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Combustion velocities were experimentally determined for nanocomposite thermite powders composed of aluminum (Al) fuel and molybdenum trioxide (MoO3) oxidizer under well-confined conditions. Pressures were also measured to provide detailed information about the reaction mechanism. Samples of three different fuel particle sizes (44, 80, and 121 nm) were analyzed to determine the influence of particle size on combustion velocity. Bulk powder density was varied from approximately 5% to 10% of the theoretical maximum density (TMD). The combustion velocities ranged from approximately 600 to 1000 m/s. Results indicate that combustion velocities increase with decreasing particle size. Pressure measurements indicate that strong convective mechanisms are integral in flame propagation. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis, characterized by excessive deposition of extracellular matrix (ECM) proteins in the myocardium, distorts the architecture of the myocardium, facilitates the progression of arrhythmia and cardiac dysfunction, and influences the clinical course and outcome in patients with heart failure. This review describes the composition and homeostasis in normal cardiac interstitial matrix and introduces cellular and molecular mechanisms involved in cardiac fibrosis. We also characterize the ECM alteration in the fibrotic response under diverse cardiac pathological conditions and depict the role of matricellular proteins in the pathogenesis of cardiac fibrosis. Moreover, the diagnosis of cardiac fibrosis based on imaging and biomarker detection and the therapeutic strategies are addressed. Understanding the comprehensive molecules and pathways involved in ECM homeostasis and remodeling may provide important novel potential targets for preventing and treating cardiac fibrosis. [ABSTRACT FROM AUTHOR]

Structural models of the α, β, and γ modifications of Nb5Si3 silicides, which are used as a reinforcing phase in composites obtained in situ based on the Nb‒Si system, have been constructed by computer simulation methods. A geometric analysis of unit cells is performed using the H-poisk program to estimate the voids existing in the structures. The results of measuring the number of voids and their sizes are reported. A conclusion about possible diffusion paths of interstitial boron, carbon, nitrogen and oxygen atoms is drawn based on the calculation results, and the solubility of these impurities in the structure of each Nb5Si3 modification is estimated. [ABSTRACT FROM AUTHOR]

Retinal detachments create two pathological surfaces, the surface of the outer neural retinal, and an apical retinal-pigmented epithelium (RPE) surface. The physicochemical properties of these two new surfaces are poorly understood. At a molecular level little is known how detachments form, how to optimize reattachment, or prevent extension of the detachment. A major limitation is lack of information about the biophysical consequences of the retina–RPE separation. The primary challenge is determining the molecular properties of the pathological interface surfaces. Here, using detached bovine retina, we show that this hurdle can be overcome through a combination of biophysical and ultrastructural approaches. The outer surface of freshly detached bovine neural retina, and isolated molecular components of the outer retina were subjected to: 1) Contact angle goniometry to determine the critical surface tension of the outer retinal surface, isolated insoluble interphotoreceptor matrix (IPM) and purified interphotoreceptor retinoid binding protein (IRBP); 2) Multiple attenuated internal reflectance infrared (MAIR-IR) spectroscopy was used to characterize the molecular composition of the retinal surface. MAIR-IR depth penetration was established through ellipsometric measurement of barium-stearate films. Light microscopy, immunohistochemistry and electron microscopy defined the structures probed spectroscopically. Furthermore, the data were correlated to IR spectra of docosahexaenoic acid, hyaluronan, chondroitin-6-sulfate and IRBP, and imaging by IR-microscopy. We found that the retinal critical surface tension is 24 mN/m, similar to isolated insoluble IPM and lower than IRBP. Barium-stearate calibration studies established that the MAIR-IR spectroscopy penetration depth was 0.2 μm. Ultrastructural observations and MAIR-IR studies of isolated outer retina components determined that the pericellular IPM coating the outer retinal surface is primarily responsible for these surface properties. The critical surface tension of detached bovine retina is dictated not by the outer segments, but by a pericellular IPM covering the outer segment tips. [ABSTRACT FROM AUTHOR]

We search for cavitation in tumescent subcutaneous tissue of a live pig under application of pulsed, 1-MHz ultrasound at 8 W cm-2 spatial peak and pulse-averaged intensity. We find no evidence of broadband acoustic emission indicative of inertial cavitation. These acoustic parameters are representative of those used in external-ultrasound-assisted lipoplasty and in physical therapy and our null result brings into question the role of cavitation in those applications. A comparison of broadband acoustic emission from a suspension of ultrasound contrast agent in bulk water with a suspension injected subcutaneously indicates that the interstitial matrix suppresses cavitation and provides an additional mechanism behind the apparent lack of in-vivo cavitation to supplement the absence of nuclei explanation offered in the literature. We also find a short-lived cavitation signal in normal, non-tumesced tissue that disappears after the first pulse, consistent with cavitation nuclei depletion in vivo. [ABSTRACT FROM AUTHOR]

Background. Patients with prediabetes are at risk for diabetes, cardiovascular events, and microvascular complications. The rtx1 (Imagine Eyes, France) permits early detection of changes in the retinal photoreceptors and vessels. Objective. Cone parameters and retinal microvasculature were analyzed with the rtx1 in 12 prediabetic patients and 22 healthy subjects. The analysis was based on cone density (DM), interphotoreceptor distance (SM), cone packing regularity, and retinal vessel parameters: wall thickness, lumen diameter (LD), wall-to-lumen ratio (WLR), and cross-sectional area of the vascular wall. Results. DM in the prediabetic group was not significantly lower than that in the control group (18,935 ± 1713 cells/mm2 and 19,900 ± 2375 cells/mm2, respectively; p=0.0928). The LD and WLR means differed significantly between the prediabetic and the control groups (LD 94.3 ± 10.9 versus 101.2 ± 15, p=0.022; WLR 0.29 ± 0.05 versus 0.22 ± 0.03, p<0.05). A multivariate regression analysis showed that the WLR was significantly correlated with BMI and total cholesterol. Conclusions. Abnormalities found in rtx1 examinations indicated early signs of arteriolar dysfunction, prior to impaired glucose tolerance progressing to diabetes. The rtx1 retinal image analysis offers noninvasive measurement of early changes in the vasculature that routine clinical examination cannot detect. [ABSTRACT FROM AUTHOR]

The article discusses central pathological feature of many diseases in altered extracellular matrix. It also discusses current investigation in haemophilia, cause of recurrent and prolonged bleeding of the basement membranes and the interstitial matrix, collagen biomarkers of basement membrane and interstitial matrix turnover in a well-characterized case-control study.

