Robust and stable transcriptional repression in Giardia using CRISPRi.

Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems

Hydrological impacts of floodplain restoration: a case study of the River Cherwell, UK

International audienceThis paper investigates the impacts on floods of hypothetical changes to river channel geometry by construction or removal of embankments to prevent water spreading onto the floodplain at high flows. A numerical model is applied to the River Cherwell between Oxford and Banbury to simulate changes to flood hydrographs. Embanking the river increases the peak flows downstream by 50-150%. Restoring the river channel through the floodplain to pre-engineered dimensions reduces peak flow by around 10-15% and increases peak water levels within the floodplain by 0.5-1.6 m. These results suggest that floodplain rehabilitation, in terms of embankment removal or returning the channel to pre-engineered dimensions, can be a valuable part of the flood management strategy of a catchment. Both measures lead to increased inundation of the floodplain, which can be positive for ecological restoration. Keywords: floodplains, hydrological impacts, rehabilitation, flood frequenc

Developing a cultural competence assessment tool for people in recovery from racial, ethnic and cultural backgrounds: the journey, challenges and lessons learned.

In 1997, Maryland implemented a new managed care mental health system. Consumer satisfaction, evaluation and cultural competency were considered high priorities for the new system. While standardized tools for measuring consumer satisfaction were readily available, no validated, reliable and standardized tool existed to measure the perception of people from minority groups receiving mental health services. The MHA*/MHP* Cultural Competency Advisory Group (CCAG) accepted the challenge of developing a consumer assessment tool for cultural competency. The CCAG, composed of people in recovery, clinicians and administrators used their collective knowledge and experiences to develop a 52-item tool that met standards for validity and reliability. Consultation from a researcher helped to further develop the tool into one possessing tremendous potential for statewide implementation within Maryland's Public Mental Health System. Recognizing the limitations of the study and the need for further research, this instrument is a work in progress. Strategies to improve the instrument are currently underway with the Mental Hygiene Administration's Systems Evaluation Center of the University of Maryland and several national researchers

Verification of Space Station Secondary Power System Stability Using Design of Experiment

This paper describes analytical methods used in verification of large DC power systems with applications to the International Space Station (ISS). Large DC power systems contain many switching power converters with negative resistor characteristics. The ISS power system presents numerous challenges with respect to system stability such as complex sources and undefined loads. The Space Station program has developed impedance specifications for sources and loads. The overall approach to system stability consists of specific hardware requirements coupled with extensive system analysis and testing. Testing of large complex distributed power systems is not practical due to size and complexity of the system. Computer modeling has been extensively used to develop hardware specifications as well as to identify system configurations for lab testing. The statistical method of Design of Experiments (DoE) is used as an analysis tool for verification of these large systems. DOE reduces the number of computer runs which are necessary to analyze the performance of a complex power system consisting of hundreds of DC/DC converters. DoE also provides valuable information about the effect of changes in system parameters on the performance of the system. DoE provides information about various operating scenarios and identification of the ones with potential for instability. In this paper we will describe how we have used computer modeling to analyze a large DC power system. A brief description of DoE is given. Examples using applications of DoE to analysis and verification of the ISS power system are provided

Polyploidy breaks speciation barriers in Australian burrowing frogs Neobatrachus

Polyploidy has played an important role in evolution across the tree of life but it is still unclear how polyploid lineages may persist after their initial formation. While both common and well-studied in plants, polyploidy is rare in animals and generally less understood. The Australian burrowing frog genus Neobatrachus is comprised of six diploid and three polyploid species and offers a powerful animal polyploid model system. We generated exome-capture sequence data from 87 individuals representing all nine species of Neobatrachus to investigate species-level relationships, the origin and inheritance mode of polyploid species, and the population genomic effects of polyploidy on genus-wide demography. We describe rapid speciation of diploid Neobatrachus species and show that the three independently originated polyploid species have tetrasomic or mixed inheritance. We document higher genetic diversity in tetraploids, resulting from widespread gene flow between the tetraploids, asymmetric inter-ploidy gene flow directed from sympatric diploids to tetraploids, and isolation of diploid species from each other. We also constructed models of ecologically suitable areas for each species to investigate the impact of climate on differing ploidy levels. These models suggest substantial change in suitable areas compared to past climate, which correspond to population genomic estimates of demographic histories. We propose that Neobatrachus diploids may be suffering the early genomic impacts of climate-induced habitat loss, while tetraploids appear to be avoiding this fate, possibly due to widespread gene flow. Finally, we demonstrate that Neobatrachus is an attractive model to study the effects of ploidy on the evolution of adaptation in animals

