51 patients (39%). Complications were necrotic drainage/ulceration in the skin and subcutaneous tissues within the radiation field (n=7), chronic colitis (n=4), strictures (n=4), osteopenia (n=2), and one each rectal perforation, urinary bladder thickening, iliosacral osteosarcoma, pelvic limb edema, and perianal pain. Two prognostic factors were identified. There was an increase in complications in dogs with perineal tumors compared with other pelvic region sites (P = 0.04), and also in dogs with larger radiation fields (P = 0.04). The finding of an association of tumor site to complications may be a spurious finding and the association between field size and complications is not unexpected although absolute difference in field size between dogs with and without complications was small. There was no association between development of complications and survival. Based on the observed complication rate, consideration can be given to reducing dose per fraction in dogs receiving definitive pelvic region irradiation to <3 Gy.

Syngeneic Schwann cell transplantation preserves vision in RCS rat without immunosuppression.

PURPOSE: To evaluate the efficacy of immunologically compatible Schwann cells transplanted without immunosuppression in the RCS rat retina to preserve vision. METHODS: Syngeneic (dystrophic RCS) Schwann cells harvested from sciatic nerves were cultured and transplanted into one eye of dystrophic RCS rats at an early stage of retinal degeneration. Allogeneic (Long-Evans) Schwann cells and unoperated eyes served as controls. Vision through transplanted and unoperated eyes was then quantified using two visual behavior tasks, one measuring the spatial frequency and contrast sensitivity thresholds of the optokinetic response (OKR) and the other measuring grating acuity in a perception task. RESULTS: Spatial frequency thresholds measured through syngeneically transplanted eyes maintained near normal spatial frequency sensitivity for approximately 30 weeks, whereas thresholds through control eyes deteriorated to less than 20% of normal over the same period. Contrast sensitivity was preserved through syngeneically transplanted eyes better than through allogeneic and unoperated eyes, at all spatial frequencies. Grating acuity measured through syngeneically transplanted eyes was maintained at approximately 60% of normal, whereas acuity of allogeneically transplanted eyes was significantly lower at approximately 40% of normal. CONCLUSIONS: The ability of immunoprivileged Schwann cell transplants to preserve vision in RCS rats indicates that transplantation of syngeneic Schwann cells holds promise as a preventive treatment for retinal degenerative disease.