Evaluation of photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients with defects in the EYS gene: a possible cost-effective cellular model for mechanism-oriented drug.

BACKGROUND: The most common gene responsible for autosomal recessive retinitis pigmentosa (RP) is EYS. The manner of decay of genetically defective EYS gene transcripts varies depending on the type of mutation using our cellular model, which consists of induced photoreceptor-directed fibroblasts from EYS-RP patients (EYS-RP cells). However, disease-specific profiles have not been clarified in EYS-RP cells. Herein we investigated comprehensive gene expression patterns and restoration of altered expression by low molecular weight molecules in EYS-RP cells. METHODS: Using induced photoreceptor-like cells by CRX, RAX, NeuroD, and OTX2, we employed qRT-PCR and DNA microarray analysis to compare expression levels of disease-related genes in EYS-RP cells. We investigated the effect of antiapoptotic or anti-endoplasmic reticulum (ER) stress/antioxidant reagents on the restoration of altered gene expression. RESULTS: Expression levels of phototransduction-related genes (blue opsin, rhodopsin, S-antigen, GNAT1, GNAT2) were lower in EYS-RP cells. CRYGD was extracted by global gene expression analysis, as a downregulated, retina-related and apoptosis-, endoplasmic reticulum (ER) stress- or aging-related gene. Pathway enrichment analysis suggested that "complement and coagulation cascades," "ECM-receptor interaction" and "PI3K-Akt signaling pathway" could be involved in EYS-RP-associated pathogenesis. Among the matching/overlapping genes involved in those pathways, F2R was suggested as an EYS-RP-associated gene. The downregulation of CRYGD and F2R was completely restored by additional 4-PBA, an inhibitor of ER stress, and partially restored by metformin or NAC. In addition, 4-PBA normalized the expression level of cleaved caspase-3. CONCLUSIONS: Our cellular model may reflect the ER stress-mediated degenerative retina and serve as a pathogenesis-oriented cost-effective rescue strategy for RP patients.

Assessment of visual function and retinal structure following acute light exposure in the light sensitive T4R rhodopsin mutant dog.

The effect of acute exposure to various intensities of white light on visual behavior and retinal structure was evaluated in the T4R RHO dog, a naturally-occurring model of autosomal dominant retinitis pigmentosa due to a mutation in the Rhodopsin gene. A total of 14 dogs (ages: 4-5.5 months) were used in this study: 3 homozygous mutant RHO(T4R/T4R), 8 heterozygous mutant RHO(T4R/+), and 3 normal wild-type (WT) dogs. Following overnight dark adaptation, the left eyes were acutely exposed to bright white light with a monocular Ganzfeld dome, while the contralateral right eye was shielded. Each of the 3 homozygous (RHO(T4R/T4R)) mutant dogs had a single unilateral light exposure (LE) to a different (low, moderate, and high) dose of white light (corneal irradiance/illuminance: 0.1 mW/cm(2), 170 lux; 0.5 mW/cm(2), 820 lux; or 1 mW/cm(2), 1590 lux) for 1 min. All 8 heterozygous (RHO(T4R/+)) mutant dogs were exposed once to the same moderate dose of light. The 3 WT dogs had their left eyes exposed 1, 2, or 3 times to the same highest dose of light. Visual function prior to LE and at 2 weeks and 33 weeks after exposure was objectively assessed in the RHO(T4R/T4R) and WT dogs by using an obstacle-avoidance course. Transit time through the obstacle course was measured under different scotopic to photopic ambient illuminations. Morphological retinal changes were evaluated by non-invasive in vivo cSLO/sdOCT imaging and histology before and at several time-points (2-36 weeks) after light exposure. The analysis of the transit time through the obstacle course showed that no differences were observed in any of mutant or WT dogs at 2 weeks and 33 weeks post LE. The RHO(T4R/T4R) retina exposed to the lowest dose of white light showed no obvious changes in ONL thickness at 2 weeks, but mild decrease was noted 36 weeks after LE. The RHO(T4R/T4R) retina that received a moderate dose (showed an obvious decrease in ONL thickness along the superior and temporal meridians at 2 weeks post LE with more severe damage at 36 weeks post LE in all four meridians. The RHO(T4R/T4R) retina exposed to the high dose showed at 2 weeks after LE extensive ONL damage in all four meridians. This light intensity did not cause any retinal damage in WT dogs even after repeated (up to 3) LE. Analysis of ONL thickness in heterozygous mutant dogs exposed to the moderate dose of light confirmed the increased sensitivity to light damage of the superior/tapetal retina, and the occurrence of an ongoing cell death process several weeks after the acute LE. In conclusion, a short single exposure to a dose of white light that is not retinotoxic in WT dogs causes in the T4R RHO retina an acute loss of ONL in the central to mid peripheral region that keeps progressing over the course of several weeks. However, this severe retinal damage does not affect visual behavior presumably because of islands of surviving photoreceptors found in the area centralis including the newly discovered canine fovea-like area, and the lack of damage to peripheral photoreceptors.