A significant portion of patients diagnosed with vitelliform macular dystrophy (VMD) do not carry causative mutations in the classic VMD genes BEST1 or PRPH2. We therefore performed a mutational screen in a cohort of 106 BEST1/PRPH2-negative VMD patients in two genes encoding secreted interphotoreceptor matrix proteoglycans-1 and -2 (IMPG1 and IMPG2). We identified two novel mutations in IMPG1 in two simplex VMD cases with disease onset in their early childhood, a heterozygous p.(Leu238Pro) missense mutation and a homozygous c.807 + 5G > A splice site mutation. The latter induced partial skipping of exon 7 of IMPG1 in an in vitro splicing assay. Furthermore, we found heterozygous mutations including three stop [p.(Glu226*), p.(Ser522*), p.(Gln856*)] and five missense mutations [p.(Ala243Pro), p.(Gly1008Asp), p.(Phe1016Ser), p.(Tyr1042Cys), p.(Cys1077Phe)] in the IMPG2 gene, one of them, p.(Cys1077Phe), previously associated with VMD. Asymptomatic carriers of the p.(Ala243Pro) and p.(Cys1077Phe) mutations show subtle foveal irregularities that could characterize a subclinical stage of disease. Taken together, our results provide further evidence for an involvement of dominant and recessive mutations in IMPG1 and IMPG2 in VMD pathology. There is a remarkable similarity in the clinical appearance of mutation carriers, presenting with bilateral, central, dome-shaped foveal accumulation of yellowish material with preserved integrity of the retinal pigment epithelium (RPE). Clinical symptoms tend to be more severe for IMPG1 mutations. [ABSTRACT FROM AUTHOR]

Olfactomedin 1 (OLFM1) is a secreted glycoprotein and member of the olfactomedin protein family, which is preferentially expressed in various areas throughout the central nervous system. To learn about the functional properties of OLFM1 in the eye, we investigated its localization in the mouse and pig eye. In addition, we analyzed the ocular phenotype of Olfm1 mutant mice in which 52 amino acids were deleted in the central part (M2 region) of OLFM1. OLFM1 was detected in cornea, sclera, retina, and optic nerve of both wild-type and Olfm1 mutant littermates. By immunohistochemistry and double labeling with the lectin peanut agglutinin, OLFM1 was found in the interphotoreceptor matrix (IPM) of mouse and pig retina where it was directly localized to the inner segments of photoreceptors. Western blotting confirmed the presence of the OLFM1 isoforms pancortin 1 (BMY) and pancortin 2 (BMZ) in the IPM. The retinal phenotype of Olfm1 mutant mice did not obviously differ from that of wild-type littermates. In addition, outer nuclear layer (ONL) and total retinal thickness were not different, and the same was true for the area of the optic nerve in cross sections. Functional changes were observed though by electroretinography, which showed significantly lower a- and b-wave amplitudes in Olfm1 mutant mice when compared to age-matched wild-type mice. When light damage experiments were performed as an experimental paradigm of photoreceptor apoptosis, significantly more TUNEL-positive cells were observed in Olfm1 mutant mice 30 h after light exposure. One week after light exposure, the ONL was significantly thinner in Olfm1 mutant mice than in wild-type littermates indicating increased photoreceptor loss. No differences were observed when rhodopsin turnover or ERK1/2 signaling was investigated. We conclude that OLFM1 is a newly identified IPM molecule that serves an important role for photoreceptor homeostasis, which is significantly compromised in the eyes of Olfm1 mutant mice. [ABSTRACT FROM AUTHOR]

Experimental evidence suggests human Müller glia exhibit neural progenitor properties in vitro. CD117 and CD44 are known to be expressed by stem cells, the survival of which appears to depend critically on interactions with hyaluronan-rich extracellular matrix (ECM). Here, we characterise Müller glia expression of CD117 and CD44 in normal adult human retina and describe how it correlates with hyaluronan distribution in ocular ECM. By using chromogen-based immunohistochemistry, CD117 expression was found in entire Müller glia cytoplasm spanning from inner to outer limiting membrane in both peripheral retina (PR) and macular retina (MR), mirroring expression of the established Müller glia marker vimentin. Unlike vimentin, CD117 was also strongly expressed by Müller glia nuclei. Relative to total inner nuclear layer (INL) nuclei, more CD117+ Müller glia nuclei were seen in PR than MR. By contrast, CD44 expression was found predominantly in Müller glia apical processes of PR; no expression was found in MR. Astral blue staining demonstrated the presence of hyaluronan in cortical vitreous and the interphotoreceptor matrix (IPM) in both MR and PR. Our findings demonstrate that: (i) both CD117 and CD44 are expressed by human adult Müller glia; (ii) CD117 is a robust nuclear and cytoplasmic immunohistochemical marker of Müller glia; and (iii) that while CD117 is expressed by the entire Müller glia in both PR and MR, CD44 is only expressed by Müller glia apices in PR. Since the apices of Müller glia are in direct contact with the hyaluronan-rich IPM, the Müller glia-IPM interface in PR is likely a favourable region for supporting progenitor or stem cell-like signalling. These observations provide novel insights into potential stem-cell favouring microenvironments in mature human retina. [ABSTRACT FROM AUTHOR]

Understanding the molecular composition of ocular tissues and fluids could inform new approaches to prevalent causes of blindness. Subretinal fluid accumulating between the photoreceptor outer segments and retinal pigment epithelium (RPE) is potentially a rich source of proteins and lipids normally cycling among outer retinal cells and choroid. Herein, intact post-translationally modified proteins (proteoforms) were extracted from subretinal fluids of five patients with rhegmatogenous retinal detachment (RRD), analyzed by tandem mass spectrometry, and compared to published data on these same proteins as synthesized by other organs. Single-nuclei transcriptomic data from non-diseased human retina/RPE were used to identify whether proteins in subretinal fluid were of potential ocular origin. Two human donor eyes with normal maculas were immunoprobed for transthyretin (TTR) with appropriate controls. The three most abundant proteins detected in subretinal fluid were albumin, TTR, and apolipoprotein A-I. Remarkably, TTR relative to the other proteins was more abundant than its serum counterpart, suggestive of TTR being synthesized predominantly locally. Six proteoforms of TTR were detected, with the relative amount of glutathionylated TTR being much higher in the subretinal fluid (12–43%) than values reported for serum (<5%) and cerebrospinal fluid (0.4–13%). Moreover, a putative glycosylated TTR dimer of 32,428 Da was detected as the fourth most abundant protein. The high abundance of TTR and putative TTR dimer in subretinal fluid was supported by analysis of available single-nuclei transcriptomic data, which showed strong and specific signal for TTR in RPE. Immunohistochemistry further showed strong diffuse TTR immunoreactivity in choroidal stroma that contrasted with vertically aligned signal in the outer segment zone of the subretinal space and negligible signal in RPE cell bodies. These results suggest that TTR in the retina is synthesized intraocularly, and glutathionylation is crucial for its normal function. Further studies on the composition, function, and quantities of TTR and other proteoforms in subretinal fluid could inform mechanisms, diagnostic methods, and treatment strategies for age-related macular degeneration, familial amyloidosis, and other retinal diseases involving dysregulation of physiologic lipid transfer and oxidative stress. • Subretinal fluid removed during retinal detachment repair is rich in transthyretin. • Transthyretin in subretinal fluid, unlike elsewhere, is highly glutathionylated. • A putative glycosylated transthyretin dimer of 32,428 Da is detected. • Transthyretin is synthesized in the RPE and secreted into interphotoreceptor matrix. [ABSTRACT FROM AUTHOR]

