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Cornea | April 2017
The Structural Role of Elastic Fibers in the Cornea Investigated Using a Mouse Model for Marfan Syndrome
Tomas L. White; Philip Lewis; Sally Hayes; James Fergusson; James Bell; Luis Farinha; Nick S. White; Lygia V. Pereira; Keith M. Meek
Tomas L. White
Structural Biophysics Research Group, School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cardiff, United Kingdom
Philip Lewis
Sally Hayes
James Fergusson
Vision Science Bioimaging Labs, School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom
Luis Farinha
Department of Genetics and Evolutionary Biology, University of São Paulo, Rua do Matão, São Paulo, Brazil
Nick S. White
Lygia V. Pereira
Keith M. Meek
Correspondence: Keith M. Meek, Structural Biophysics Research Group, School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cardiff CF24 4HQ, United Kingdom; meekkm@cardiff.ac.uk.
Investigative Ophthalmology & Visual Science April 2017, Vol.58, 2106-2116. doi:https://doi.org/10.1167/iovs.16-21358
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Tomas L. White, Philip Lewis, Sally Hayes, James Fergusson, James Bell, Luis Farinha, Nick S. White, Lygia V. Pereira, Keith M. Meek; The Structural Role of Elastic Fibers in the Cornea Investigated Using a Mouse Model for Marfan Syndrome. Invest. Ophthalmol. Vis. Sci. 2017;58(4):2106-2116. doi: https://doi.org/10.1167/iovs.16-21358.
© ARVO (1962-2015); The Authors (2016-present)
Purpose: The presence of fibrillin-rich elastic fibers in the cornea has been overlooked in recent years. The aim of the current study was to elucidate their functional role using a mouse model for Marfan syndrome, defective in fibrillin-1, the major structural component of the microfibril bundles that constitute most of the elastic fibers.
Methods: Mouse corneas were obtained from animals with a heterozygous fibrillin-1 mutation (Fbn1+/−) and compared to wild type controls. Corneal thickness and radius of curvature were calculated using optical coherence tomography microscopy. Elastic microfibril bundles were quantified and visualized in three-dimensions using serial block face scanning electron microscopy. Transmission electron microscopy was used to analyze stromal ultrastructure and proteoglycan distribution. Center-to-center average interfibrillar spacing was determined using x-ray scattering.
Results: Fbn1+/− corneas were significantly thinner than wild types and displayed a higher radius of curvature. In the Fbn1+/− corneas, elastic microfibril bundles were significantly reduced in density and disorganized compared to wild-type controls, in addition to containing a higher average center-to-center collagen interfibrillar spacing in the center of the cornea. No other differences were detected in stromal ultrastructure or proteoglycan distribution between the two groups. Proteoglycan side chains appeared to colocalize with the microfibril bundles.
Conclusions: Elastic fibers have an important, multifunctional role in the cornea as highlighted by the differences observed between Fbn1+/− and wild type animals. We contend that the presence of normal quantities of structurally organized elastic fibers are required to maintain the correct geometry of the cornea, which is disrupted in Marfan syndrome.
The optical and biomechanical properties of the human cornea are largely governed by the collagen-rich stroma, a layer that represents approximately 90% of the total thickness. Within the stroma, the specific arrangement of superimposed lamellae confers requisite mechanical properties,1 while the spatial arrangement of individual collagen fibrils within the lamellae enables transparency.2,3 As well as collagen, elastic tissue has a fundamental biomechanical role in many dynamic tissues,4 for example, lungs and the intima of blood vessels, where it routinely experiences strains in excess of 100% in response to changes in blood pressure and vasoactivity.5 Similarly, the sclera contains an elastic fiber system6,7 allowing the eye to deform slightly as a result of internal and external pressure, before regaining its original shape. Despite the cornea being part of the outer tunic of the eye, along with the sclera, the presence of elastic tissue in the corneal stroma has been overlooked in recent years. The development of more advanced imaging techniques, such as serial block-face scanning electron microscopy (SBF SEM), has enabled this area to be revisited. We recently have used this technique to characterize a complex network of elastic fibers in human cornea.8 These fibers consist of bundles of microfibrils, the main component of which is the fibrillin-1 protein.9,10 In the human cornea they appear as elastin-containing sheets at the limbus before extending into the peripheral and central stroma as narrower fibrillin-rich microfibril bundles, most of which appear to be devoid of elastin cores. These microfibril bundles run parallel to the surface of the cornea and are concentrated in the posterior stroma, immediately anterior to Descemet's membrane.8 Serial block-face scanning electron microscopy also has been used to demonstrate the presence of fibrillin-rich microfibril bundles in the mouse corneal stroma,9 although their functional role remains unclear.