Monte Carlo loop refinement and virtual screening of the thyroid-stimulating hormone receptor transmembrane domain.

Metropolis Monte Carlo (MMC) loop refinement has been performed on the three extracellular loops (ECLs) of rhodopsin and opsin-based homology models of the thyroid-stimulating hormone receptor transmembrane domain, a class A type G protein-coupled receptor. The Monte Carlo sampling technique, employing torsion angles of amino acid side chains and local moves for the six consecutive backbone torsion angles, has previously reproduced the conformation of several loops with known crystal structures with accuracy consistently less than 2 Å. A grid-based potential map, which includes van der Waals, electrostatics, hydrophobic as well as hydrogen-bond potentials for bulk protein environment and the solvation effect, has been used to significantly reduce the computational cost of energy evaluation. A modified sigmoidal distance-dependent dielectric function has been implemented in conjunction with the desolvation and hydrogen-bonding terms. A long high-temperature simulation with 2 kcal/mol repulsion potential resulted in extensive sampling of the conformational space. The slow annealing leading to the low-energy structures predicted secondary structure by the MMC technique. Molecular docking with the reported agonist reproduced the binding site within 1.5 Å. Virtual screening performed on the three lowest structures showed that the ligand-binding mode in the inter-helical region is dependent on the ECL conformations.

Optogenetic assessment of horizontal interactions in primary visual cortex.

Columnar organization of orientation selectivity and clustered horizontal connections linking orientation columns are two of the distinctive organizational features of primary visual cortex in many mammalian species. However, the functional role of these connections has been harder to characterize. Here we examine the extent and nature of horizontal interactions in V1 of the tree shrew using optical imaging of intrinsic signals, optogenetic stimulation, and multi-unit recording. Surprisingly, we find the effects of optogenetic stimulation depend primarily on distance and not on the specific orientation domains or axes in the cortex, which are stimulated. In addition, across a wide range of variation in both visual and optogenetic stimulation we find linear addition of the two inputs. These results emphasize that the cortex provides a rich substrate for functional interactions that are not limited to the orientation-specific interactions predicted by the monosynaptic distribution of horizontal connections.

Rapid diversification in the North American minnow genus Nocomis.

Diversification rates among stream fishes are likely governed by geographical factors that impact connectivity among and between stream systems. Herein we investigate rates of diversification within species of River Chubs, Nocomis, a monophyletic group of stream fishes distributed throughout the drainage basins of the Mississippi River, Atlantic Slope and Gulf Coast. We used nucleotide sequences of two protein-coding nuclear genes (IRBP and rhodopsin) and one mitochondrial gene (cytochrome b) to generate a phylogenetic hypothesis of population-level relationships within and among species. Results of molecular analyses suggest that morphological and meristic treatments of Nocomis have underestimated species diversity within the group. Tree-based Bayesian methods and diversification statistics were implemented to model the rate of evolutionary change along lineages and estimate divergence dates among phylogroups. Significantly elevated rates of cladogenesis are observed among coastally distributed populations 2-3 million years before present. This period was dominated by dramatic sea level fluctuations that suggest a period of climatic instability. Climatic instability and other factors may have driven the burst of rapid diversification observed in Nocomis. Results generated in this study reinforce faunistic and geologic arguments for the hypothesized existence of extinct rivers, such as White's River and the Appalachian River.

Assessment of cone survival in response to CNTF, GDNF, and VEGF165b in a novel ex vivo model of end-stage retinitis pigmentosa.

PURPOSE: To develop a robust ex vivo model for evaluating cone survival in end-stage retinitis pigmentosa (RP) and apply this to quantify the effects of putative neuroprotective compounds. METHODS: Rhodopsin knockout mice were crossed with OPN1-GFP reporter mice so that GFP-positive cones could be identified against the background of a rod-specific degeneration. Retinal explants were harvested from 10-week-old mice and maintained in organotypic culture. Ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), or vascular endothelial growth factor 165b (VEGF(165b)) was administered daily to treatment groups at three doses (200 ng/mL, 100 ng/mL, or 50 ng/mL; n = 5 explants per group). Fluorescence microscopy was performed on days 1, 3, 5, 7, 9, and 12 to document the number of GFP-expressing cones. RESULTS: Cone survival could be assessed reliably and reproducibly in this model, and cone degeneration was significantly greater in the absence of rods, in keeping with clinical observations of RP. Daily administration of 200 ng/mL CNTF led to significantly increased cone survival compared with sham-treated controls. The effect was dose dependent; 100 ng/mL CNTF reduced cone loss but to a lesser extent, and 200 ng/mL GDNF showed significant protection against cone loss at later time points (day 9-12) but was much less effective than CNTF at all doses. VEGF(165b) showed no neuroprotective effect in this model at any dose. CONCLUSIONS: This model allows precise quantification of the neuroprotective effects of various compounds on cone survival and may therefore provide a robust method of screening neuroprotective compounds before application in vivo.

