Capturing a rhodopsin receptor signalling cascade across a native membrane.

G protein-coupled receptors (GPCRs) are cell-surface receptors that respond to various stimuli to induce signalling pathways across cell membranes. Recent progress has yielded atomic structures of key intermediates1,2 and roles for lipids in signalling3,4. However, capturing signalling events of a wild-type receptor in real time, across a native membrane to its downstream effectors, has remained elusive. Here we probe the archetypal class A GPCR, rhodopsin, directly from fragments of native disc membranes using mass spectrometry. We monitor real-time photoconversion of dark-adapted rhodopsin to opsin, delineating retinal isomerization and hydrolysis steps, and further showing that the reaction is significantly slower in its native membrane than in detergent micelles. Considering the lipids ejected with rhodopsin, we demonstrate that opsin can be regenerated in membranes through photoisomerized retinal-lipid conjugates, and we provide evidence for increased association of rhodopsin with unsaturated long-chain phosphatidylcholine during signalling. Capturing the secondary steps of the signalling cascade, we monitor light activation of transducin (Gt) through loss of GDP to generate an intermediate apo-trimeric G protein, and observe Gαt•GTP subunits interacting with PDE6 to hydrolyse cyclic GMP. We also show how rhodopsin-targeting compounds either stimulate or dampen signalling through rhodopsin-opsin and transducin signalling pathways. Our results not only reveal the effect of native lipids on rhodopsin signalling and regeneration but also enable us to propose a paradigm for GPCR drug discovery in native membrane environments.

Ultrafast spectra and kinetics of human green-cone visual pigment at room temperature.

Rhodopsin is the pigment that enables night vision, whereas cone opsins are the pigments responsible for color vision in bright-light conditions. Despite their importance for vision, cone opsins are poorly characterized at the molecular level compared to rhodopsin. Spectra and kinetics of the intermediate states of human green-cone visual pigment (mid-wavelength sensitive, or MWS opsin) were measured and compared with the intermediates and kinetics of bovine rhodopsin. All the major intermediates of the MWS opsin were recorded in the picosecond to millisecond time range. Several intermediates in MWS opsin appear to have characteristics similar to the intermediates of bovine rhodopsin; however, there are some marked differences. One of the most striking differences is in their kinetics, where the kinetics of the MWS opsin intermediates are slower compared to those of the bovine rhodopsin intermediates.

Retinylidene chromophore hydrolysis from mammalian visual and non-visual opsins.

Rhodopsin (Rho) and cone opsins are essential for detection of light. They respond via photoisomerization, converting their Schiff-base-adducted 11-cis-retinylidene chromophores to the all-trans configuration, eliciting conformational changes to activate opsin signaling. Subsequent Schiff-base hydrolysis releases all-trans-retinal, initiating two important cycles that maintain continuous vision-the Rho photocycle and visual cycle pathway. Schiff-base hydrolysis has been thoroughly studied with photoactivated Rho but not with cone opsins. Using established methodology, we directly measured the formation of Schiff-base between retinal chromophores with mammalian visual and nonvisual opsins of the eye. Next, we determined the rate of light-induced chromophore hydrolysis. We found that retinal hydrolysis from photoactivated cone opsins was markedly faster than from photoactivated Rho. Bovine retinal G protein-coupled receptor (bRGR) displayed rapid hydrolysis of its 11-cis-retinylidene photoproduct to quickly supply 11-cis-retinal and re-bind all-trans-retinal. Hydrolysis within bRGR in native retinal pigment epithelium microsomal membranes was >6-times faster than that of bRGR purified in detergent micelles. N-terminal-targeted antibodies significantly slowed bRGR hydrolysis, while C-terminal antibodies had no effect. Our study highlights the much faster photocycle of cone opsins relative to Rho and the crucial role of RGR in chromophore recycling in daylight. By contrast, in our experimental conditions, bovine peropsin did not form pigment in the presence of all-trans-retinal nor with any mono-cis retinal isomers, leaving uncertain the role of this opsin as a light sensor.

Chromophore hydrolysis and release from photoactivated rhodopsin in native membranes.