Marfan syndrome (MFS) is an autosomal dominant disease of connective tissue caused by mutations in the FBN1 gene encoding the fibrillin-1 protein.11,12 The condition results in ocular, skeletal, and cardiovascular abnormalities, caused by altered biomechanical properties in tissues containing elastic material. The most common symptom seen in the eye is ectopia lentis, induced by biomechanically weak ciliary zonules that hold the lens in dynamic suspension. Mouse models for MFS have been developed with variable phenotypic severity to study the pathogenic mechanisms underlying the disease.13–15 Lima et al.15 developed a mouse model (mgΔloxPneo) with a mutant Fbn1 allele that produces an internally truncated fibrillin-1. Heterozygotes present defective microfibrillar deposition, which results in kyphosis and cardiovascular abnormalities (deterioration of the aortic wall and aneurysms) equivalent to those of MFS patients.
The corneas of mgΔloxPneo mice have not been characterized previously. In an attempt to elucidate the functional role of elastic fibers in the cornea, we have used a combination of SBF SEM, transmission electron microscopy (TEM), optical coherence tomography (OCT) microscopy and x-ray scattering to examine corneas from the mgΔloxPneo mouse model for MFS in comparison with wild type (WT) controls. Corneas of mgΔloxPneo mice are referred to as Fbn1+/− throughout this study. It also should be noted that the term "elastic fibers" here refers to bundles of fibrillin-rich microfibrils, or oxytalan fibers, as opposed to true elastic or elaunin fibers, which contain fibrillar and amorphous components.
Mouse Eyes
Whole eye globes from Fbn1+/− and WT mice were obtained from the Department of Genetics and Evolutionary Biology, University of São Paulo, Brazil. Development of the Fbn1+/− mouse model has been described previously.15 Fbn1+/− mice, along with WT controls, were euthanized at 3 months of age. Whole eye globes were removed immediately following death and fixed in 0.5% paraformaldehyde before being transported to the United Kingdom in a box cooled with ice packs and stored at 4°C. All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
OCT Microscopy
Eyes from eight WT and nine Fbn1+/− mice were imaged using a near infrared (NIR) bespoke OCT microscope (Fig. 1). The digitized spectra of reflected light from every point of a 2D (XY) scan over the specimen were processed into a 3D volume image by spectral resampling and Fourier transformation. The instrument was controlled by LabView (National Instruments, Newbury, UK) software and spectra and image data were processed using MATLAB (Mathworks, Cambridge, UK) and ImageJ software.16
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Optical coherence tomography microscopy setup. Illumination from an amplified spontaneous emission light source with a central wavelength of 1040 nm and a bandwidth of 70 nm (1-M-ASE-HPE-S; NP Photonics, Tucson, AZ, USA) was coupled through a 2 × 2 optical fiber coupler (FC; FOBC-2-64+/−100-20-L-H64f-2; AFW Technologies, Hallam, Victoria, Australia) to an imaging head and reference arm. The imaging head comprised an achromatic off-axis parabolic reflector (not shown) to collimate the fiber output beam to approximately 2 mm diameter (RC02APC-P01; Thorlabs, Ely, UK), close-coupled 2D (XY) optical scanners (6210HBM60/6102103R; Cambridge Technology Division, GSI Group GmbH, Muenchner, Germany) and a broad-band NIR telecentric scan lens (LSM02BB; Thorlabs). The reference arm consisted of a polarization controller (PC; FPC560, Thorlabs), a second reflecting collimator (not shown; RC08APC-01; Thorlabs), an adjustable aperture (Ap), a precision NIR retroreflector (RR; 1 Arcsec Gold, Edmund Optics, York, UK) and a glass compensation block (CB; LSM02DC; Thorlabs) to correct for NIR dispersion in the scan lens. Reflected light from the specimen and reference arm was combined in a spectrometer.58 The camera has been upgraded to a 47khz Goodrich SU-LDH-1.7 (UTC Aerospace Systems, Arlington, VA, USA).
