Efficient targeted DNA editing and replacement in <i>Chlamydomonas reinhardtii</i> using Cpf1 ribonucleoproteins and single-stranded DNA

Significance Our findings establish a method of efficient, targeted genome editing in Chlamydomonas reinhardtii . We demonstrate an approach to bypass inefficient gene targeting via homologous recombination and achieve homology-directed DNA replacement in C. reinhardtii . In addition, we report CRISPR/Cpf1-mediated DNA editing efficiencies being boosted 500-fold through the use of single-stranded oligodeoxynucleotides (ssODNs) as repair templates. It remains to be determined whether Cpf1-induced staggered DNA cleavage enhances ssODN-mediated gene editing in a wider range of species and whether the underlying repair pathway(s) responsible is more broadly conserved. </jats:p

The comparative evaluation of ERTS-1 imagery for resource inventory in land use planning

The author has identified the following significant results. Multidiscipline team interpretation and mapping of resources for Crook County is nearly complete on 1:250,000 scale enlargements of ERTS-1 imagery. Maps of geology, landforms, soils and vegetation-land use are being interpreted to show limitations, suitabilities and geologic hazards for land use planning. Mapping of lineaments and structures from ERTS-1 imagery has shown a number of features not previously mapped in Oregon. A timber inventory of Ochoco National Forest has been made. Inventory of forest clear-cutting practices has been successfully demonstrated with ERTS-1 color composites. Soil tonal differences in fallow fields shown on ERTS-1 correspond with major soil boundaries in loess-mantled terrain. A digital classification system used for discriminating natural vegetation and geologic materials classes has been successful in separation of most major classes around Newberry Cauldera, Mt. Washington and Big Summit Prairie. Computer routines are available for correction of scanner data variations; and for matching scales and coordinates between digital and photographic imagery. Methods of Diazo film color printing of computer classifications and elevation-slope perspective plots with computer are being developed

The comparative evaluation of ERTS-1 imagery for resource inventory in land use planning

The author has identified the following significant results. Multidiscipline team interpretation and mapping of resources for Crook County is complete on 1:250,000 scale enlargements of ERTS imagery and 1:120,000 hi-flight photography. Maps of geology, soils, vegetation-land use and land resources units were interpreted to show limitations, suitabilities, and geologic hazards for land use planning. Mapping of lineaments and structures from ERTS imagery has shown a number of features not previously mapped in Oregon. A multistage timber inventory of Ochoco National Forest was made, using ERTS images as the first stage. Inventory of forest clear-cutting practices was successfully demonstrated with color composites. Soil tonal differences in fallow fields correspond with major soil boundaries in loess-mantled terrain. A digital classification system used for discriminating natural vegetation and geologic material classes was successful in separating most major classes around Newberry Caldera, Mt. Washington, and Big Summit Prairie

Tennis Anyone? Teaching Experimental Design by Designing and Executing a Tennis Ball Experiment

Understanding the abstract principles of statistical experimental design can challenge undergraduate students, especially when learned in a lecture setting. This article presents a concrete and easily replicated example of experimental design principles in action through a hands-on learning activity for students enrolled in an experimental design course. The activity, conducted during five 50-min classes, requires the students to work as a team to design and execute a simple and safe factorial experiment and collect and analyze the data. During three in-class design meetings, the students design and plan all aspects of the experiment, including choosing the response variable and factors, making a list of needed supplies, creating a randomized run schedule with the MINITAB DOE utility, and writing a statistical analysis plan. A feasibility study is conducted in the fourth class. During the fifth and last class, the students conduct the experiment. Each student writes a lab report including all background research, methods, analyses, and conclusions, as well as a reflection on the learning experience. Students’ reflections indicate the active-learning experience was enjoyable and boosted course engagement and perceived conceptual understanding

Sodium-activated potassium channels shape peripheral auditory function and activity of the primary auditory neurons in mice

