Incorporation of chromaffin granule membranes into large-size vesicles suitable for patch-clamp recording

AbstractIncubation of chromaffin granules with excess liposomes at pH 6.0 resulted in the formation of cell-size structures, which were purified by centrifugation on sucrose gradients. Experiments with fluorescein-labeled granules indicated incorporation of granule membrane to these structures. The preparation contained various vesicular structures with a diameter up to 15 μm. The largest elements were studied by the ‘patch-clamp’ technique. ‘Cell-attached’ and ‘whole-cell’ recordings indicated the presence of currents corresponding to unitary conductances ranging from 100 to 500 pS

Eye and taurine

Taurine is the most abundant amino acid in retina. Although unclear, its role is mainly related to its powerful antioxidant properties. The taurine concentrations in tissues are regulated by an exogenous intake through the nutrition. This taurine intake is highly dependent on the function of taurine transporter. In addition, an endogenous synthesis accounts for the physiological taurine amounts. Previous studies had shown that taurine nutritional deprivation in cat was responsible for severe retinal damages at the photoreceptor layer. By discovering the taurine depletion was incriminated in the retinal toxicity of vigabatrin, we recently demonstrated in different models of retinal degeneration that taurine was involved in the retinal ganglion cells survival. Accordingly, Taurine may play a crucial role in the prevention of retinal degeneration such as retinopathies and glaucomas.La taurine est l’acide aminé le plus abondant dans la rétine. Son rôle, encore mal connu est essentiellement lié à son pouvoir anti-oxydant. Sa concentration tissulaire dépend d’un apport nutritionnel en taurine exogène et du fonctionnement de son transporteur. De plus, une synthèse endogène de taurine participe au maintien de son taux physiologique. D’anciennes études ont montré que la privation nutritionnelle de taurine chez le chat est responsable de dommages rétiniens graves, affectant la couche des photorécepteurs. En découvrant que la toxicité du vigabatrin est liée à une déplétion en taurine, nous avons récemment montré que la taurine participe également à la survie des cellules ganglionnaires rétiniennes dans différents modèles de dégénérescence rétinienne. La taurine pourrait ainsi être impliquée dans la prévention des dégénérescences rétiniennes telles que les rétinopathies et les glaucomes

The protein kinases AtMAP3Kε1 and BnMAP3Kε1 are functional homologues of S. pombe cdc7p and may be involved in cell division

We identified an Arabidopsis thaliana gene, AtMAP3Kε1, and a Brassica napus cDNA, BnMAP3Kε1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT–PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kε1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.This work was mainly supported by a EU grant (SIME project BIOTEC-RTD-CEE PL 960275). The authors also acknowledge the financial support of the MERS and CNRS to UMR 8618, and DGESIG PB98–0678

Panretinal, high-resolution color photography of the mouse fundus

PURPOSE. To analyze high-resolution color photographs of the mouse fundus. METHODS. A contact fundus camera based on topical endoscopy fundus imaging (TEFI) was built. Fundus photographs of C57 and Balb/c mice obtained by TEFI were qualitatively analyzed. RESULTS. High-resolution digital imaging of the fundus, including the ciliary body, was routinely obtained. The reflectance and contrast of retinal vessels varied significantly with the amount of incident and reflected light and, thus, with the degree of fundus pigmentation. The combination of chromatic and spherical aberration favored blue light imaging, in term of both field and contrast. CONCLUSIONS. TEFI is a small, low-cost system that allows highresolution color fundus imaging and fluorescein angiography in conscious mice. Panretinal imaging is facilitated by the presence of the large rounded lens. TEFI significantly improves the quality of in vivo photography of retina and ciliary process of mice. Resolution is, however, affected by chromatic aberration, and should be improved by monochromatic imaging. (Invest Ophthalmol Vis Sci

PRIMA subretinal wireless photovoltaic microchip implantation in non-human primate and feline models