A small well made from mounting putty was created on a glass slide to hold the eye in the correct position for scanning (with the cornea facing upwards). The eye was hydrated with drops of 0.5% paraformaldehyde before being imaged. Data sets of 1000 images at 1000 × 1000 pixel resolution (axial scaling of 2.66 μm, lateral scaling 3.09 μm per pixel in air) were obtained from eye globes, focused on the cornea and anterior chamber. Data sets were reconstructed into 3D models using Amira 6.0.1 software (FEI, Mérignac, France).
Central corneal thickness was calculated by firstly taking the average intensity of approximately 10 images from the center of each data set. The images had an axial scaling of 2.66 μm per pixel in air; therefore, for any part of the image that is in the tissue, the scaling is reduced by the refractive index of the material (1.4 in the case of the mouse cornea).17 The number of pixels from the anterior to the posterior surface at the center of the cornea was measured and converted to actual thickness using the following formula:
The radius of curvature was measured from the arc formed at the anterior surface of the central cornea (Fig. 2) using the following equation:
Schematic of a mouse eye to illustrate the parameters used in the calculation of corneal radius of curvature from optical coherence tomography images. The radius of curvature was calculated by applying Pythagoras theorem to the red triangle. The height (h) of the arc was maintained at 300 μm in all cases, with only the width (w) varying between specimens.
The height (h) of the arc being measured for each sample was consistent at 300 μm to ensure that the same size arcs were being measured, with only the width (w) varying. All thickness and radius of curvature measurements were performed using ImageJ software.16
Serial Block Face Scanning Electron Microscopy
Three mouse corneas from each group were dissected from whole globes, cut into small segments, and fixed in modified Karnovsky's fixative18 (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer at pH 7.2) for 2 hours. Tissue was processed according to methods described previously.8 Briefly, this consisted of postfixation with 1% osmium tetroxide, incubation in 0.5% tannic acid, staining with 2% aqueous uranyl acetate, dehydration in an ethanol series, and further staining with 2% ethanoic uranyl acetate and a saturated solution of lead acetate19 before being embedded in CY212 (TAAB) epoxy resin. Specimens were examined using a Zeiss Sigma VP FEG SEM (Carl Zeiss Meditec, Jena, Germany) equipped with a Gatan 3View2 system, where data sets of up to 1000 images were acquired every 50 nm at 4000 × 4000 pixel resolution. Three-dimensional (3D) reconstructions of data sets were created with Amira 6.0.1 software, using automated and semiautomated thresholding.
Elastic fiber quantification throughout the entire central stroma was carried out on one cornea from each group to estimate the amount of elastic tissue in a normal mouse cornea, and to discover the extent of elastic fiber reduction in the Fbn1+/− cornea. Additionally, obtaining these data provided 3D information throughout the entire depth of the cornea. Quantification of elastic fiber density was done as detailed previously for human corneas.8 Data sets were acquired through the entire depth of WT and Fbn1+/− corneas at 2000 × 2000 pixel resolution. Each full-thickness set of data was analyzed in groups of approximately 250 images (equating to a depth of approximately 12.5 μm), where percentages of tissue volume occupied by elastic fibers in each group were calculated using Amira 6.0.1 software.
Embedded tissue that had been stained en bloc for SBF SEM also was used for TEM. A Leica UC6 ultra-microtome (Leica Biosystems, Wetzlar, Germany) was used to cut 90 nm-thick gold sections that were floated on distilled water before being mounted on copper grids and visualized using a JEOL 1010 TEM (JEOL, Welwyn Garden City, UK).