Multiple functions for Interphotoreceptor Retinoid-Binding Protein (IRBP) may explain its localization in the retina, vitreous and pineal gland and association with retinitis pigmentosa and myopia. We have been engaged in uncovering the structure-function relationships of this interesting protein long thought to bind visual-cycle retinoids and fatty acids in the subretinal space. Although hydrophobic domains capable of binding such ligands have now been found, we ask what other structural domains might be present that could predict new functions? Interestingly, IRBP possesses a fold similar to C-terminal processing proteases (CTPases) but is missing the PDZ domain. Here we present structural evidence that this fold may have a role in a recently observed autoproteolytic activity of the two-module zebrafish (z) IRBP (Ghosh et al. Exp. Eye Res., 2015). When the structure of Scenedesmus obliquus D1 CTPase (D1P) is superimposed with the first module of zIRBP (z1), the PDZ domain of D1P occupies roughly the same position in the amino acid sequence as the inter-domain tether in z1, between residues P71 and P85. The catalytic triad K397, S372 and E375 of D1P is located at the inter-domain interfacial cleft, similarly as the tetrad K241, S243, D177 and T179 of z1 residues, presumed to have proteolytic function. Packing of two adjacent symmetry-related molecules within the z1 crystal show that the helix α8 penetrates the interfacial cleft underneath the inter-domain tether, forming a simple intermolecular “knot”. The full-length zIRBP is cleaved at or immediately after T309, which is located at the end of α8 and is the ninth residue of the second module z2. We propose that the helix α8 within intact zIRBP bends at P301, away from the improbable knotted fold, and positions the cleavage site T309 near the putative catalytic tetrad of the neighboring zIRBP to be proteolytically cleaved. The conservation of this functional catalytic domain suggests that possible physiological roles of IRBP as a hydrolase needs to be considered. [ABSTRACT FROM AUTHOR]

Background: Crystalloid fluid requires 30 min for complete distribution throughout the extracellular fluid space and tends to cause long-standing peripheral edema. A kinetic analysis of the distribution of Ringer's acetate with increasing infusion rates was performed to obtain a better understanding of these characteristics of crystalloids.Methods: Data were retrieved from six studies in which 76 volunteers and preoperative patients had received between 300 ml and 2375 ml of Ringer's acetate solution at a rate of 20-80 ml/min (0.33-0.83 ml/min/kg). Serial measurements of the blood hemoglobin concentration were used as inputs in a kinetic analysis based on a two-volume model with micro-constants, using software for nonlinear mixed effects.Results: The micro-constants describing distribution (k12) and elimination (k10) were unchanged when the rate of infusion increased, with half-times of 16 and 26 min, respectively. In contrast, the micro-constant describing how rapidly the already distributed fluid left the peripheral space (k21) decreased by 90% when the fluid was infused more rapidly, corresponding to an increase in the half-time from 3 to 30 min. The central volume of distribution (V(c)) doubled.Conclusion: The return of Ringer's acetate from the peripheral fluid compartment to the plasma was slower with high than with low infusion rates. Edema is a normal consequence of plasma volume expansion with this fluid, even in healthy volunteers. The results are consistent with the view that the viscoelastic properties of the interstitial matrix are responsible for the distribution and redistribution characteristics of crystalloid fluid. [ABSTRACT FROM AUTHOR]

Numerous preclinical and clinical studies have focused on the periodontal regenerative functions of enamel matrix derivative (EMD), a heat-treated preparation derived from enamel matrix proteins (EMPs) of developing porcine teeth. In this study, periodontal ligament (PDL) stem cells (PDLSCs) were isolated, and the effects of EMD on the extracorporeal induction process and the characteristics of PDLSC sheets were investigated for their potential as a more effective stem-cell therapy. EMD-enhanced cell sheets could be induced by complete medium supplemented with 50 μg/mL vitamin C and 100 μg/mL EMD. The EMD-enhanced cell sheets appeared thicker and more compact than the normal PDLSC sheets, demonstrated more layers of cells (3–7 layers), secreted richer extracellular matrix (ECM), showed varying degrees of increases in mRNA expression of periodontal tissue-specific genes (COL I, POSTN), calcification-related genes (RUNX2, OPN, OCN) and a cementum tissue-specific gene (CAP), and possessed a better mineralization ability in terms of osteogenic differentiation in vitro. These EMD-enhanced cell sheets may represent a potential option for stem-cell therapy for PDL regeneration. [ABSTRACT FROM AUTHOR]

Oxidative stress is involved in activating photoreceptor death in several retinal degenerations. Docosahexaenoic acid ( DHA), the major polyunsaturated fatty acid in the retina, protects cultured retina photoreceptors from apoptosis induced by oxidative stress and promotes photoreceptor differentiation. Here, we investigated whether eicosapentaenoic acid ( EPA), a metabolic precursor to DHA, had similar effects and whether retinal neurons could metabolize EPA to DHA. Adding EPA to rat retina neuronal cultures increased opsin expression and protected photoreceptors from apoptosis induced by the oxidants paraquat and hydrogen peroxide (H2O2). Palmitic, oleic, and arachidonic acids had no protective effect, showing the specificity for DHA. We found that EPA supplementation significantly increased DHA percentage in retinal neurons, but not EPA percentage. Photoreceptors and glial cells expressed Δ6 desaturase ( FADS2), which introduces the last double bond in DHA biosynthetic pathway. Pre-treatment of neuronal cultures with CP-24879 hydrochloride, a Δ5/Δ6 desaturase inhibitor, prevented EPA-induced increase in DHA percentage and completely blocked EPA protection and its effect on photoreceptor differentiation. These results suggest that EPA promoted photoreceptor differentiation and rescued photoreceptors from oxidative stress-induced apoptosis through its elongation and desaturation to DHA. Our data show, for the first time, that isolated retinal neurons can synthesize DHA in culture. [ABSTRACT FROM AUTHOR]

Rod and cone photoreceptors are highly similar in many respects but they have important functional and molecular differences. Here, we investigate genome-wide patterns of DNA methylation and chromatin accessibility in mouse rods and cones and correlate differences in these features with gene expression, histone marks, transcription factor binding, and DNA sequence motifs. Loss of NR2E3 in rods shifts their epigenomes to a more cone-like state. The data further reveal wide differences in DNA methylation between retinal photoreceptors and brain neurons. Surprisingly, we also find a substantial fraction of DNA hypo-methylated regions in adult rods that are not in active chromatin. Many of these regions exhibit hallmarks of regulatory regions that were active earlier in neuronal development, suggesting that these regions could remain undermethylated due to the highly compact chromatin in mature rods. This work defines the epigenomic landscapes of rods and cones, revealing features relevant to photoreceptor development and function. [ABSTRACT FROM AUTHOR]

Detailed immunocytochemical analyses comparing wild-type (WT), GRF1-knockout (KO), GRF2-KO and GRF1/2 double-knockout (DKO) mouse retinas uncovered the specific accumulation of misplaced, 'ectopic' cone photoreceptor nuclei in the photoreceptor segment (PS) area of retinas from GRF2-KO and GRF1/2-DKO, but not of WT or GRF1-KO mice. Localization of ectopic nuclei in the PS area of GRF2-depleted retinas occurred postnatally and peaked between postnatal day (P)11 and P15. Mechanistically, the generation of this phenotype involved disruption of the outer limiting membrane and intrusion into the PS layer by cone nuclei displaying significant perinuclear accumulation of signaling molecules known to participate in nuclear migration and cytoskeletal reorganization, such as PAR3, PAR6 and activated, phosphorylated forms of PAK, MLC2 and VASP. Electroretinographic recordings showed specific impairment of cone-mediated retinal function in GRF2-KO and GRF1/2-DKO retinas compared with WT controls. These data identify defective cone nuclear migration as a novel phenotype in mouse retinas lacking GRF2 and support a crucial role of GRF2 in control of the nuclear migration processes required for proper postnatal development and function of retinal cone photoreceptors. [ABSTRACT FROM AUTHOR]