For sustained vision, photoactivated rhodopsin (Rho*) must undergo hydrolysis and release of all-trans-retinal, producing substrate for the visual cycle and apo-opsin available for regeneration with 11-cis-retinal. The kinetics of this hydrolysis has yet to be described for rhodopsin in its native membrane environment. We developed a method consisting of simultaneous denaturation and chromophore trapping by isopropanol/borohydride, followed by exhaustive protein digestion, complete extraction, and liquid chromatography-mass spectrometry. Using our method, we tracked Rho* hydrolysis, the subsequent formation of N-retinylidene-phosphatidylethanolamine (N-ret-PE) adducts with the released all-trans-retinal, and the reduction of all-trans-retinal to all-trans-retinol. We found that hydrolysis occurred faster in native membranes than in detergent micelles typically used to study membrane proteins. The activation energy of the hydrolysis in native membranes was determined to be 17.7 ± 2.4 kcal/mol. Our data support the interpretation that metarhodopsin II, the signaling state of rhodopsin, is the primary species undergoing hydrolysis and release of its all-trans-retinal. In the absence of NADPH, free all-trans-retinal reacts with phosphatidylethanolamine (PE), forming a substantial amount of N-ret-PE (∼40% of total all-trans-retinal at physiological pH), at a rate that is an order of magnitude faster than Rho* hydrolysis. However, N-ret-PE formation was highly attenuated by NADPH-dependent reduction of all-trans-retinal to all-trans-retinol. Neither N-ret-PE formation nor all-trans-retinal reduction affected the rate of hydrolysis of Rho*. Our study provides a comprehensive picture of the hydrolysis of Rho* and the release of all-trans-retinal and its reentry into the visual cycle, a process in which alteration can lead to severe retinopathies.

Comparison of two extended depth of focus intraocular lenses with a monofocal lens: a multi-centre randomised trial.

PURPOSE: The AT LARA 829MP is a next-generation extended depth of focus (EDOF) intraocular lens (IOL) providing continuous vision over a range of distances. The aim of this prospective multi-centre randomised trial was to compare two EDOF IOLs and one monofocal IOL. METHODS: Cataract patients between 50 and 80 years were randomised for bilateral implantation with either the AT LARA 829MP (EDOF), the TECNIS Symfony (EDOF) or the CT ASPHINA 409MP (monofocal). Follow-up was at 1 to 2 weeks, 1 month and 4 to 6 months. RESULTS: A total of 211 patients were randomised and included in the final analysis. Monocular depth of focus was significantly better for AT LARA 829MP eyes compared with that for TECNIS Symfony at all thresholds (p = 0.024, 0.001 and 0.006, for 0.1, 0.2 and 0.3 logMAR respectively) with no significant difference for binocular depth of focus. LARA eyes had significantly better monocular depth of focus at all levels compared with ASPHINA eyes (all p < 0.0001), while there was no significant difference between Symfony and ASPHINA eyes at 0.1 logMAR and 0.2 logMAR. Both EDOF IOLs were significantly better than the monofocal ASPHINA at all levels for binocular depth of focus (LARA: all p < 0.0001; Symfony: all p = 0.002). Distance visual acuity was similar for all IOLs at 6 months; intermediate and near visual acuity were significantly better for the EDOF IOLs than for the monofocal (p < 0.0001). Refraction improved in all groups relative to baseline. Contrast sensitivity was higher with the CT ASPHINA 409MP but both EDOF lenses had a better spectacle independence rate. At 6 months, all IOLs were well centred with no cases of tilt. No general safety issues were raised for any of the groups. CONCLUSION: The two EDOF intraocular lenses investigated provided good visual outcomes with comparable visual acuity at all distances. The AT LARA 829MP provided the widest monocular depth of focus at 0.1 and 0.2 logMAR, with a clear superiority compared with the monofocal IOL. TECNIS Symfony was superior to the monofocal control at 0.3 logMAR. Spectacle independence and patient satisfaction were comparable. TRIAL REGISTRATION: Trial registered on https://clinicaltrials.gov/ under the identification NCT03172351 (date of registration 1 June May 2017).

CONCOMITANT MUTATIONS IN INHERITED RETINAL DYSTROPHIES: Why the Reproductive and Therapeutic Counseling Should Be Addressed Cautiously.

