Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond

Precise genome editing with CRISPR/Cas paves the way for many biochemical, biotechnological, and medical applications, and consequently, it may enable treatment of already known and still-to-be-found genetic diseases. Meanwhile, another rapidly emerging field—structural DNA nanotechnology—provides a customizable and modular platform for accurate positioning of nanoscopic materials, for e.g., biomedical uses. This addressability has just recently been applied in conjunction with the newly developed gene engineering tools to enable impactful, programmable nanotechnological applications. As of yet, self-assembled DNA nanostructures have been mainly employed to enhance and direct the delivery of CRISPR/Cas, but lately the groundwork has also been laid out for other intriguing and complex functions. These recent advances will be described in this perspective

A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation

BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12860-014-0041-5) contains supplementary material, which is available to authorized users

Inflammatory mediator bradykinin increases population of sensory neurons expressing functional T-type Ca2+ channels

T-type Ca2+ channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca2+ currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca2+ channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca2+ currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect CaV3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a 'reserve pool' of CaV3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions

Periodic eclipses of the young star PDS 110 discovered with WASP and KELT photometry

We report the discovery of eclipses by circumstellar disc material associated with the young star PDS 110 in the Ori OB1a association using the SuperWASP and Kilodegree Extremely Little Telescope surveys. PDS 110 (HD 290380, IRAS 05209-0107) is a rare Fe/Ge-type star, an similar to 10 Myr-old accreting intermediate-mass star showing strong infrared excess (L-IR/L-bol similar or equal to 0.25). Two extremely similar eclipses with a depth of 30 per cent and duration similar to 25 d were observed in 2008 November and 2011 January. We interpret the eclipses as caused by the same structure with an orbital period of 808 +/- 2 d. Shearing over a single orbit rules out diffuse dust clumps as the cause, favouring the hypothesis of a companion at similar to 2 au. The characteristics of the eclipses are consistent with transits by an unseen low-mass (1.8-70M(Jup)) planet or brown dwarf with a circumsecondary disc of diameter similar to 0.3 au. The next eclipse event is predicted to take place in 2017 September and could be monitored by amateur and professional observatories across the world

The use of contextualised standardised client simulation to develop clinical reasoning in final year veterinary students

Clinical reasoning is an important skill for veterinary students to develop before graduation. Simulation has been studied in medical education as a method for developing clinical reasoning in students, but evidence supporting it is limited. This study involved the creation of a contextualized, standardized client simulation session that aimed to improve the clinical reasoning ability and confidence of final-year veterinary students. Sixty-eight participants completed three simulated primary-care consultations, with the client played by an actor and the pet by a healthy animal. Survey data showed that all participants felt that the session improved their clinical decision-making ability. Quantitative clinical reasoning self-assessment, performed using a validated rubric, triangulated this finding, showing an improvement in students’ perception of several components of their clinical reasoning skill level from before the simulation to after it. Blinded researcher analysis of the consultation video recordings found that students showed a significant increase in ability on the history-taking and making-sense-of-data (including formation of a differential diagnosis) components of the assessment rubric. Thirty students took part in focus groups investigating their experience with the simulation. Two themes arose from thematic analysis of these data: variety of reasoning methods and “It’s a different way of thinking.” The latter highlights differences between the decision making students practice during their time in education and the decision making they will use once they are in practice. Our findings suggest that simulation can be used to develop clinical reasoning in veterinary students, and they demonstrate the need for further research in this area

Communication in production animal medicine: modelling a complex interaction with the example of dairy herd health medicine

<p>Abstract</p> <p>Background</p> <p>The importance of communication skills in veterinary medicine is increasingly recognised. Appropriate communication skills towards the client are of utmost importance in both companion animal practice and production animal field and consultancy work. The need for building a relationship with the client, alongside developing a structure for the consultation is widely recognised and applies to both types of veterinary practice.</p> <p>Results</p> <p>Veterinary advisory practice in production animal medicine is, however, characterised by a more complex communication on different levels. While the person-orientated communication is a permanent process between veterinarian and client with a rather personal perspective and defines the roles of interaction, the problem-orientated communication deals with emerging difficulties; the objective is to solve an acute health problem. The solution - orientated communication is a form of communication in which both veterinarian and client address longstanding situations or problems with the objective to improve herd health and subsequently productivity performance. All three forms of communication overlap.</p> <p>Conclusions</p> <p>Based on this model, it appears useful for a veterinary practice to offer both a curative and an advisory service, but to keep these two separated when deemed appropriate. In veterinary education, the strategies and techniques necessary for solution orientated communication should be included in the teaching of communication skills.</p

The TIP30 Protein Complex, Arachidonic Acid and Coenzyme A Are Required for Vesicle Membrane Fusion

Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4) and Endophilin B1 (Endo B1) that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H+)-ATPases (V-ATPases) to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA), producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes

Notch signaling in mouse blastocyst development and hatching

Research Areas: Developmental BiologyBackground: Mammalian early embryo development requires a well-orchestrated interplay of cell signaling pathways. Notch is a major regulatory pathway involved in cell-fate determination in embryonic and adult scenarios. However, the role of Notch in embryonic pre-implantation development is controversial. In particular, Notch role on blastocyst development and hatching remains elusive, and a complete picture of the transcription and expression patterns of Notch components during this time-period is not available. Results: This study provided a comprehensive view on the dynamics of individual embryo gene transcription and protein expression patterns of Notch components (receptors Notch1–4; ligands Dll1 and Dll4, Jagged1–2; and effectors Hes1–2), and their relationship with transcription of gene markers of pluripotency and differentiation (Sox2, Oct4, Klf4, Cdx2) during mouse blastocyst development and hatching. Transcription of Notch1–2, Jagged1–2 and Hes1 was highly prevalent and dynamic along stages of development, whereas transcription of Notch3–4, Dll4 and Hes2 had a low prevalence among embryos. Transcription levels of Notch1, Notch2, Jagged2 and Hes1 correlated with each other and with those of pluripotency and differentiation genes. Gene transcription was associated to protein expression, except for Jagged2, where high transcription levels in all embryos were not translated into protein. Presence of Notch signaling activity was confirmed through nuclear NICD and Hes1 detection, and downregulation of Hes1 transcription following canonical signaling blockade with DAPT. In vitro embryo culture supplementation with Jagged1 had no effect on embryo developmental kinetics. In contrast, supplementation with Jagged2 abolished Jagged1 transcription, downregulated Cdx2 transcription and inhibited blastocyst hatching. Notch signaling blockade by DAPT downregulated transcription of Sox2, and retarded embryo hatching. Conclusion: Transcription of Notch genes showed a dynamic pattern along blastocyst development and hatching. Data confirmed Notch signaling activity, and lead to the suggestion that Notch canonical signaling may be operating through Notch1, Notch3, Jagged1 and Hes1. Embryo culture supplementation with Jagged1 and Jagged2 unveiled a possible regulatory effect between Jagged1, Cdx2 and blastocyst hatching. Overall, results indicate that a deregulation in Notch signaling, either by its over or under-activation, affects blastocyst development and hatching.info:eu-repo/semantics/publishedVersio

HATS-47b, HATS-48Ab, HATS-49b and HATS-72b: Four Warm Giant Planets Transiting K Dwarfs

We report the discovery of four transiting giant planets around K dwarfs. The planets HATS-47b, HATS-48Ab, HATS49b, and HATS-72b have masses of 0.369+ 0.0210.031MJ, 0.243+ 0.0300.022 MJ, 0.353+ 0.0270.038 MJ, and 0.1254. 0.0039 MJ, respectively, and radii of 1.117. 0.014 RJ, 0.800. 0.015 RJ, 0.765. 0.013 RJ, and 0.7224. 0.0032 RJ, respectively. The planets orbit close to their host stars with orbital periods of 3.9228 days, 3.1317 days, 4.1480 days, and 7.3279 days, respectively. The hosts are main-sequence K dwarfs with masses of 0.674+ 0.0120.016.M, 0.7279. 0.0066.M, 0.7133. 0.0075.M, and 0.7311. 0.0028, and with V-band magnitudes of V = 14.829. 0.010, 14.35. 0.11, 14.998. 0.040 and 12.469. 0.010. The super-Neptune HATS-72b (a.k.a. WASP-191b and TOI 294.01) was independently identified as a transiting planet candidate by the HATSouth, WASP, and TESS surveys, and we present a combined analysis of all of the data gathered by each of these projects (and their follow-up programs). An exceptionally precise mass is measured for HATS-72b thanks to high-precision radial velocity (RV) measurements obtained with VLT/ESPRESSO, FEROS, HARPS, and Magellan/PFS. We also incorporate TESS observations of the warm Saturn-hosting systems HATS-47 (a.k.a. TOI.1073.01), HATS-48A, and HATS-49. HATS-47 was independently identified as a candidate by the TESS team, while the other two systems were not previously identified from the TESS data. The RV orbital variations are measured for these systems using Magellan/PFS. HATS-48A has a resolved 5.. 4 neighbor in Gaia.DR2, which is a common-proper-motion binary star companion to HATS-48A with a mass of 0.22.M and a current projected physical separation of similar to 1400 au

Saposin C Coupled Lipid Nanovesicles Specifically Target Arthritic Mouse Joints for Optical Imaging of Disease Severity

Rheumatoid arthritis is a chronic inflammatory disease affecting approximately 1% of the population and is characterized by cartilage and bone destruction ultimately leading to loss of joint function. Early detection and intervention of disease provides the best hope for successful treatment and preservation of joint mobility and function. Reliable and non-invasive techniques that accurately measure arthritic disease onset and progression are lacking. We recently developed a novel agent, SapC-DOPS, which is composed of the membrane-associated lysosomal protein saposin C (SapC) incorporated into 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipid nanovesicles. SapC-DOPS has a high fusogenic affinity for phosphatidylserine-enriched microdomains on surfaces of target cell membranes. Incorporation of a far-red fluorophore, CellVue Maroon (CVM), into the nanovesicles allows for in vivo non-invasive visualization of the agent in targeted tissue. Given that phosphatidylserine is present only on the inner leaflet of healthy plasma membranes but is “flipped” to the outer leaflet upon cell damage, we hypothesized that SapC-DOPS would target tissue damage associated with inflammatory arthritis due to local surface-exposure of phosphatidylserine. Optical imaging with SapC-DOPS-CVM in two distinct models of arthritis, serum-transfer arthritis (e.g., K/BxN) and collagen-induced arthritis (CIA) revealed robust SapC-DOPS-CVM specific localization to arthritic paws and joints in live animals. Importantly, intensity of localized fluorescent signal correlated with macroscopic arthritic disease severity and increased with disease progression. Flow cytometry of cells extracted from arthritic joints demonstrated that SapC-DOPS-CVM localized to an average of 7–8% of total joint cells and primarily to CD11b+Gr-1+ cells. Results from the current studies strongly support the application of SapC-DOPS-CVM for advanced clinical and research applications including: detecting early arthritis onset, assessing disease progression real-time in live subjects, and providing novel information regarding cell types that may mediate arthritis progression within joints