Interphotoreceptor retinoid-binding protein (IRBP) has a remarkable role in targeting and protecting all- trans and 11- cis retinol, and 11- cis retinal during the rod and cone visual cycles. Little is known about how the correct retinoid is efficiently delivered and removed from the correct cell at the required time. It has been proposed that different fatty composition at that the outer-segments and retinal-pigmented epithelium have an important role is regulating the delivery and uptake of the visual cycle retinoids at the cell-interphotoreceptor-matrix interface. Although this suggests intriguing mechanisms for the role of local fatty acids in visual-cycle retinoid trafficking, nothing is known about the structural basis of IRBP-fatty acid interactions. Such regulation may be mediated through IRBP's unusual repeating homologous modules, each containing about 300 amino acids. We have been investigating structure-function relationships of Zebrafish IRBP (zIRBP), which has only two tandem modules (z1 and z2), as a model for the more complex four-module mammalian IRBP's. Here we report the first X-ray crystal structure of a teleost IRBP, and the only structure with a bound ligand. The X-ray structure of z1, determined at 1.90 Å resolution, reveals a two-domain organization of the module (domains A and B). A deep hydrophobic pocket with a single bound molecule of oleic acid was identified within the N-terminal domain A. In fluorescence titrations assays, oleic acid displaced all- trans retinol from zIRBP. Our study, which provides the first structure of an IRBP with bound ligand, supports a potential role for fatty acids in regulating retinoid binding. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is characterized by irreversible damage of photoreceptors in the central posterior part of the retina, called the macula and is the most common cause of vision loss in those aged over 50. A growing body of evidence shows that cumulative long-term exposure to UV radiation may be harmful to the retina and possibly leads to AMD irrespective of age. In spite of many research efforts, cellular and molecular mechanisms leading to UV-induced retinal damage and possibly retinal diseases such as AMD are not completely understood. In the present study we explored damage mechanisms accounting for UV-induced retinal phototoxicity in the rats exposed to UVA and UVB irradiation using a proteomics approach. Our study showed that UV irradiation induces profound changes in the retinal proteomes of the rats associated with the disruption of energy homeostasis, oxidative stress, DNA damage response and structural and functional impairments of the interphotoreceptor matrix components and their cell surface receptors such as galectins. Two small leucine-rich proteoglycans, biglycan and lumican, were identified as phototoxicity biomarkers associated with UV-induced disruption of interphotoreceptor matrix (IPM). In addition, UVB induced activation of Src kinase, which could account for cytoskeletal rearrangements in the retina was observed at the proteomics level. Pharmacological intervention either to target Src kinase with the aim of preventing cytoskeletal rearrangements in the retinal pigment epithelium (RPE) and neuronal retina or to help rebuild damaged IPM may provide fresh avenues of treatment for patients suffering from AMD. [ABSTRACT FROM AUTHOR]

Blood flow in the retina is intrinsically regulated to meet the metabolic demands of its constituent cells. Flickering light or stationary contrast reversals induce an increase in blood flow within seconds of the stimulus onset. This phenomenon is thought to compensate for an increase in ganglion cell activity and energy consumption. Ganglion cell activity is in turn dependent on signals from photoreceptors, bipolar cells, horizontal cells and amacrine cells. The physiological properties of these neurons determine how each type is affected by a particular light characteristic. Neuronal activity then triggers the release of signalling molecules that dilate local blood vessels and increase blood flow. Nitric oxide has been implicated as an important mediator, but metabolites of arachidonic acid may also be involved. Detailed elucidation of these mechanisms, together with advances in imaging technology, may facilitate the use of neurovascular tests to improve the detection of retinal damage in pathological conditions. [ABSTRACT FROM AUTHOR]

Dysfunction of the retinal pigmented epithelium (RPE) results in degeneration of photoreceptors and vision loss and is correlated with common blinding disorders in humans. Although many protein-coding genes are known to be expressed in RPE and are important for its development and maintenance, virtually nothing is known about the in vivo roles of non-coding transcripts. The expression patterns of microRNAs (miRNAs) have been analyzed in a variety of ocular tissues, and a few were implicated to play role in RPE based on studies in cell lines. Here, through RPE-specific conditional mutagenesis of Dicer1 or Dgcr8 in mice, the importance of miRNAs for RPE differentiation was uncovered. miRNAs were found to be dispensable for maintaining RPE fate and survival, and yet they are essential for the acquisition of important RPE properties such as the expression of genes involved in the visual cycle pathway, pigmentation and cell adhesion. Importantly, miRNAs of the RPE are required for maturation of adjacent photoreceptors, specifically for the morphogenesis of the outer segments. The alterations in the miRNA and mRNA profiles in the Dicer1-deficient RPE point to a key role of miR-204 in regulation of the RPE differentiation program in vivo and uncover the importance of additional novel RPE miRNAs. This study reveals the combined regulatory activity of miRNAs that is required for RPE differentiation and for the development of the adjacent neuroretina. [ABSTRACT FROM AUTHOR]

Purpose To examine the expression of interstitial extracellular matrix components and their role during retinal development. Material and methods Fibronectin (FN), collagen IV (Coll IV) and laminin 5 (Lam 5) expression in rat retinas from developmental stages E17 to adult were studied. In addition, PN5 full-thickness retinas were cultured for 7 days with dispase, which selectively cleaves FN and Coll IV, at either 0.5 U/ml or 5.0 U/ml for 3 or 24 h. Eyecups and retinal cultures were examined morphologically using hematoxylin and eosin staining and immunohistochemistry. Results Coll IV, Lam 5 and FN were all transiently expressed in the interstitial matrix of the retinal layers during development. The retinal layers in dispase treated explants was severely disturbed in a dose and time dependent manner. Conclusions FN, Lam 5 and Coll IV, are present in the interstitial extracellular matrix during rat retinal development. Enzymatic cleavage of FN and Coll IV early in the lamination process disrupts the retinal layers implicating their pivotal role in this process. [ABSTRACT FROM AUTHOR]

The interphotoreceptor matrix (IPM) is a highly organized structure with interconnected domains surrounding cone and rod photoreceptor cells and extends throughout the subretinal space. Based on known roles of the extracellular matrix in other tissues, the IPM is thought to have several prominent functions including serving as a receptor for growth factors, regulating retinoid transport, participating in cytoskeletal organization in surrounding cells, and regulation of oxygen and nutrient transport. In addition, a number of studies suggest that the IPM also may play a significant role in the etiology of retinal degenerative disorders. In this review, we describe the present knowledge concerning the structure and function of the IPM under physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