Potassium (K+) channels shape the response properties of neurons. Although enormous progress has been made to characterize K+ channels in the primary auditory neurons, the molecular identities of many of these channels and their contributions to hearing in vivo remain unknown. Using a combination of RNA sequencing and single molecule fluorescent in situ hybridization, we localized expression of transcripts encoding the sodium-activated potassium channels K(Na)1.1(SLO2.2/Slack) and K(Na)1.2 (SLO2.1/Slick) to the primary auditory neurons (spiral ganglion neurons, SGNs). To examine the contribution of these channels to function of the SGNs in vivo, we measured auditory brainstem responses in K(Na)1.1/1.2 double knockout (DKO) mice. Although auditory brainstem response (wave I) thresholds were not altered, the amplitudes of suprathreshold responses were reduced in DKO mice. This reduction in amplitude occurred despite normal numbers and molecular architecture of the SGNs and their synapses with the inner hair cells. Patch clamp electrophysiology of SGNs isolated from DKO mice displayed altered membrane properties, including reduced action potential thresholds and amplitudes. These findings show that K(Na)1 channel activity is essential for normal cochlear function and suggest that early forms of hearing loss may result from physiological changes in the activity of the primary auditory neurons

Studies in Natural Product Chemistry

The first section of this thesis is concerned with a biogenetic-type approach to the synthesis of the aconitine-lycoctonine group of diterpene alkaloids. The synthetic precursor, atisine, was transformed in an eleven step sequence into a keto-tosylate. This material underwent a novel, stereospecific pyrolytic rearrangement to give a key intermediate in the proposed atisane-aconane biogenesis, whose constitution and stereochemistry were confirmed by anx-ray crystallographic analysis, conducted on a heavy atom derivative. Our efforts to convert this intermediate into the desired aconitine-lycoctonine skeleton, a task which had already been accomplished in principle by other workers, met with limited success. N. M. R. studies on some acetamides, obtained in the foregoing synthesis, revealed an interesting example of restricted rotation around the bond. Variable temperature work enabled a crude barrier to rotation to be extracted. The second section of the thesis, also in the realm of diterpenes, concerns the synthesis of the cassane skeleton, in a biogenetically-patterned fashion, from isopimaric acid. The route from isopimaric acid to an important intermediate enone is described. Despite numerous attempts,we could not induce the Wagner-Meerwein rearrangement in this enone,which would have resulted in the desired cassane skeleton

03. David E.I. Pyott: Letter of Support

UCI Law letter of support from David E.I. Pyott of Allergan

Potential for gene editing in antiviral resistance

The discovery of CRISPR/Cas systems and their subsequent application in genome modifications and in gene expression control have fundamentally changed both basic and applied research. They have already been employed to generate novel virus resistance traits either by modifying host factors in the plant genome or by directly inducing targeted virus degradation. Here we summarise the latest developments in this field and discuss the potential applications and concerns around this technology

Temperature modulates virus-induced transcriptional gene silencing via secondary small RNAs

Virus-induced gene silencing (VIGS) can be harnessed to sequence-specifically degrade host transcripts and induce heritable epigenetic modifications referred to as virus-induced posttranscriptional gene silencing (ViPTGS) and virus-induced transcriptional gene silencing (ViTGS), respectively. Both ViPTGS and ViTGS enable manipulation of endogenous gene expression without the need for transgenesis. Although VIGS has been widely used in many plant species, it is not always uniform or highly efficient. The efficiency of VIGS is affected by developmental, physiological and environmental factors. Here, we use recombinant tobacco rattle viruses (TRV) to study the effect of temperature on ViPTGS and ViTGS using GFP as a reporter gene of silencing in N. benthamiana 16c plants.We found that unlike ViPTGS, ViTGS was impaired at high temperature. Using a novel mismatch-siRNA tool, which precisely distinguishes virus-derived (primary) from target-generated (secondary) siRNAs, we demonstrated that the lack of secondary siRNA production/amplification was responsible for inefficient ViTGS at 29°C. Moreover, inefficient ViTGS at 29°C inhibited the transmission of epigenetic gene silencing to the subsequent generations.Our finding contributes to understanding the impact of environmental conditions on primary and secondary siRNA production and may pave the way to design/optimise ViTGS for transgene-free crop improvement. <br/