CRISPR-mediated optogene expression from a cell-specific endogenous promoter in retinal ON-bipolar cells to restore vision

Retinitis pigmentosa, an inherited form of retinal degeneration, is characterized by a progressive loss of rods and subsequent degeneration of cones, leading to blindness. However, the remaining neural portion of the retina (bipolar and ganglion cells) remains anatomically and functionally intact for an extended time. A possible treatment to restore the light sensitivity of the retina consists of rendering the remaining retinal cells photosensitive using optogenetic tools like, for example, Opto-mGluR6, a light-sensitive mGluR6 receptor. We have previously demonstrated that AAV vector-mediated expression of Opto-mGluR6 in ON-bipolar cells restores visual function in otherwise blind mice. However, classical gene supplementation therapy still suffers from high off-target expression rates and uncontrollable target gene expression levels that may lead to either cytotoxicity or lack of functional restoration. To address these issues and achieve cell-specific and endogenously controlled Opto-mGluR6 expression, we employed the CRISPR/Cas technology—in particular, homology-independent targeted integration (HITI) and microhomology-dependent targeted integration (MITI)—to knock-in the Opto-mGluR6 gene behind the ON-bipolar cell-specific GRM6 promoter. We compared four Cas systems in vitro and show that SpCas9 for HITI and LbCpf1 for MITI are well suited to promoting knock-in. As AAV2-mediated ON-bipolar cell transduction resulted in inefficiency, we evaluated Exo-AAVs as delivery vehicles and found Exo-AAV1 efficient for targeting ON-bipolar cells. We demonstrate that intravitreal injection of Exo-AAV1 carrying vectors that promote MITI significantly improved visual acuity in otherwise blind rd1 mice. We conclude by confirming and providing a qualitative evaluation of the MITI-mediated knock-in in the correct genomic locus

The secret world of shrimps: polarisation vision at its best

Animal vision spans a great range of complexity, with systems evolving to detect variations in optical intensity, distribution, colour, and polarisation. Polarisation vision systems studied to date detect one to four channels of linear polarisation, combining them in opponent pairs to provide intensity-independent operation. Circular polarisation vision has never been seen, and is widely believed to play no part in animal vision. Polarisation is fully measured via Stokes' parameters--obtained by combined linear and circular polarisation measurements. Optimal polarisation vision is the ability to see Stokes' parameters: here we show that the crustacean \emph{Gonodactylus smithii} measures the exact components required. This vision provides optimal contrast-enhancement, and precise determination of polarisation with no confusion-states or neutral-points--significant advantages. We emphasise that linear and circular polarisation vision are not different modalities--both are necessary for optimal polarisation vision, regardless of the presence of strongly linear or circularly polarised features in the animal's environment.Comment: 10 pages, 6 figures, 2 table

Electrophysiological evidence for linear polarization sensitivity in the compound eyes of the stomatopod crustacean Gonodactylus chiragra

Gonodactyloid stomatopod crustaceans possess polarization vision, which enables them to discriminate light of different e-vector angle. Their unusual apposition compound eyes are divided by an equatorial band of six rows of enlarged, structurally modified ommatidia, the mid-band (MB). The rhabdoms of the two most ventral MB rows 5 and 6 are structurally designed for polarization vision. Here we show, with electrophysiological recordings, that the photoreceptors R1-R7 within these two MB rows in Gonodactylus chiragra are highly sensitive to linear polarized light of two orthogonal directions (PS=6.1). They possess a narrow spectral sensitivity peaking at 565 nm. Unexpectedly, photoreceptors within the distal rhabdomal tier of MB row 2 also possess highly sensitive linear polarization receptors, which are in their spectral and polarization characteristics similar to the receptors of MB rows 5 and 6. Photoreceptors R1-R7 within the remainder of the MB exhibit low polarization sensitivity (PS=2.3). Outside the MB, in the two hemispheres, R1-R7 possess medium linear polarization sensitivity (PS=3.8) and a broad spectral sensitivity peaking at around 500 nm, typical for most crustaceans. Throughout the retina the most distally situated UV-sensitive R8 cells are not sensitive to linear polarized light

Ultraviolet polarisation sensitivity in the stomatopod crustacean Odontodactylus scyllarus

The ommatidia of crustacean eyes typically contain two classes of photoreceptors with orthogonally oriented microvilli. These receptors provide the basis for two-channel polarisation vision in the blue–green spectrum. The retinae of gonodactyloid stomatopod crustaceans possess a great variety of structural specialisations for elaborate polarisation vision. One type of specialisation is found in the small, distally placed R8 cells within the two most ventral rows of the mid-band. These ultraviolet-sensitive photoreceptors produce parallel microvilli, a feature suggestive for polarisation-sensitive photoreceptors. Here, we show by means of intracellular recordings combined with dye-injections that in the gonodactyloid species Odontodactylus scyllarus, the R8 cells of mid-band rows 5 and 6 are sensitive to linear polarised ultraviolet light. We show that mid-band row 5 R8 cells respond maximally to light with an e-vector oriented parallel to the mid-band, whereas mid-band row 6 R8 cells respond maximally to light with an e-vector oriented perpendicular to the mid-band. This orthogonal arrangement of ultraviolet-sensitive receptor cells could support ultraviolet polarisation vision. R8 cells of rows 5 and 6 are known to act as quarter-wave retarders around 500 nm and thus are the first photoreceptor type described with a potential dual role in polarisation vision

The psychological effects of terrorism are moderated by cultural worldviews

Terrorism cannot be easily studied experimentally for obvious reasons. We report the results of a laboratory study (N = 149) testing the effect of cultural worldviews on feelings of threat and hostility toward Muslims in France that include in the design the deadly terrorist attack of January 7th 2015 in Paris as a naturally occurring independent variable. The results replicate past research by showing that in a natural context, people felt more threatened and more hostile toward Muslims after the terrorist attack than before. However, the reverse occurred in an experimental condition that made the French cultural worldview of colorblind equality salient: People felt less threatened and less hostile after the terrorist attack than before. These results provide, for the first time in the context of a real terrorist attack, support for Terror Management Theory's proposal that cultural worldviews are an effective buffer against terror

Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumour microenvironment

Adoptive cell transfer utilizing tumour-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against haematological malignancies, but significant clinical success has not yet been achieved in solid tumours due in part to the strong immunosuppressive tumour microenvironment. Here, we show that suppression of CTL killing by CD4+CD25+Foxp+ regulatory T cell (Treg) is in part mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Highly selective optical control of Ca2+ signalling in adoptively transferred CTLs enhances T cell activation and IFN-γ production in vitro, leading to a significant reduction in tumour growth in mice. Altogether, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses at the tumour sites

Optogenetic acidification of synaptic vesicles and lysosomes

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes

Microstructural and Electrical Features of Yttrium Stabilised Zirconia with ZnO as Sintering Additive

Adding ZnO reduces sintering temperature of yttria stabilized zirconia. Adding up to 0.5 wt% of ZnO is possible to densify to 8 mol% yttria stabilized zirconia (TZ8Y) to 95% of relative density at 1300 °C, besides, the electrical conductivity increases about 30% at 800 °C when compared to pure TZ8Y with the same relative density and average grain size. These results show that TZ8Y co-doped with ZnO can be a potential electrolyte to solid oxide fuel cells and electrolyzer cells