The current knowledge and description of guest molecules within clathrate hydrates only accounts for occupancy within regular polyhedral water cages. Experimental measurements and simulations, examining the tert-butylamine+H2+H2O hydrate system, now suggest that H2 can also be incorporated within hydrate crystal structures by occupying interstitial sites, that is, locations other than the interior of regular polyhedral water cages. Specifically, H2 is found within the shared heptagonal faces of the large (43596273) cage and in cavities formed from the disruption of smaller (4454) water cages. The ability of H2 to occupy these interstitial sites and fluctuate position in the crystal lattice demonstrates the dynamic behavior of H2 in solids and reveals new insight into guest-guest and guest-host interactions in clathrate hydrates, with potential implications in increasing overall energy storage properties. [ABSTRACT FROM AUTHOR]

The retinal pigment epithelium (RPE) constitutes a monolayer of cuboidal cells that interact apically with the interphotoreceptor matrix (IPM) and outer segments of the photoreceptor cells and basally with the subjacent Bruch's membrane. This highly polarized structure is maintained by the cytoskeleton of individual cells and their interactions at the basolateral junctional complexes that stabilize this epithelial structure. This RPE complex network of filaments, tubules and associated proteins is modeled by the cellular environment, the RPE intercellular interactions, and by its interactions with the extracellular matrix. This is a review of the key features of the RPE cytoskeleton in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Abstract: Interphotoreceptor retinoid-binding protein (IRBP), which is critical to photoreceptor survival and function, is comprised of homologous tandem modules each ∼300 amino acids, and contains 10 cysteines, possibly 8 as free thiols. Purification of IRBP has historically been difficult due to aggregation, denaturation and precipitation. Our observation that reducing agent 1,4-dithiothreitol dramatically prevents aggregation prompted investigation of possible functions for IRBP's free thiols. Bovine IRBP (bIRBP) was purified from retina saline washes by a combination of concanavalin A, ion exchange and size exclusion chromatography. Antioxidant activity of the purified protein was measured by its ability to inhibit oxidation of 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonate] by metmyoglobin. Homology modeling predicted the relationship of the retinoid binding sites to cysteine residues. As a free radical scavenger, bIRBP was more active than ovalbumin, thioredoxin, and vitamin E analog Trolox. Alkylation of free cysteines by N-ethylmaleimide inhibited bIRBP's antioxidant activity, but not its ability to bind all-trans retinol. Structural modeling predicted that Cys 1051 is at the mouth of the module 4 hydrophobic ligand-binding site. Its free radical scavenging activity points to a new function for IRBP in defining the redox environment in the subretinal space. [Copyright &y& Elsevier]

Tissue engineering has been widely applied in different areas of regenerative medicine, including retinal regeneration. Typically, artificial biopolymers require additional surface modification (e.g. with arginine-glycine-aspartate-containing peptides or adsorption of protein, such as fibronectin), before cell seeding. Here, we describe an alternative approach for scaffold design: the manufacture of hybrid interphotoreceptor matrix-poly (ε-caprolactone) scaffolds, in which the insoluble extracellular matrix of the retina is incorporated into a biodegradable polymer well suited for transplantation. The incorporation of interphotoreceptor matrix did not change the topography of polycaprolactone film, although it led to a slight increase in hydrophilic properties (water contact angle measurements). This hybrid scaffold provided sufficient stimuli for human retinal progenitor cell adhesion and inhibited proliferation, leading to differentiation toward photoreceptor cells (expression of Crx, Nrl, rhodopsin, ROM1). This scaffold may be used for transplantation of retinal progenitor cells and their progeny to treat retinal degenerative disorders. [ABSTRACT FROM AUTHOR]

Highlights: [•] Interphotoreceptor retinoid-binding protein (IRBP) binds a variety of ligands. [•] A surface plasmon resonance biosensor was employed to study these interactions. [•] IRBP is able to bind carotenoids and retinoids with similar affinities. [•] IRBP has 10times less affinity for ocular fatty acids. [•] IRBP may function as a carrier of carotenoids across the interphotoreceptor matrix. [ABSTRACT FROM AUTHOR]

Adaptation is at the heart of sensation and nowhere is it more salient than in early visual processing. Light adaptation in photoreceptors is doubly dynamical: it depends upon the temporal structure of the input and it affects the temporal structure of the response. We introduce a non-linear dynamical adaptation model of photoreceptors. It is simple enough that it can be solved exactly and simulated with ease; analytical and numerical approaches combined provide both intuition on the behavior of dynamical adaptation and quantitative results to be compared with data. Yet the model is rich enough to capture intricate phenomenology. First, we show that it reproduces the known phenomenology of light response and short-term adaptation. Second, we present new recordings and demonstrate that the model reproduces cone response with great precision. Third, we derive a number of predictions on the response of photoreceptors to sophisticated stimuli such as periodic inputs, various forms of flickering inputs, and natural inputs. In particular, we demonstrate that photoreceptors undergo rapid adaptation of response gain and time scale, over ∼ 300 ms—i. e., over the time scale of the response itself—and we confirm this prediction with data. For natural inputs, this fast adaptation can modulate the response gain more than tenfold and is hence physiologically relevant. [ABSTRACT FROM AUTHOR]

Capture of light in the photoreceptor outer segment initiates a cascade of chemical events that inhibit neurotransmitter release, ultimately resulting in vision. The massed response of the photoreceptor population can be measured non-invasively by electrical recordings, but responses from individual cells cannot be measured without dissecting the retina. Here we used optical imaging to observe individual human cones in the living eye as they underwent bleaching of photopigment and associated phototransduction. The retina was simultaneously stimulated and observed with high intensity visible light at 1 kHz, using adaptive optics. There was marked variability between individual cones in both photosensitivity and pigment optical density, challenging the conventional assumption that photoreceptors act as identical subunits (coefficient of variation in rate of photoisomerization = 23%). There was also a pronounced inverse correlation between these two parameters (p<10−7); the temporal evolution of image statistics revealed this to be a dynamic relationship, with cone waveguiding efficiency beginning a dramatic increase within 3 ms of light onset. Beginning as early as 2 ms after light onset and including half of cells by ∼7 ms, cone intensity showed reversals characteristic of interference phenomena, with greater delays in reversal corresponding to cones with more photopigment (p<10−3). The timing of these changes is argued to best correspond with either the cessation of dark current, or to related events such as changes in intracellular cGMP. Cone intensity also showed fluctuations of high frequency (332±25 Hz) and low amplitude (3.0±0.85%). Other groups have shown similar fluctuations that were directly evoked by light; if this corresponds to the same phenomenon, we propose that the amplitude of fluctuation may be increased by the use of a bright flash followed by a brief pause, to allow recovery of cone circulating current. [ABSTRACT FROM AUTHOR]