Purpose To evaluate the surgical technique for subretinal implantation of two sizes of PRIMA photovoltaic wireless microchip in two animal models, and refine these surgical procedures for human trials. Methods Cats and Macaca fascicularis primates with healthy retina underwent vitrectomy surgery and were implanted with subretinal wireless photovoltaic microchip at the macula/central retina. The 1.5mm PRIMA chip was initially studied in feline eyes. PRIMA implant (2mm,1.5mm sizes) arrays were studied in primates. Feasibility of subretinal chip implantation was evaluated with a newly-developed surgical technique, with surgical complications and adverse events recorded. Results The 1.5mm implant was placed in the central retina of 11 feline eyes, with implantation duration 43-106 days. The 1.5mm implant was correctly positioned into central macula of 11 primate eyes, with follow-up periods of minimum 6 weeks (n = 11), 2 years (n = 2), and one eye for 3 years. One primate eye underwent multi-chip 1.5mm implantation using two 1.5mm chips. The 2mm implant was delivered to 4 primate eyes. Optical coherence tomography confirmed correct surgical placement of photovoltaic arrays in the subretinal space in all 26 eyes. Intraoperative complications in primate eyes included retinal tear, macular hole, retinal detachment, and vitreous hemorrhage that resolved spontaneously. Postoperatively, there was no case of significant ocular inflammation in the 1.5mm implant group. Conclusions We report subretinal implantation of 1.5mm and 2mm photovoltaic arrays in the central retina of feline and central macula of primate eyes with a low rate of device-related complications. The in vivo PRIMA implantation technique has been developed and refined for use for a 2mm PRIMA implant in ongoing human trials

Bilateral Visual Improvement with Unilateral Gene Therapy Injection for Leber Hereditary Optic Neuropathy

REVERSE is a randomized, double-masked, sham-controlled, multicenter, phase III clinical trial that evaluated the efficacy of a single intravitreal injection of rAAV2/2 ND4 in subjects with visual loss from Leber hereditary optic neuropathy (LHON). A total of 37 subjects carrying the m.11778G>A (MT-ND4) mutation and with duration of vision loss between 6 to 12 months were treated. Each subject’s right eye was randomly assigned in a 1:1 ratio to treatment with rAAV2/2 ND4 (GS010) or sham injection. The left eye received the treatment not allocated to the right eye. Unexpectedly, sustained visual improvement was observed in both eyes over the 96-week follow-up period. At Week 96, rAAV2/2 ND4-treated eyes showed a mean improvement in best-corrected visual acuity (BCVA) of -0.308 LogMAR (+15 ETDRS letters). A mean improvement of 0.259 (0.068) LogMAR (+13 ETDRS letters) was observed in the sham treated eyes. Consequently, the primary endpoint, defined as the difference in the change in BCVA from baseline to Week 48 between the two treatment groups, was not met (p = 0.894, ANCOVA). At Week 96, 25 subjects (68%) had a clinically relevant recovery in BCVA from baseline in at least one eye and 29 subjects (78%) had an improvement in vision in both eyes. A non-human primate study was conducted to investigate this bilateral improvement. Evidence of transfer of viral vector DNA from the injected eye to the anterior segment, retina and optic nerve of the contralateral non-injected eye supports a plausible mechanistic explanation for the unexpected bilateral improvement in visual function after unilateral injection

Epigenetic Silencing of IRF7 and/or IRF5 in Lung Cancer Cells Leads to Increased Sensitivity to Oncolytic Viruses

Defective IFN signaling results in loss of innate immunity and sensitizes cells to enhanced cytolytic killing after Vesticular Stomatitis Virus (VSV) infection. Examination of the innate immunity status of normal human bronchial epithelial cells Beas2B and 7 lung cancer cells revealed that the abrogation of IFN signaling in cancer cells is associated with greater sensitivity to VSV infection. The disruption of the IFN pathway in lung cancer cell lines and primary tumor tissues is caused by epigenetic silencing of critical interferon responsive transcription factors IRF7 and/or IRF5. Although 5-aza-2′-deoxycytidine treatment fails to reactivate IRF7 and IRF5 expression or protect cells from VSV infection, manipulating IFN signaling by altering IRF expression changes the viral susceptibility of these cells. Lung cancer cells can be partially protected from viral killing using IRF5+IRF7 overexpression, whereas IFN pathway disruption by transfection of siRNAs to IRF5+IRF7 increases cells' vulnerability to viral infection. Therefore, IRF5 and IRF7 are key transcription factors in IFN pathway that determine viral sensitivity of lung cancer cells; the epigenetically impaired IFN pathway in lung cancer tissues provides potential biomarkers for successful selective killing of cancer cells by oncolytic viral therapy

Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

The authors are grateful to Manuel Simonutti, Julie Dégardin, Jennifer Da Silva, Samantha Beck and Caroline Carvalho for their valuable help in phenotyping (platform of Institut de la Vision) and to Isabelle Renault, Léa Biedermann and André Tiffoche for animal care (platform of Institut de la Vision). The authors thank Stéphane Fouquet for his support in developing a custom-made Image J macro to measure thickness of retinal layers.This work was supported by Fondation Valentin Hauy (IA, EO), Retina France (IA, EO), e-rare RHORCOD (IA), Fondation de l’Oeil—Fondation de France (IA), Foundation Voir et Entendre (CZ), Foundation Fighting Blindness (FFB) (CD-CL-0808-0466-CHNO) (IA), and the FFB center grant (CD-CL-0808-0466-CHNO), Ville de Paris and Region Ile de France, Labex Lifesenses (reference ANR-10-LABX-65) supported by French state funds managed by the ANR within the Investissements d’Avenir programme (ANR-11-IDEX-0004-0), the Regional Council of Ile de France (I09–1727/R) (EO), the National Institute of Health grants EY10609 (MIN), EY001919 (MML) and EY006842 (MML) and the Foundation Fighting Blindness (MIN and MML).Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3’ UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.Yeshttp://www.plosone.org/static/editorial#pee

Fonction et régulation des oxydoréductases à disulfure (étude comparative des interactions entre le cycle isoalloxazine et son environnement)

Ce manuscrit présente une étude sur les facteurs structuraux qui modulent les différences d'activité enzymatique et de comportement vis-à-vis de métaux lourds de certaines oxydoréductases à disulfure et nicotinamide (Glutathion réductase (GR), Thiorédoxine réductase (TrxR), et NADH peroxydase (NPX)). Des approches complémentaires combinant des expériences de biochimie à des spectroscopies d'absorption électronique et diffusion Raman de résonance (DRR) ont été utilisées. La réduction de la GR à deux électrons par le substrat (NADPH) ou par le produit (GSH)de la réaction aboutit à la formation de deux espèces différentes. L'étude par DRR de ces deux états réduits a montré que le noyau isoalloxazine du FAD imposait à l'enzyme un contrôle redox et une régulation de son activité via une modulation de la force d'une liaison hydrogène sur l'atome N5. L'inhibition provoquée par l'association de sels métalliques (Hg2+, Cd2+) au dithiol du site actif de la GR est expliquée par le découplage des deux centres redox de la GR (dithiol et FAD) et par une forte distorsion du noyau isoalloxazine, qui engendre des modifications profondes dans les interactions FAD-protéine. Les effets sont tout aussi importants sur la TrxR puisque l'association de ces sels déplace fortement la seconde réduction du FAD de 1 à 3 équivalents de NADPH. Elle permet également de stabiliser beaucoup plus facilement une forme semiquinonique bleue, qui serait un intermédiaire dans une voie de régulation pour cette classe d'enzymes. Les spectres de DRR des différentes espèces redox de la NPX montrent que le cycle isoalloxazine possède connue dans la GR une densité électronique faible. Elle permet la stabilisation de l'acide cystéine-sulfénique et celle du complexe de transfert de charge formé après la réduction de l'enzyme par le NADH.This manuscript presents a study on the characterization of structural factors modulating enzymatic activity and reactivity with metallic ions from members of the pyridine nucleotide disulfide-oxidoreductase family (Glutathione reductase (GR), Thioredoxin reductase (TrxR) and NADH peroxidase (NPX). Complementary techniques combining biochemical experiments, electronic absorption and resonance Raman (RR) spectroscopies have been used. The two-electron reduction of GR by the substrate (NADPH) or the product (GSH) of the enzyme reaction gives rise to the formation of two different species. RR studies of these two-electron reduced states show that isoalloxazine ring of the FAD imposes a redox control and a regulation, of enzyme activity via a hydrogen bond modulation on the N5 atom of FAD. The enzyme inhibition induced by the binding of metallic ions (Hg2+, Cd2+) on the active site dithiol of GR is explained by both an elimination of the redox coupling between the two active centers of GR (dithiol and FAD) and a strong out-of-plane distortion of isoalloxazine moiety that generates strong alterations in FAD-protein interactions. Association of the heavy metal to TrxR produces a displacement of the second redaction step of FAD from 1 to 3 equivalents of NADPH. Moreover, the formation of a blue semiquinone is stabilized. The stabilization of this radical is proposed to be a regulation way for this enzyme class. RR spectra obtained for different redox states of NPX show that the isoalloxazine ring has a low electronic density, as in the case of GR. A direct consequence is the stabilization, of the cysteine-sulfenic acid as well as a charge transfer complex formed after the enzyme redaction by NADH. For the three enzymes, this study showed an intimate electronic coupling between the two redox centers for the two electron-reduced enzymes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF