High efficiency In Vivo genome engineering with a simplified 15-RVD GoldyTALEN design

published_or_final_versio

Functional conservation of a forebrain enhancer from the elephant shark (Callorhinchus milii) in zebrafish and mice

Background: The phylogenetic position of the elephant shark (Callorhinchus milii ) is particularly relevant to study the evolution of genes and gene regulation in vertebrates. Here we examine the evolution of Dlx homeobox gene regulation during vertebrate embryonic development with a particular focus on the forebrain. We first identified the elephant shark sequence orthologous to the URE2 cis -regulatory element of the mouse Dlx1/Dlx2 locus (herein named CmURE2). We then conducted a comparative study of the sequence and enhancer activity of CmURE2 with that of orthologous regulatory sequences from zebrafish and mouse. Results: The CmURE2 sequence shows a high percentage of identity with its mouse and zebrafish counterparts but is overall more similar to mouse URE2 (MmURE2) than to zebrafish URE2 (DrURE2). In transgenic zebrafish and mouse embryos, CmURE2 displayed enhancer activity in the forebrain that overlapped with that of DrURE2 and MmURE2. However, we detected notable differences in the activity of the three sequences in the diencephalon. Outside of the forebrain, CmURE2 shows enhancer activity in areas such as the pharyngeal arches and dorsal root ganglia where its' counterparts are also active. Conclusions: Our transgenic assays show that part of the URE2 enhancer activity is conserved throughout jawed vertebrates but also that new characteristics have evolved in the different groups. Our study demonstrates that the elephant shark is a useful outgroup to study the evolution of regulatory mechanisms in vertebrates and to address how changes in the sequence of cis -regulatory elements translate into changes in their regulatory activity

Clinical outcomes of combined transepithelial phototherapeutic keratectomy and conventional corneal collagen crosslinking (t-PTK+CXL) versus conventional corneal collagen crosslinking (CXL) for progressive keratoconus at St. Olavs Hospital, Trondheim University Hospital: A retrospective comparative study

Purpose: To compare the outcomes of corneal collagen cross-linking (CXL) for the treatment of progressive keratoconus using 2 different techniques for epithelial removal: transepithelial phototherapeutic keratectomy (t-PTK) + CXL versus mechanical epithelial debridement. Design: retrospective comparison study. Participants: Two hundred and twenty patients (298 eyes) with progressive keratoconus were included. Method: All patients underwent uncomplicated CXL treatment. Hundred and ten patients (155 eyes) underwent epithelial removal using t-PTK (group 1) and ninety-five patients (143 eyes) underwent mechanical epithelial debridement using a Amoils rotating brush (group 2) during CXL treatment. Visual and refractive outcomes were evaluated with keratometric and aberrometric values preoperatively at 12±6 months postoperatively. Main outcome measures: Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest refractive spherical equivalent (MRSE), and keratometry readings as steepest radius of anterior curvature (Kmax), and corneal astigmatism (AST.). Additionally, aberometric values root mean square (RMS) and higher order aberrations (HOAs). Secondary outcomes; subjective spherical power, subjective astigmatic power, steepest meridian (K2), flattest meridian (K1) and spherical aberrations (Z40). Results: In group 1, Snellen decimal mean UDVA and mean CDVA improved from 0.41 ±0.24 and 0.96 ±0.20 preoperatively to 0.67 ±0.32 (p .05). In group 1, Kmax improved from 53.15 ±4.3 dioptres (D) preoperatively to 50.99 ±4.68 D (p <.001), and in group 2 Kmax improved from 53.92 ±8.26 dioptres (D) preoperatively to 53.74 ±8.47 D (p <.001). RMS Total and RMS HOAs both showed significance between baseline and follow-up (p <.001) and between treatments in the same follow-up interval (p <.001). Conclusions: Epithelial removal using t-PTK during CXL results in better visual and refractive outcomes compared with mechanical epithelial debridement. However, further studies are needed to determine the true differences between treatments

Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages. © 2013 Rogers et al

Predicting the binding preference of transcription factors to individual DNA k-mers

Motivation: Recognition of specific DNA sequences is a central mechanism by which transcription factors (TFs) control gene expression. Many TF-binding preferences, however, are unknown or poorly characterized, in part due to the difficulty associated with determining their specificity experimentally, and an incomplete understanding of the mechanisms governing sequence specificity. New techniques that estimate the affinity of TFs to all possible k-mers provide a new opportunity to study DNA–protein interaction mechanisms, and may facilitate inference of binding preferences for members of a given TF family when such information is available for other family members. Results: We employed a new dataset consisting of the relative preferences of mouse homeodomains for all eight-base DNA sequences in order to ask how well we can predict the binding profiles of homeodomains when only their protein sequences are given. We evaluated a panel of standard statistical inference techniques, as well as variations of the protein features considered. Nearest neighbour among functionally important residues emerged among the most effective methods. Our results underscore the complexity of TF–DNA recognition, and suggest a rational approach for future analyses of TF families. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.Canadian Institutes of Health ResearchOntario Research FundNational Institutes of Health (U.S.)National Human Genome Research Institute (U.S.

Tools for peer assessment in an e-learning environment

Our exploration of peer assessment in the formative feedback of themes within ITL111 Digital Competence for teachers (15 ECTS) and GEO102 Physical Geography (15 ECTS) is based on support from tools within the LMS, sets of learning based outcomes, rubrics and Six Thinking Hats. The overall effect is improved quality of the student assignments and deeper learning. The best results were registered with the use of rubrics where the students were presented with clearly defined criteria for expected performance on a sample of different themes within the course. In order to perform the peer review, the students had to acquire the basic knowledge of the various themes. In addition, seeing how others solved the assignment provided the student with reflections on the themes that would improve the student\u27s own final portfolio

Fin development in a cartilaginous fish and the origin of vertebrate limbs

Recent fossil finds and experimental analysis of chick and mouse embryos highlighted the lateral fin fold theory, which suggests that two pairs of limbs in tetrapods evolved by subdivision of an elongated single fin1. Here we examine fin development in embryos of the primitive cartilaginous fish, Scyliorhinus canicula (dogfish) using scanning electron microscopy and investigate expression of genes known to be involved in limb positioning, identity and patterning in higher vertebrates. Although we did not detect lateral fin folds in dogfish embryos, Engrailed-1 expression suggests that the body is compartmentalized dorso-ventrally. Furthermore, specification of limb identity occurs through the Tbx4 and Tbx5 genes, as in higher vertebrates. In contrast, unlike higher vertebrates, we did not detect Shh transcripts in dogfish fin-buds, although dHand (a gene involved in establishing Shh) is expressed. In S. canicula, the main fin axis seems to lie parallel to the body axis. 'Freeing' fins from the body axis and establishing a separate 'limb' axis has been proposed to be a crucial step in evolution of tetrapod limbs2, 3. We suggest that Shh plays a critical role in this process

Jätkutoe roll vanglast vabanenud isikute taasühiskonnastamisel

http://www.ester.ee/record=b514900