Abstract: Evidence is reviewed from a wide range of studies relevant to the evolution of vertebrate photoreceptors and phototransduction, in order to permit the synthesis of a scenario for the major steps that occurred during the evolution of cones, rods and the vertebrate retina. The ancestral opsin originated more than 700 Mya (million years ago) and duplicated to form three branches before cnidarians diverged from our own lineage. During chordate evolution, ciliary opsins (C-opsins) underwent multiple stages of improvement, giving rise to the ‘bleaching’ opsins that characterise cones and rods. Prior to the ‘2R’ rounds of whole genome duplication near the base of the vertebrate lineage, ‘cone’ photoreceptors already existed; they possessed a transduction cascade essentially the same as in modern cones, along with two classes of opsin: SWS and LWS (short- and long-wave-sensitive). These cones appear to have made synaptic contact directly onto ganglion cells, in a two-layered retina that resembled the pineal organ of extant non-mammalian vertebrates. Interestingly, those ganglion cells appear to be descendants of microvillar photoreceptor cells. No lens was associated with this two-layered retina, and it is likely to have mediated circadian timing rather than spatial vision. Subsequently, retinal bipolar cells evolved, as variants of ciliary photoreceptors, and greatly increased the computational power of the retina. With the advent of a lens and extraocular muscles, spatial imaging information became available for central processing, and gave rise to vision in vertebrates more than 500 Mya. The ‘2R’ genome duplications permitted the refinement of cascade components suitable for both rods and cones, and also led to the emergence of five visual opsins. The exact timing of the emergence of ‘true rods’ is not yet clear, but it may not have occurred until after the divergence of jawed and jawless vertebrates. [Copyright &y& Elsevier]

The close packing of vertebrate photoreceptors presents a challenge to the exchange of molecules between the outer segments, retinal pigmented epithelium (RPE), and Müller glia. An extracellular hyaluronan scaffold separates these cells while soluble interphotoreceptor matrix (IPM) proteins traffic visual cycle retinoids, fatty acids, and other molecules between them. In the IPM, retinoids and fatty acids are carried by interphotoreceptor retinoid-binding protein (IRBP). The fact that much of the retina's IRBP can be extracted by saline wash has led to the notion that IRBP does not bind to the retina, but freely distributes itself within the subretinal space. In this study, we challenge this idea by asking if there are specialized IPM domains that bind IRBP, perhaps facilitating its ability to target delivery/uptake of its ligands. Xenopus is an ideal animal model to study the role of the IPM in RPE-photoreceptor interactions. Here, we took advantage of the large size of its photoreceptors, ability to detach the retina in light, sustainability of the retina in short term organ culture, and the availability of recombinant full-length Xenopus IRBP and antisera directed against Xenopus IRBP. We compared the distribution of wash resistant native IRBP, and that of IRBP-Alexa 647 binding in Xenopus retina. IRBP and cone opsin were localized using anti-Xenopus IRBP serum, and monoclonal COS-1 respectively. Cone matrix sheath proteoglycans were localized with wheat germ agglutinin (WGA), and diffuse IPM proteoglycans with peanut agglutinin (PNA). Wholemounts and frozen sections were compared by immunofluorescence from retinas detached under Ringer's followed by additional washes, or detached directly under 4% paraformaldehyde without Ringer's wash. Undetached Lowicryl embedded retinas were subjected to IRBP immunogold electron microscopy (EM). Immunogold labeled a diffuse network of filamentous structures, and a separate distinct flocculant material directly coating the outer segments, filling the rod periciliary ridge, and associated with Müller microvilli. By immunofluorescence, Ringer's wash removed most of the diffuse IRBP, but not that coating the outer segments. IRBP-Alexa 647 bound to the cone outer segments and Müller villi region, and comparably less to rod outer segments. Co-incubation with unlabeled IRBP markedly reduced this binding; ovalbumin-Alexa 647 and Alexa 647 dye alone showed no binding. Our data suggest that the pericellular matrix of the cone outer segments and Müller microvilli provide specialized domains that facilitate IRBP's functions. [ABSTRACT FROM AUTHOR]

Purpose: To investigate changes in cone photoreceptors with adaptive optics (AO) fundus imaging and spectral domain optical coherence tomography (SD-OCT) in a case of occult macular dystrophy (OMD). Patient and methods: Both eyes of a 42-year-old woman diagnosed with OMD were examined. We used an AO fundus camera to obtain images of cone photoreceptors in the macula of the OMD subject and five healthy control subjects. Correlations between the AO images and the SD-OCT images were examined. Cone photoreceptors in eight areas in the macula of OMD and healthy control subjects were analyzed and compared. Results: SD-OCT showed a loss of the cone outer-segment tips line outside of the fovea in both eyes of the subject with OMD. The left eye with decreased visual acuity showed a discontinuous photoreceptor inner-segment and outer-segment line and cone outer-segment tips line at the fovea in SD-OCT and loss of cone mosaics as a dark spot in the AO image. In panoramic AO images and cone-density maps, less cone density was observed in a ring-like region outside the fovea than in the peripheral retina. In most of the areas examined, the cone densities were lower in the OMD eyes than in the healthy control eyes. Conclusions: Cone densities in the macula of the OMD patient were greatly decreased. AO images were found to be useful to evaluate morphologic changes in cone photoreceptors in patients with OMD. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the variation in human cone photoreceptor packing density with various demographic or clinical factors, cone packing density was measured using a Canon prototype adaptive optics scanning laser ophthalmoscope and compared as a function of retinal eccentricity, refractive error, axial length, age, gender, race/ethnicity and ocular dominance. We enrolled 192 eyes of 192 subjects with no ocular pathology. Cone packing density was measured at three different retinal eccentricities (0.5 mm, 1.0 mm, and 1.5 mm from the foveal center) along four meridians. Cone density decreased from 32,200 to 11,600 cells/mm2 with retinal eccentricity (0.5 mm to 1.5 mm from the fovea, P < 0.001). A trend towards a slightly negative correlation was observed between age and density (r = −0.117, P = 0.14). There was, however, a statistically significant negative correlation (r = −0.367, P = 0.003) between axial length and cone density. Gender, ocular dominance, and race/ethnicity were not important determinants of cone density (all, P > 0.05). In addition, to assess the spatial arrangement of the cone mosaics, the nearest-neighbor distances (NNDs) and the Voronoi domains were analyzed. The results of NND and Voronoi analysis were significantly correlated with the variation of the cone density. Average NND and Voronoi area were gradually increased (all, P ≤ 0.001) and the degree of regularity of the cone mosaics was decreased (P ≤ 0.001) with increasing retinal eccentricity. In conclusion, we demonstrated cone packing density decreases as a function of retinal eccentricity and axial length and the results of NND and Voronoi analysis is a useful index for cone mosaics arrangements. The results also serve as a reference for further studies designed to detect or monitor cone photoreceptors in patients with retinal diseases. [Copyright &y& Elsevier]

We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. Protease- and protease inhibitor--specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. Interestingly, the peri-islet BM and underlying IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies. [ABSTRACT FROM AUTHOR]

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2+/Δ307), decrease (Crb1rd8/rd8, Gnat1-/-, Rho-/-), or remain constant (PDE6βrd1/rd1, Prph2rd2/rd2) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho-/-), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment. [ABSTRACT FROM AUTHOR]