Cupromeronic Blue Stain
Small segments of tissue from two WT and two Fbn1+/− corneas fixed in Karnovsky's fixative were washed in 25 mM sodium acetate buffer containing 0.1 M MgCl2 (pH 5.7) for 3 hours at room temperature on a rotator. Segments were stained with the 25 mM sodium acetate buffer containing 0.1 M MgCl2 and 0.05% cupromeronic blue at room temperature overnight. Tissue was rinsed (3 × 5 minutes) with the sodium acetate buffer (without cupromeronic blue) before being exposed to 0.5% aqueous sodium tungstate (3 × 10 minutes) and 50% EtOH + 0.5% sodium tungstate for 15 minutes.20,21 This was followed by dehydration through an ascending ethanol series (70%, 90%, 100% EtOH) and propylene oxide. The tissue was incubated in a 1:1 mixture of propylene oxide and epoxy resin (araldite monomer CY212, DDSA hardener, BDMA accelerator) for 1 hour before 100% epoxy resin infiltration for 1.5 days. Cornea segments were placed into plastic molds with fresh epoxy resin and polymerized at 60°C for 36 hours. Ultrathin (approximately 90 nm) gold sections were cut, collected on copper grids, and allowed to dry overnight. Sections were counterstained with 1% aqueous uranyl acetate for 15 minutes, washed with distilled water for 5 minutes, and visualized with TEM.
Four WT and four Fbn1+/− corneas, obtained from individual mice, were dissected from whole globes that had been fixed and stored in 4% paraformaldehyde. Small-angle x-ray scatter patterns were collected on beamline I22 (Diamond Light Source, Oxfordshire, UK), using an x-ray beam measuring approximately 200 × 200 μm with a wavelength of 0.1 nm. Before x-ray exposure, each cornea was wrapped in catering cling film to prevent dehydration and placed inside an airtight Perspex sample holder containing two transparent Mylar sheet windows. The sample holder then was positioned on the beamline such that the center of the anterior surface of the cornea was perpendicular to the incident x-ray beam. X-ray scatter patterns resulting from a 0.4-second exposure were collected at 0.2 or 0.3 mm intervals along the vertical transect of each specimen. X-ray scatter patterns were collected on an x-ray detector positioned 6 m behind the specimen. Our previous small-angle x-ray scattering studies have shown that the hydration of mouse corneas examined in this way does not significantly change.22,23
The x-ray scatter patterns were analyzed as described previously24,25 using a custom-made graphic user interface written in MATLAB (MathWorks). Each pattern was radially integrated to generate an x-ray intensity profile, from which background scatter was removed by fitting and subtracting a power-law background function. Scattering patterns were centered using the pattern of powdered silver behanate, and calibrated against the 67 nm meridional spacing of hydrated rat tail tendon. Average interfibrillar Bragg spacing (IFS) of corneal collagen fibrils was determined from the calibrated position of the first-order equatorial reflection. Measurements of IFS were made at each sampling site along the vertical axis of the cornea.
Tests for normality and equal variance were performed using Shapiro-Wilk and Levene's tests, before an independent t-test being done to determine whether any differences existed between Fbn1+/− and WT corneas for central corneal thickness, radius of curvature, elastic fiber density, and IFS. Correlation between the corneal thickness and radius of curvature variables was tested using Pearson's correlation test. P < 0.05 was determined as statistically significant. All statistical testing was done using IBM SPSS software (IBM, Armonk, NY, USA).
Corneal Thickness and Radius of Curvature Measurements
Individual images taken from the center of each OCT microscopy data set revealed a difference in central corneal thickness between WT and Fbn1+/− corneas (Fig. 3). Additionally, Fbn1+/− cornea appeared to be flatter compared to the more curved WT. The center-mid corneas were segmented, along with the iris and pupil. As a result of poor contrast, the peripheral cornea was omitted from the 3D reconstructions.
A series of 1000 optical coherence tomography images were obtained from each eye and reconstructed into 3D models. By viewing individual images (top), it appeared as though the WT cornea (left) was thicker and more curved than the Fbn1+/− cornea (right). The cornea (purple), lens (yellow), and iris (pink) were represented in 3D (bottom) for both groups.