PURPOSE: To highlight the challenge of correct reproductive and therapeutic counseling in complex pedigrees with different inherited retinal dystrophies (IRD). METHODS: Two hundred eight patients diagnosed with nonsyndromic IRD underwent full ophthalmologic examination and molecular analysis using targeted next-generation sequencing. RESULTS: Five families (4%) carried mutations in more than one gene that contribute to different IRD. Family fRPN-NB had a dominant mutation in SNRNP200, which was present in nine affected individuals and four unaffected, and a mutation in RP2 among 11 family members. Family fRPN-142 carried a mutation in RPGR that cosegregated with the disease in all affected individuals. In addition, the proband also harbored two disease-causing mutations in the genes BEST1 and SNRNP200. Family fRPN-169 beared compound heterozygous mutations in USH2A and a dominant mutation in RP1. Genetic testing of fRPN-194 determined compound heterozygous mutations in CNGA3 and a dominant mutation in PRPF8 only in the proband. Finally, fRPN-219 carried compound heterozygous mutations in the genes ABCA4 and TYR. CONCLUSION: These findings reinforce the complexity of IRD and underscore the need for the combination of high-throughput genetic testing and clinical characterization. Because of these features, the reproductive and therapeutic counseling for IRD must be approached with caution.

Human red and green cone opsins are O-glycosylated at an N-terminal Ser/Thr-rich domain conserved in vertebrates.

There are fundamental differences in the structures of outer segments between rod and cone photoreceptor cells in the vertebrate retina. Visual pigments are the only essential membrane proteins that differ between rod and cone outer segments, making it likely that they contribute to these structural differences. Human rhodopsin is N-glycosylated on Asn2 and Asn15, whereas human (h) red and green cone opsins (hOPSR and hOPSG, respectively) are N-glycosylated at Asn34 Here, utilizing a monoclonal antibody (7G8 mAB), we demonstrate that hOPSR and hOPSG from human retina also are O-glycosylated with full occupancy. We determined that 7G8 mAB recognizes the N-terminal sequence 21DSTQSSIF28 of hOPSR and hOPSG from extracts of human retina, but only after their O-glycans have been removed with O-glycosidase treatment, thus revealing this post-translational modification of red and green cone opsins. In addition, we show that hOPSR and hOPSG from human retina are recognized by jacalin, a lectin that binds to O-glycans, preferentially to Gal-GalNAc. Next, we confirmed the presence of O-glycans on OPSR and OPSG from several vertebrate species, including mammals, birds, and amphibians. Finally, the analysis of bovine OPSR by MS identified an O-glycan on Ser22, a residue that is semi-conserved (Ser or Thr) among vertebrate OPSR and OPSG. These results suggest that O-glycosylation is a fundamental feature of red and green cone opsins, which may be relevant to their function or to cone cell development, and that differences in this post-translational modification also could contribute to the different morphologies of rod and cone photoreceptors.

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium.

Photoisomerization of the 11-cis-retinal chromophore of rod and cone visual pigments to an all-trans-configuration is the initiating event for vision in vertebrates. The regeneration of 11-cis-retinal, necessary for sustained visual function, is an endergonic process normally conducted by specialized enzyme systems. However, 11-cis-retinal also can be formed through reverse photoisomerization from all-trans-retinal. A nonvisual opsin known as retinal pigment epithelium (RPE)-retinal G-protein-coupled receptor (RGR) was previously shown to mediate visual chromophore regeneration in photic conditions, but conflicting results have cast doubt on its role as a photoisomerase. Here, we describe high-level production of 11-cis-retinal from RPE membranes stimulated by illumination at a narrow band of wavelengths. This activity was associated with RGR and enhanced by cellular retinaldehyde-binding protein (CRALBP), which binds the 11-cis-retinal produced by RGR and prevents its re-isomerization to all-trans-retinal. The activity was recapitulated with cells heterologously expressing RGR and with purified recombinant RGR. Using an RGR variant, K255A, we confirmed that a Schiff base linkage at Lys-255 is critical for substrate binding and isomerization. Single-cell RNA-Seq analysis of the retina and RPE tissue confirmed that RGR is expressed in human and bovine RPE and Müller glia, whereas mouse RGR is expressed in RPE but not in Müller glia. These results provide key insights into the mechanisms of physiological retinoid photoisomerization and suggest a novel mechanism by which RGR, in concert with CRALBP, regenerates the visual chromophore in the RPE under sustained light conditions.

Increasing the Stability of Recombinant Human Green Cone Pigment.