We report a case of an interstitial chromosome 20q11.21 microdeletion in a 7-year-old male child presenting with mild intellectual disability and facial dysmorphisms. Array comparative genomic hybridization (CGH) has shown that the deletion resulted in the loss of 68 genes, among which 5 genes (COX4I2, MYLK2, ASXL1, DNMT3B, and SNTA1) are disease causing. The size of the deletion was estimated to span 2.6Mb. Only three cases of deletions encompassing this chromosomal region have been reported. The phenotype of the index patient was found to resemble the mildest cases of Bohring-Opitz syndrome that is caused by ASXL1 mutations.An in silico evaluation of the deleted genomic region has shown that benign genomic variations have never been observed to affect the ASXL1 gene, in contrast to the other disease-causing genes. As a result, it was suggested that ASXL1 loss is likely to be the main cause of the phenotypic manifestations. The present case report indicates that a loss of the disease-causing gene can produce a milder phenotype of a single gene condition. [ABSTRACT FROM AUTHOR]

Interneuronal subtype diversity lies at the heart of the distinct molecular properties and synaptic connections that shape the formation of the neuronal circuits that are necessary for the complex spatial and temporal processing of sensory information. Here, we investigate the role of Irx6, a member of the Iroquois homeodomain transcription factor family, in regulating the development of retinal bipolar interneurons. Using a knock-in reporter approach, we show that, in the mouse retina, Irx6 is expressed in type 2 and 3a OFF bipolar interneurons and is required for the expression of cell type-specific markers in these cells, likely through direct transcriptional regulation. In Irx6 mutant mice, presumptive type 3a bipolar cells exhibit an expansion of their axonal projection domain to the entire OFF region of the inner plexiform layer, and adopt molecular features of both type 2 and 3a bipolar cells, highlighted by the ectopic upregulation of neurokinin 3 receptor (Nk3r) and Vsx1. These findings reveal Irx6 as a key regulator of type 3a bipolar cell identity that prevents these cells from adopting characteristic features of type 2 bipolar cells. Analysis of the Irx6;Vsx1 double null retina suggests that the terminal differentiation of type 2 bipolar cells is dependent on the combined expression of the transcription factors Irx6 and Vsx1, but also points to the existence of Irx6;Vsx1-independent mechanisms in regulating OFF bipolar subtype-specific gene expression. This work provides insight into the generation of neuronal subtypes by revealing a mechanism in which opposing, yet interdependent, transcription factors regulate subtype identity. [ABSTRACT FROM AUTHOR]

Retinal degenerative diseases lead to blindness due to loss of the 'image capturing' photoreceptors, while neurons in the 'image-processing' inner retinal layers are relatively well preserved. Electronic retinal prostheses seek to restore sight by electrically stimulating the surviving neurons. Most implants are powered through inductive coils, requiring complex surgical methods to implant the coil-decoder-cable-array systems that deliver energy to stimulating electrodes via intraocular cables. We present a photovoltaic subretinal prosthesis, in which silicon photodiodes in each pixel receive power and data directly through pulsed near-infrared illumination and electrically stimulate neurons. Stimulation is produced in normal and degenerate rat retinas, with pulse durations of 0.5-4 ms, and threshold peak irradiances of 0.2-10 mW mm?2, two orders of magnitude below the ocular safety limit. Neural responses were elicited by illuminating a single 70 µm bipolar pixel, demonstrating the possibility of a fully integrated photovoltaic retinal prosthesis with high pixel density. [ABSTRACT FROM AUTHOR]

The effects of co-evolution with lytic phage on bacterial virulence-related traits are largely unknown. In this study we investigate the incidence of the mucoid phenotype of the bacterium Pseudomonas fluorescens SBW25 in response to co-evolution with the lytic phage phi2 (φ2). The mucoid phenotype of Pseudomonas spp. is due to overproduction of alginate and is a considerable virulence factor contributing to the intractability of infections most notably in cystic fibrosis (CF) lung, but also in pathogenic infections of plants. Our data show that this phenotype can evolve as an adaptive response to phage predation and is favoured under specific abiotic conditions, in particular a homogenous spatial structure and a high rate of nutrient replacement. The mucoid phenotype remains partially sensitive to phage infection, which facilitates 'apparent competition' with phage-sensitive competitors, partially offsetting the costs of alginate production. Although P. fluorescens SBW25 is not a pathogen, several key characteristics typical of Pseudomonas aeruginosa clinical isolates from CF lung were noted, including loss of motility on mucoid conversion and a high rate of spontaneous reversion to the wild-type phenotype. Although the genetic mechanisms of this phenotype remain unknown, they do not include mutations at many of the commonly reported loci implicated in mucoid conversion, including mucA and algU. These data not only further our understanding of the potential role phage have in the ecology and evolution of bacteria virulence in both natural and clinical settings, but also highlight the need to consider both biotic and abiotic variables if bacteriophages are to be used therapeutically. [ABSTRACT FROM AUTHOR]

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)-2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis. [ABSTRACT FROM AUTHOR]

Background: Recently, a transgenic rabbit with rhodopsin Pro 347 Leu mutation was generated as a model of retinitis pigmentosa (RP), which is characterized by a gradual loss of vision due to photoreceptor degeneration. The purpose of the current study is to noninvasively visualize and assess time-dependent changes in the retinal structures of a rabbit model of retinal degeneration by using speckle noise-reduced spectral-domain optical coherence tomography (SD-OCT). Methodology/Principal Findings: Wild type (WT) and RP rabbits (aged 4-20 weeks) were investigated using SD-OCT. The total retinal thickness in RP rabbits decreased with age. The thickness of the outer nuclear layer (ONL) and between the external limiting membrane and Bruch's membrane (ELM-BM) were reduced in RP rabbits around the visual streak, compared to WT rabbits even at 4 weeks of age, and the differences increased with age. However, inner nuclear layer (INL) thickness in RP rabbits did not differ from that of WT during the observation period. The ganglion cell complex (GCC) thickness in RP rabbits increased near the optic nerve head but not around the visual streak in the later stages of the observation period. Hyper-reflective change was widely observed in the inner segments (IS) and outer segments (OS) of the photoreceptors in the OCT images of RP rabbits. Ultrastructural findings in RP retinas included the appearance of small rhodopsin-containing vesicles scattered in the extracellular space around the photoreceptors. Conclusions/Significance: In the current study, SD-OCT provided the pattern of photoreceptor degeneration in RP rabbits and the longitudinal changes in each retinal layer through the evaluation of identical areas over time. The time-dependent changes in the retinal structure of RP rabbits showed regional and time-stage variations. In vivo imaging of RP rabbit retinas by using SD-OCT is a powerful method for characterizing disease dynamics and for assessing the therapeutic effects of experimental interventions. [ABSTRACT FROM AUTHOR]