Using the OCT microscopy images, central corneal thickness and radius of curvature were measured for WT and Fbn1+/− corneas (Fig. 4). Independent t-tests showed that WT corneas were significantly thicker than Fbn1+/− corneas (P < 0.001), with an average thickness of 183.6 μm (±15.2), compared to 154.8 μm (±9.1) in the fibrillin-1–deficient mice. Wild type corneal thickness reached >190 μm, whereas Fbn1+/− corneas often were <150 μm, meaning a difference of approximately 40 μm (approximately 1/3 of the stromal thickness in mice). Radius of curvature also was significantly different between the two groups (P < 0.05), with an average radius of 1.94 mm (±0.049) in WT, and 2.00 mm (±0.057) in Fbn1+/− corneas. This was more subtle than the difference in thickness, with many radii lying between 1.94 and 1.99 mm. Statistical analysis revealed that no significant correlation existed between radius of curvature and corneal thickness (P = 0.20).
Central corneal thickness plotted against radius of curvature. Each point on the graph represents measurements from a single mouse cornea (8 WT, 9 Fbn1+/−). Average central corneal thickness was significantly lower (P < 0.001) in Fbn1+/− corneas (154.8 μm) compared to WT controls (183.6 μm). Conversely, average corneal radius of curvature was significantly higher (P < 0.05) in Fbn1+/− animals (2.00 mm) compared to WT equivalents (1.94 mm).
3D Arrangement and Quantification of Elastic Fibers
The image data obtained from the full thickness fiber quantification were used to create 3D reconstructions of WT and Fbn1+/− corneas (Fig. 5). These models represent the first approximately 50 μm of stroma anterior to Descemet's membrane. The reconstructions also portray how the block was not cut perfectly parallel to the surface, resulting in stroma that runs obliquely from Descemet's membrane. An abundance of elastic fibers (colored gold) can be seen running throughout the stroma of both groups. Zooming into an approximately 12.5 μm region of stroma (Fig. 4, insets), and viewing from above, shows a clear difference in the concentration of elastic fibers, with WT cornea being more densely populated. Fibers from both groups run in all directions, primarily parallel to the surface of the cornea.
The 3D arrangement of elastic fibers (gold) in the first approximately 50 μm of stroma above Descemet's membrane (blue) in WT (A) and Fbn1+/− (B) mouse cornea. Additionally, some of the residing keratocytes have been segmented out (pink). Approximately 12.5 μm of stroma from each group was focused on and viewed from above (insets), portraying the difference in fiber density. The stroma of WT cornea (1) contains a significantly higher concentration of elastic fibers compared to Fbn1+/− stroma (2). All elastic fibers ran parallel to the surface of the cornea.
Approximately 2300 images were obtained from epithelium to Descemet's membrane in each corneal stroma, equating to a depth of 115 μm. The percentage of volume occupied by elastic fibers for each group of 250 images (equating to approximately 12.5 μm depth) was calculated for Fbn1+/− and WT corneas (Fig. 6). Elastic fiber density was highest in the posterior Fbn1+/− cornea before gradually decreasing to very low levels in the anterior stroma. The density of elastic fibers in the WT cornea showed a similar trend, but reached its highest point in the mid stroma before gradually decreasing toward the anterior. When comparing the two samples, the overall levels of elastic fibers were significantly lower (approximately 50%; P < 0.05) in the Fbn1+/− cornea (Fbn1+/− mean, 0.33% [± 0.16]; WT, mean 0.64% [± 0.27]), with the most dramatic difference being seen in the mid stroma, where the WT cornea had approximately 5 times more elastic fibers.
Volume of elastic fibers as a function of depth in the corneal stroma of one Fbn1+/− and one WT mouse cornea. Each point on the graph represents approximately 250 serial en face images summed between that depth and the one before it in the graph. All the volume elements were rendered and the percentage of the volume element occupied by elastic fibers was calculated. The Fbn1+/− cornea contained significantly less elastic fibers on average (approximately 50 %; P < 0.05) compared to the WT control, although this varied at different depths of stroma, with a 5-fold difference seen in the midstroma.