Three types of cone cells exist in the human retina, each containing a different pigment responsible for the initial step of phototransduction. These pigments are distinguished by their specific absorbance maxima: 425 nm (blue), 530 nm (green), and 560 nm (red). Each pigment contains a common chromophore, 11-cis-retinal covalently bound to an opsin protein via a Schiff base. The 11-cis-retinal protonated Schiff base has an absorbance maxima at 440 nm in methanol. Unfortunately, the chemistry that allows the same chromophore to interact with different opsin proteins to tune the absorbance of the resulting pigments to distinct λmax values is poorly understood. Rhodopsin is the only pigment with a native structure determined at high resolution. Homology models for cone pigments have been generated, but experimentally determined structures are needed for a precise understanding of spectral tuning. The principal obstacle to solving the structures of cone pigments has been their innate instability in recombinant constructs. By inserting five different thermostabilizing proteins (BRIL, T4L, PGS, RUB, and FLAV) into the recombinant green opsin sequence, constructs were created that were up to 9-fold more stable than WT. Using cellular retinaldehyde-binding protein (CRALBP), we developed a quick means of assessing the stability of the green pigment. CRALBP testing also confirmed an additional 48-fold increase in pigment stability when varying the detergent used. These results suggest an efficient protocol for routine purification and stabilization of cone pigments that could be used for high-resolution determination of their structures, as well as for other studies.

Complex binding pathways determine the regeneration of mammalian green cone opsin with a locked retinal analogue.

Phototransduction is initiated when the absorption of light converts the 11-cis-retinal chromophore to its all-trans configuration in both rod and cone vertebrate photoreceptors. To sustain vision, 11-cis-retinal is continuously regenerated from its all-trans conformation through a series of enzymatic steps comprising the "visual or retinoid" cycle. Abnormalities in this cycle can compromise vision because of the diminished supply of 11-cis-retinal and the accumulation of toxic, constitutively active opsin. As shown previously for rod cells, attenuation of constitutively active opsin can be achieved with the unbleachable analogue, 11-cis-6-membered ring (11-cis-6mr)-retinal, which has therapeutic effects against certain degenerative retinal diseases. However, to discern the molecular mechanisms responsible for this action, pigment regeneration with this locked retinal analogue requires delineation also in cone cells. Here, we compared the regenerative properties of rod and green cone opsins with 11-cis-6mr-retinal and demonstrated that this retinal analogue could regenerate rod pigment but not green cone pigment. Based on structural modeling suggesting that Pro-205 in green cone opsin could prevent entry and binding of 11-cis-6mr-retinal, we initially mutated this residue to Ile, the corresponding residue in rhodopsin. However, this substitution did not enable green cone opsin to regenerate with 11-cis-6mr-retinal. Interestingly, deletion of 16 N-terminal amino acids in green cone opsin partially restored the binding of 11-cis-6mr-retinal. These results and our structural modeling indicate that a more complex binding pathway determines the regeneration of mammalian green cone opsin with chromophore analogues such as 11-cis-6mr-retinal.

Successful Optimization of Adalimumab Therapy in Refractory Uveitis Due to Behçet's Disease.

PURPOSE: To assess efficacy, safety, and cost-effectiveness of adalimumab (ADA) therapy optimization in a large series of patients with uveitis due to Behçet disease (BD) who achieved remission after the use of this biologic agent. DESIGN: Open-label multicenter study of ADA-treated patients with BD uveitis refractory to conventional immunosuppressants. SUBJECTS: Sixty-five of 74 patients with uveitis due to BD, who achieved remission after a median ADA duration of 6 (range, 3-12) months. ADA was optimized in 23 (35.4%) of them. This biologic agent was maintained at a dose of 40 mg/subcutaneously/2 weeks in the remaining 42 patients. METHODS: After remission, based on a shared decision between the patient and the treating physician, ADA was optimized. When agreement between patient and physician was reached, optimization was performed by prolonging the ADA dosing interval progressively. Comparison between optimized and nonoptimized patients was performed. MAIN OUTCOME MEASURES: Efficacy, safety, and cost-effectiveness in optimized and nonoptimized groups. To determine efficacy, intraocular inflammation (anterior chamber cells, vitritis, and retinal vasculitis), macular thickness, visual acuity, and the sparing effect of glucocorticoids were assessed. RESULTS: No demographic or ocular differences were found at the time of ADA onset between the optimized and the nonoptimized groups. Most ocular outcomes were similar after a mean ± standard deviation follow-up of 34.7±13.3 and 26±21.3 months in the optimized and nonoptimized groups, respectively. However, relevant adverse effects were only seen in the nonoptimized group (lymphoma, pneumonia, severe local reaction at the injection site, and bacteremia by Escherichia coli, 1 each). Moreover, the mean ADA treatment costs were lower in the optimized group than in the nonoptimized group (6101.25 euros/patient/year vs. 12 339.48; P < 0.01). CONCLUSION: ADA optimization in BD uveitis refractory to conventional therapy is effective, safe, and cost-effective.