Multiphoton adaptation of a commercial low-cost confocal microscope for live tissue imaging.

The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.

Phylogeny and biogeography of bees of the tribe Osmiini (Hymenoptera: Megachilidae).

The Osmiini (Megachilidae) constitute a taxonomically and biologically diverse tribe of bees. To resolve their generic and suprageneric relationships, we inferred a phylogeny based on three nuclear genes (Elongation factor 1-alpha, LW-rhodopsin and CAD) applying both parsimony and Bayesian methods. Our phylogeny, which includes 95 osmiine species representing 18 of the 19 currently recognized genera, is well resolved with high support for most basal nodes. The core osmiine genera were found to form a well-supported monophyletic group, but four small genera, Noteriades, Afroheriades,Pseudoheriades and possibly Ochreriades, formerly included in the Osmiini, do not appear to belong within this tribe. Our phylogeny results in the following taxonomic changes: Stenosmia and Hoplosmia are reduced to subgeneric rank in Hoplitis and Osmia, respectively, Micreriades is recognized as a subgenus in Hoplitis and the subgenus Nasutosmia is transferred from Hoplitis to Osmia. We inferred a biogeographic scenario for the Osmiini applying maximum likelihood inference and models of character evolution. We provide evidence that the Osmiini originated in the Palearctic, and that extensive exchanges occurred between the Palearctic and the Nearctic. The latter finding may relate to the fact that many osmiine species nest in wood or in stems, facilitating dispersal by overseas transport of the nests.

Key issues in the computational simulation of GPCR function: representation of loop domains.

Some key concerns raised by molecular modeling and computational simulation of functional mechanisms for membrane proteins are discussed and illustrated for members of the family of G protein coupled receptors (GPCRs). Of particular importance are issues related to the modeling and computational treatment of loop regions. These are demonstrated here with results from different levels of computational simulations applied to the structures of rhodopsin and a model of the 5-HT2A serotonin receptor, 5-HT2AR. First, comparative Molecular Dynamics (MD) simulations are reported for rhodopsin in vacuum and embedded in an explicit representation of the membrane and water environment. It is shown that in spite of a partial accounting of solvent screening effects by neutralization of charged side chains, vacuum MD simulations can lead to severe distortions of the loop structures. The primary source of the distortion appears to be formation of artifactual H-bonds, as has been repeatedly observed in vacuum simulations. To address such shortcomings, a recently proposed approach that has been developed for calculating the structure of segments that connect elements of secondary structure with known coordinates, is applied to 5-HT2AR to obtain an initial representation of the loops connecting the transmembrane (TM) helices. The approach consists of a simulated annealing combined with biased scaled collective variables Monte Carlo technique, and is applied to loops connecting the TM segments on both the extra-cellular and the cytoplasmic sides of the receptor. Although this initial calculation treats the loops as independent structural entities, the final structure exhibits a number of interloop interactions that may have functional significance. Finally, it is shown here that in the case where a given loop from two different GPCRs (here rhodopsin and 5-HT2AR) has approximately the same length and some degree of sequence identity, the fold adopted by the loops can be similar. Thus, in such special cases homology modeling might be used to obtain initial structures of these loops. Notably, however, all other loops in these two receptors appear to be very different in sequence and structure, so that their conformations can be found reliably only by ab initio, energy based methods and not by homology modeling.

Assessment of otocephalan and protacanthopterygian concepts in the light of multiple molecular phylogenies.

The rise of cladistics in ichthyology has dramatically improved our knowledge of teleostean basal interrelationships. However, some questions have remained open, among them the reliability of the Otocephala, a clade grouping clupeomorphs and ostariophysans, and the relationships of the Esocoidei. These two questions have been investigated in the light of new DNA sequences (from 28S and rhodopsin genes) and sequences from data banks (cytochrome b, 12-16S, 18S, MLL and RAG1). The ability of each of these markers to resolve basal teleostean interrelationships is assessed, and the cytochrome b was not found appropriate. Practical (i.e. different taxonomic samplings) and epistemological grounds led us to perform multiple separated phylogenetic analyses, in order to estimate the reliability of the above clades from their repeatability among trees from independent sequence data. The Otocephala are found monophyletic from most of the datasets; otherwise, they are not significantly contradicted from the others, which exhibit unresolved relationships. We conclude that the evidence provided here favours the sister-group relationship of clupeomorphs and ostariophysans. Morphological evidence including fossils is discussed, concluding that morphological works have not yet provided sufficient data to support this group. Salmonids and esocoids are found sister-groups from every molecular dataset in which these groups were sampled. Based on these convincing results, the Protacanthopterygii of Johnson and Patterson [1] are redefined, including the Esocoidei.