Activation energy for fluorine transport in amorphous silicon

The transport of ion implanted F in amorphous Si is studied using secondary ion mass spectroscopy and transmission electron microscopy. Significant redistribution of F is observed at temperatures in the range 600°C to 700°C. The measured F depth-profiles are modelled using a simple Gaussian solution to the diffusion equation, and the diffusion coefficient is deduced at each temperature. An activation energy of 2.2eV±0.4eV for F transport is extracted from an Arrhenius plot of the diffusion coefficients. It is shown that the F transport is influenced by implantation induced defects

Bioenergy II: Characterization of the Pesticide Properties of Tobacco Bio-Oil

Pyrolysis converts biomass such as agricultural and forestry waste into bio-oil, preserving some chemicals while creating other, new ones. Nicotine, a chemical present in tobacco leaves and a known pesticide, was found to remain intact during pyrolysis. As expected, insecticidal properties were observed for tobacco bio-oil. Pesticide characteristics of tobacco bio-oil have been observed on the Colorado potato beetle (CPB), a pest currently resistant to all major insecticides, as well as a few bacteria and fungi that do not currently respond well to chemical treatment. Unexpectedly, nicotine-free fractions of the bio-oil were also found to be highly lethal to the beetles and successful at inhibiting the growth of select microorganisms. Through GC-MS, it was found that the active, nicotine-free fractions were rich in phenolics, chemicals likely created from lignin during pyrolysis. While bio-oils in general are known to contain phenolic chemicals, such as cresols, to our best knowledge, quantitative analysis has not been performed to determine if these chemicals are solely responsible for the observed pesticide activities. Based on GC-MS results, ten of the most abundant chemicals, eight of which were phenolic chemicals, were identified and examined through bio-assays. A mixture of these chemicals at the concentration levels found in the bio-oil did not account for the bio-oil activity towards the microorganisms. Tobacco bio-oil may have potential as a pesticide, however, further analyses using liquid chromatography is necessary to identify the remaining active chemicals

Characterization of the Pesticide Properties of Tobacco Bio-oil

Pyrolysis converts biomass such as agricultural and forestry waste into bio-oil. Our interest in the chemical analysis of bio-oil began with tobacco, which is rich in nicotine (a known pesticide). Initial inhibition assays performed with the bio-oil on the Colorado potato beetle, a pest currently resistant to all major insecticides, showed high pesticide activity as expected. Surprisingly, the nicotine-free phases of the bio-oil were also found to be highly lethal to the beetles. Thus, it was hypothesized that some of the alkaloids in plants were preserved during pyrolysis, and gave rise to the activity. Pesticide characteristics of tobacco and coffee bio-oils have been recorded on a number of insects as well as a variety of bacteria and fungi that do not currently respond well to chemical treatment; e.g., Streptomyces Scabies (a common potato scab disease). The current focus is to isolate and identify the components responsible for the pest inhibition, and in turn fully characterize their properties as a novel source of natural pesticides. The procedure begins with a crude separation or fractionation by distillation or extraction to simplify the chemical composition. The fractions are then screened by the activity assay. Analytical separation and mass spectral detection (GC-MS and LC-MS) are then used to generate chemical fingerprints for comparative analysis against libraries of known compounds to identify the active component(s). A mixture of chemical standards is generated from these identified, potentially active, components. This mixture is tested by the activity assay, and chemicals are sequentially removed from this mixture to identify the active components and potential synergistic effects between these components. Thus, a potential pesticide originating from agriculturally-based bio-oil is identified