Human infrared vision is triggered by two-photon chromophore isomerization.

Vision relies on photoactivation of visual pigments in rod and cone photoreceptor cells of the retina. The human eye structure and the absorption spectra of pigments limit our visual perception of light. Our visual perception is most responsive to stimulating light in the 400- to 720-nm (visible) range. First, we demonstrate by psychophysical experiments that humans can perceive infrared laser emission as visible light. Moreover, we show that mammalian photoreceptors can be directly activated by near infrared light with a sensitivity that paradoxically increases at wavelengths above 900 nm, and display quadratic dependence on laser power, indicating a nonlinear optical process. Biochemical experiments with rhodopsin, cone visual pigments, and a chromophore model compound 11-cis-retinyl-propylamine Schiff base demonstrate the direct isomerization of visual chromophore by a two-photon chromophore isomerization. Indeed, quantum mechanics modeling indicates the feasibility of this mechanism. Together, these findings clearly show that human visual perception of near infrared light occurs by two-photon isomerization of visual pigments.

Aflibercept as First-Line Therapy in Patients with Treatment-Naïve Neovascular Age-Related Macular Degeneration: Prospective Case Series Analysis in Real-Life Clinical Practice.

PURPOSE: To assess the 13-month effectiveness and safety of aflibercept in naïve patients with neovascular age-related macular degeneration (nvAMD) in a real-life clinical setting. METHODS: Thirty-two treatment-naïve patients with nvAMD participated in a prospective two-center study. Patients received intravitreal injections of aflibercept (Eylea®), a loading dose of three monthly injections (2 mg/0.05 ml) every 4 weeks for the first 3 months, followed by intravitreal injections every 2 months. RESULTS: At 3 and 13 months, the mean best-corrected visual acuity improved significantly as compared with baseline (logMAR 0.53 ± 0.30 and 0.55 ± 0.32 vs. 0.30 ± 0.24, respectively, p < 0.001). At 3 and 13 months, 46.8% of patients (15/32) gained ≥15 ETDRS letters. The mean decrease in central macular thickness was also significant at 3 months (252 ± 35 µm) and at 13 months (249 ± 38 µm) as compared with pretreatment values (383 ± 76 µm) (p < 0.01). Also, 50% resolution of pigment epithelial detachment (PED) was observed in 8 out of 9 eyes (88.9%) with PED at baseline. Intravitreal injections were well tolerated and no adverse events were recorded. CONCLUSION: Aflibercept was effective and safe for treating nvAMD in naïve patients in routine daily practice.

Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans.

High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with (15)N,(13)C by providing them with isotopically labeled bacteria. (2)H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the "test" GPCR to demonstrate the viability of this approach. Although the worms' cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization.

A hybrid structural approach to analyze ligand binding by the serotonin type 4 receptor (5-HT4).

Hybrid structural methods have been used in recent years to understand protein-protein or protein-ligand interactions where high resolution crystallography or NMR data on the protein of interest has been limited. For G protein-coupled receptors (GPCRs), high resolution structures of native structural forms other than rhodopsin have not yet been achieved; gaps in our knowledge have been filled by creative crystallography studies that have developed stable forms of receptors by multiple means. The neurotransmitter serotonin (5-hydroxytryptamine) is a key GPCR-based signaling molecule affecting many physiological manifestations in humans ranging from mood and anxiety to bowel function. However, a high resolution structure of any of the serotonin receptors has not yet been solved. Here, we used structural mass spectrometry along with theoretical computations, modeling, and other biochemical methods to develop a structured model for human serotonin receptor subtype 4(b) in the presence and absence of its antagonist GR125487. Our data confirmed the overall structure predicted by the model and revealed a highly conserved motif in the ligand-binding pocket of serotonin receptors as an important participant in ligand binding. In addition, identification of waters in the transmembrane region provided clues as to likely paths mediating intramolecular signaling. Overall, this study reveals the potential of hybrid structural methods, including mass spectrometry, to probe physiological and functional GPCR-ligand interactions with purified native protein.