Background: Excessive exposure to light enhances the progression and severity of some human retinal degenerative diseases. While retinal microglia are likely to be important in neuron damage associated with these diseases, the relationship between photoreceptor damage and microglial activation remains poorly understood. Some recent studies have indicated that the chemokine fractalkine is involved in the pathogenesis of many neurodegenerative diseases. The present study was performed to investigate the cross-talk between injured photoreceptors and activated retinal microglia, focusing on the role of fractalkine and its receptor CX3CR1 in light-induced photoreceptor degeneration. Methodology/Principal Findings: Both in vivo and in vitro experiments were involved in the research. In vivo, Sprague-Dawley rats were exposed to blue light for 24 hours. In vitro, the co-culture of primary retinal microglia and a photoreceptor cell line (661W cell) was exposed to blue light for five hours. Some cultures were pretreated by the addition of anti-CX3CR1 neutralizing antibody or recombinant fractalkine. Expression of fractalkine/CX3CR1 and inflammatory cytokines was detected by immunofluorescence, real-time PCR, Western immunoblot analysis, and ELISA assay. TUNEL method was used to detect cell apoptosis. In addition, chemotaxis assay was performed to evaluate the impact of soluble fractalkine on microglial migration. Our results showed that the expression of fractalkine that was significantly upregulated after exposure to light, located mainly at the photoreceptors. The extent of photoreceptor degeneration and microglial migration paralleled the increased level of fractalkine/CX3CR1. Compared with the control, the expression of inflammatory cytokines was significantly downregulated in the anti-CX3CR1 neutralizing antibody-treated group, and the number of photoreceptors was also well preserved. The addition of recombinant full-length fractalkine or soluble fractalkine resulted in fewer TUNEL-positive photoreceptors and an increased number of migratory microglia respectively. Conclusions/Significance: These findings demonstrate that fractalkine/CX3CR1 interaction may play an important role in the photoreceptor-microglia cross-talk in light-induced photoreceptor degeneration. [ABSTRACT FROM AUTHOR]

Many studies have demonstrated that epigenetic mechanisms are important in the regulation of gene expression during embryogenesis, gametogenesis, and other forms of tissue-specific gene regulation. We sought to explore the possible role of epigenetics, specifically DNA methylation, in the establishment and maintenance of cell type-restricted gene expression in the retina. To assess the relationship between DNA methylation status and expression level of retinal genes, bisulfite sequence analysis of the 1000 bp region around the transcription start sites (TSS) of representative rod and cone photoreceptor-specific genes and gene expression analysis were performed in the WERI and Y79 human retinoblastoma cell lines. Next, the homologous genes in mouse were bisulfite sequenced in the retina and in non-expressing tissues. Finally, bisulfite sequencing was performed on isolated photoreceptor and non-photoreceptor retinal cells isolated by laser capture microdissection. Differential methylation of rhodopsin (RHO), retinal binding protein 3 (RBP3, IRBP) cone opsin, short-wavesensitive (OPN1SW), cone opsin, middle-wave-sensitive (OPN1MW), and cone opsin, long-wave-sensitive (OPN1LW) was found in the retinoblastoma cell lines that inversely correlated with gene expression levels. Similarly, we found tissue-specific hypomethylation of the promoter region of Rho and Rbp3 in mouse retina as compared to non-expressing tissues, and also observed hypomethylation of retinal-expressed microRNAs. The Rho and Rbp3 promoter regions were unmethylated in expressing photoreceptor cells and methylated in non-expressing, non-photoreceptor cells from the inner nuclear layer. A third regional hypomethylation pattern of photoreceptor-specific genes was seen in a subpopulation of non-expressing photoreceptors (Rho in cones from the Nrl -/- mouse and Opn1sw in rods). These results demonstrate that a number of photoreceptor-specific genes have cell-specific differential DNA methylation that correlates inversely with their expression level. Furthermore, these cell-specific patterns suggest that DNA methylation may play an important role in modulating photoreceptor gene expression in the developing mammalian retina. [ABSTRACT FROM AUTHOR]

The neural retinal leucine zipper (Nrl) knockout mouse is a widely used model to study cone photoreceptor development, physiology, and molecular biology in the absence of rods. In the Nrl-/- retina, rods are converted into functional cone-like cells. The Nrl-/- retina is characterized by large undulations of the outer nuclear layer (ONL) commonly known as rosettes. Here we explore the mechanism of rosette development in the Nrl-/- retina. We report that rosettes first appear at postnatal day (P)8, and that the structure of nascent rosettes is morphologically distinct from what is seen in the adult retina. The lumen of these nascent rosettes contains a population of aberrant cells protruding into the subretinal space that induce infolding of the ONL. Morphologically adult rosettes do not contain any cell bodies and are first detected at P15. The cells found in nascent rosettes are photoreceptors in origin but lack inner and outer segments. We show that the adherens junctions between photoreceptors and Müller glia which comprise the retinal outer limiting membrane (OLM) are not uniformly formed in the Nrl-/- retina and thus allow protrusion of a population of developing photoreceptors into the subretinal space where their maturation becomes delayed. These data suggest that the rosettes of the Nrl-/- retina arise due to defects in the OLM and delayed maturation of a subset of photoreceptors, and that rods may play an important role in the proper formation of the OLM. [ABSTRACT FROM AUTHOR]

A defining feature of malignant tumor progression is cellular penetration through the basement membrane and interstitial matrices that separate various cellular compartments. Accumulating evidence supports the notion that invasive cells employ specialized structures termed invadopodia to breach these structural barriers. Invadopodia are actin-based, lipid-raft-enriched membrane protrusions containing membrane-type-1 matrix metalloproteinase (MT1-MMP; also known as matrix metalloproteinase 14; MMP14) and several signaling proteins. CD147 (emmprin, basigin), an immunoglobulin superfamily protein that is associated with tumor invasion and metastasis, induces the synthesis of various matrix metalloproteinases in many systems. In this study we show that upregulation of CD147 is sufficient to induce MT1-MMP expression, invasiveness and formation of invadopodia-like structures in non-transformed, non-invasive, breast epithelial cells. We also demonstrate that CD147 and MT1-MMP are in close proximity within these invadopodialike structures and co-fractionate in membrane compartments with the properties of lipid rafts. Moreover, manipulation of CD147 levels in invasive breast carcinoma cells causes corresponding changes in MT1-MMP expression, invasiveness and invadopodia formation and activity. These findings indicate that CD147 regulates invadopodia formation and activity, probably through assembly of MT1-MMP-containing complexes within lipid-raft domains of the invadopodia. [ABSTRACT FROM AUTHOR]

Vacancy-type defects created by single-energy implantation of Czochralski-grown single-crystal silicon by 4MeV silicon ions at doses of 1012 and 1013 cm-2 have been compared with those created by an energy chain of implants of 0.4, 0.9, 1.5, 2.2 and 4MeV ions, each at one-fifth of the single-energy dose. Measurements were taken for as-implanted samples and after annealing to temperatures up to 600 °C. In contrast to the expectation that a more uniform depth distribution of interstitials and vacancies would lead to a more efficient recombination and consequently fewer surviving vacancies, vacancy-related damage survived in the chain-implanted samples to higher temperatures, before almost complete annealing at 600 °C. It is therefore concluded that it is the absolute initial monovacancy concentration, rather than any initial separation of vacancy- and interstitial-rich regions, that determines the probability of survival as divacancies, and that there exists a threshold divacancy concentration of 1-2×1018 cm-3 for clustering at 400-500 °C. [ABSTRACT FROM AUTHOR]

Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE), and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds) mouse model. Global gene expression profiling was performed on flowsorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold) differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P) 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model. [ABSTRACT FROM AUTHOR]

Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death. [ABSTRACT FROM AUTHOR]