Infliximab reduces Zaprinast-induced retinal degeneration in cultures of porcine retina.

BACKGROUND: cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4 to 5% of retinitis pigmentosa (RP), a rare form of retinal dystrophy. Growing evidence suggests that inflammation is involved in the progression of RP. The aims of this study were to corroborate the presence of high TNFα concentration in the eyes of RP patients and to evaluate whether the blockade of TNFα with Infliximab, a monoclonal anti-TNFα antibody, prevented retinal degeneration induced by PDE6 inhibition in cultures of porcine retina. METHODS: Aqueous humor from 30 patients with RP and 13 healthy controls were used to quantify the inflammatory mediators IL-6, TNFα, IL-1ß, IL-10 by a multiplex enzyme-linked immunosorbent assay (ELISA) system. Retinal explants from pig were exposed to Zaprinast, a PDE6 inhibitor, for 24 hours in the absence or the presence of Infliximab. Cell death was evaluated by TUNEL assay. The number and distribution of caspase-3 positive cells, indirect poly(ADP)ribose polymerase (PARP) activation and glial fibrillary acidic protein (GFAP) content were visualized by immunolabeling. Antioxidant total capacity, nitrites and thiobarbituric acid reactive substances (TBARS) formation were determined to evaluate antioxidant-oxidant status. RESULTS: IL-6 and TNFα concentrations were higher in the aqueous humor of RP patients than in controls. Infliximab prevented retinal degeneration, as judging by the reduced presence of TUNEL-positive cells, the reduction of caspase-3 activation and also reduction of glial activation, in an ex vivo model of porcine retina. Additionally, Infliximab partially reduced oxidative stress in retinal explants exposed to Zaprinast. CONCLUSIONS: Inflammatory mediators IL-6 and TNFα were elevated in the aqueous humor of RP patients corroborating previous studies suggesting sustained chronic inflammation. Our study suggests that TNFα is playing an important role in cell death in an ex vivo model of retinal degeneration by activating different cell pathways at different cell layers of the retina that should be further studied.

Anti-TNF-α therapy in patients with refractory uveitis due to Behçet's disease: a 1-year follow-up study of 124 patients.

OBJECTIVE: The aim of this study was to assess the efficacy of anti-TNF-α therapy in refractory uveitis due to Behçet's disease (BD). METHODS: We performed a multicentre study of 124 patients with BD uveitis refractory to conventional treatment including high-dose corticosteroids and at least one standard immunosuppressive agent. Patients were treated for at least 12 months with infliximab (IFX) (3-5 mg/kg at 0, 2 and 6 weeks and then every 4-8 weeks) or adalimumab (ADA) (usually 40 mg every 2 weeks). The main outcome measures were degree of anterior and posterior chamber inflammation, visual acuity, macular thickness and immunosuppression load. RESULTS: Sixty-eight men and 56 women (221 affected eyes) were studied. The mean age was 38.6 years (s.d. 10.4). HLA-B51 was positive in 66.1% of patients and uveitis was bilateral in 78.2%. IFX was the first biologic agent in 77 cases (62%) and ADA was first in 47 (38%). In most cases anti-TNF-α drugs were used in combination with conventional immunosuppressive drugs. At the onset of anti-TNF-α therapy, anterior chamber and vitreous inflammation was observed in 57% and 64.4% of patients, respectively. In both conditions the damage decreased significantly after 1 year. At baseline, 50 patients (80 eyes) had macular thickening [optical coherence tomography (OCT) >250 µm] and 35 (49 eyes) had cystoid macular oedema (OCT>300 µm) that improved from 420 µm (s.d. 119.5) at baseline to 271 µm (s.d. 45.6) at month 12 (P < 0.01). The best-corrected visual acuity and the suppression load also showed significant improvement. After 1 year of follow-up, 67.7% of patients were inactive. Biologic therapy was well tolerated in most cases. CONCLUSION: Anti-TNF-α therapy is effective and relatively safe in refractory BD uveitis.

Structural basis for the function and inhibition of an influenza virus proton channel.

The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

Lack of association between the protein tyrosine phosphatase non-receptor type 22 R263Q and R620W functional genetic variants and endogenous non-anterior uveitis.

OBJECTIVE: Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility. METHODS: We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test. RESULTS: Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information. CONCLUSIONS: Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans.

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.