Elastic fiber distribution was further analyzed at higher resolution in a region of stroma immediately anterior to Descemet's membrane (Fig. 7). Elastic fibers in the WT cornea were structurally organized, consisting of long fibers that travelled in straight lines with uniform thickness (Figs. 7A, 7B), occasionally bifurcating (Fig. 7C), with the two thinner fibers continuing to run in the same direction following the division. Elastic fibers in the Fbn1+/− cornea appeared disorganized, running in multiple directions (Figs. 7D, 7E) when compared to the preferred orientation seen in WT cornea fibers. Five or six individual fibers were seen to originate from a broader region of elastic material at the center of the 3D reconstruction before travelling in several different directions (Fig. 7D). Furthermore, the elastic fiber in Fbn1+/− cornea formed more extensive connections, with approximately 10 individual fibers seen sprouting from a thicker fiber (Fig. 7F).
High resolution 3D reconstructions immediately anterior to Descemet's membrane (blue). WT (A–C) cornea shows an organized elastic fiber network (gold) running in a preferred direction (blue arrows), and occasionally bifurcating (red arrow). In contrast, fibers from Fbn1+/− cornea (D–F) appear more disorganized, originating from a thickened area of tissue (yellow arrow) before travelling in several different directions. Fibers were seen to divide into many branches (white arrows).
Elastic fibers in the mouse cornea of both groups were clearly visible in TEM micrographs (Fig. 8). The tannic acid-based staining method used stains elastic fibers as well as collagen.26,27 Elastic fibers were orientated longitudinally (Fig. 8A) and transversely (Fig. 8B), with individual 10 to 12 nm microfibrils visible within the transverse fibers; however, the 56 nm pseudoperiodicity seen in human corneal elastic fibers8 was not visible in the mouse elastic fibers. The diameter of the elastic fibers varied between 100 and 270 nm with an average diameter of approximately 187 nm in WT and approximately 145 nm in the Fbn1+/− corneas (data not shown). Other than a higher quantity of elastic fibers in the WT cornea, no structural differences were apparent when comparing the anterior, middle, and posterior stroma of the two groups (Fig. 9). The stroma of the fibrillin-1–deficient mouse cornea contained organized lamellae, with comparable lamella thickness to WT cornea.
Transmission electron microscopy images of elastic fibers in the WT mouse cornea stained with tannic acid. Electron dense elastic fibers (black arrows) are orientated longitudinally (A) and transversely (B), with individual 10 to 12 nm microfibrils visible upon close inspection; these fibers can be easily distinguished from the surrounding collagen fibrils.
Transmission electron microscopy images obtained at various depths in the corneal stroma. Comparing anterior, middle, and posterior stroma of WT (left) and Fbn1+/− (right) shows no obvious differences in ultrastructure. Both corneas appear well organized with comparable lamellae thickness. Elastic fibers are visible in each image (white arrows). Scale bars: 2 μm.
Cupromeronic blue staining was done to compare proteoglycan quantity/distribution between the two groups (Fig. 10). Comparing the two sets of micrographs by eye suggested that there were no obvious differences in the quantity or spatial arrangement of proteoglycans between the two groups. Proteoglycan filaments were observed in the interfibrillar spaces, in association with collagen fibrils. These filaments formed bridges between transversely and longitudinally arranged collagen fibrils. Transverse elastic fibers were observed as circular blank spaces in the micrographs due to the omission of tannic acid from the staining protocol (Fig. 11). This assumption is based on the fact that the size and shape of the spaces closely match those of the elastic fibers seen in Figure 8. Observing the fiber spaces at high magnification indicated that there is a potential association between proteoglycan filaments and fibrillin-1, as suggested by long chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycan (GAG) chains appearing to travel into the elastic fiber (Fig. 11, inset).
Proteoglycans stained with cupromeronic blue visualized with transmission electron microscopy. WT (top) and Fbn1+/− (bottom) cornea, showing transversely (right) and longitudinally (left) oriented collagen fibrils with numerous associated GAG chains (white arrows) in the interfibrillar spaces, bridging the fibrils. No differences were apparent between the two groups.
Proteoglycan-elastic fiber interaction shown with cupromeronic blue staining. An elastic fiber space in the stroma viewed at high magnification (inset) appeared to show proteoglycan filaments (white arrows) travelling into the transverse fiber and colocalizing with fibrillin-1 microfibrils.
Average center–center fibril spacings for WT and Fbn1+/− corneas are shown in Figure 12. In all specimens, IFS in the center of the cornea was clearly reduced compared to peripheral regions, where IFS increased by approximately 25% to 40%. This "U" shaped distribution also is seen when measuring mouse corneal fibril diameters.25 Corneal samples measured approximately 3 mm in diameter, indicating that each vertical scan was taken through the center, with the only exception being one WT cornea in which patterns were recorded at 0.3 mm intervals, as opposed to 0.2 mm intervals. Data from this sample suggested that the vertical transect was off center. D-periodicity at the center of each sample was consistent at 64 to 65 nm. Average IFS at the center of Fbn1+/− corneas was higher (71.9 ± 5.6 nm) than the WT equivalents (63.7 ± 5.8 nm). Following the omission of the central IFS value from the off-center specimen, an independent t-test revealed that the difference in IFS was statistically significant (P ≤ 0.05), despite the low sample number (n = 3–4 for each group).
Radial plots of interfibrillar spacing (IFS) in WT and Fbn1+/− mouse cornea. These graphs show the center–center average IFS in the center and periphery of WT and Fbn1+/− corneas. Each plot represents a single cornea, and displays a "U" shaped distribution, with higher IFS at the periphery. At the center of the cornea, average IFS was significantly higher (P ≤ 0.05) in Fbn1+/− corneas (71.9 nm) compared to WT equivalents (63.7 nm).
This study has provided evidence that the mgΔloxPneo mouse model for MFS is valuable for studying the corneal pathogenesis of the disease and the functional role of fibrillin-1 in the cornea. Importantly, quantification of elastic fibers in the fibrillin-1–deficient cornea showed a significant reduction (approximately 50%) in overall levels of fibrillin-1–containing elastic fibers throughout the entire thickness of the stroma compared to the WT control. These findings are consistent with studies from Lima et al.,15 who showed a diffused network of fibrillin-1–containing microfibrils in cultured foetal Fbn1+/− fibroblasts when compared to WT. Similar findings were observed in human MFS patients, where decreased extracellular deposition of fibrillin-1 by fibroblasts has been reported.28–30
Optical coherence tomography microscopy image measurements revealed that central corneal thickness was significantly reduced in Fbn1+/− mice compared to WT. Radius of curvature also was significantly different between the two groups, although there was no significant correlation between the two variables, which may be a symptom of underpowering the study. A significant reduction in central corneal thickness has been reported in human patients with MFS.31–33 A significant difference in keratometry readings also has been reported, suggesting a flattened cornea in MFS.31,33–35 Increased radius of curvature and increase in axial globe length in MFS cornea has been observed in previous studies.34,36 Therefore, based on these previous studies, and results obtained in the current study, we contend that the elastic fiber system has an important role in maintaining the curvature of the cornea. A reduction/malformation of taut elastic fibers in MFS would result in the cornea being pulled outwards at the periphery by an expanding globe following a release in internal stress, leading to a thin and flattened cornea. Elastin and elastin-free microfibril bundles are located in the sclera.6,37 This would imply an alteration in biomechanical properties in the sclera, as well as in the cornea, in MFS patients' eyes. With one of the main functions of the sclera being to maintain fixed axial dimensions to ensure a stable retinal image,38 and with absolute quantities of elastic tissue in the sclera unknown, it is likely that increased globe dimensions and subsequent flattened cornea associated with MFS are caused by a combination of biomechanical changes in the cornea and sclera. However, differences in corneal curvature in MFS patients, with no significant difference in axial length has been reported,39 which demonstrates the phenotypic variability of the disease. With no correlation between radius of curvature and central corneal thickness between Fbn1+/− and WT mice detected, it is unlikely that corneal stretching alone is responsible for the observed changes in thickness. The biomechanical behavior of MFS cornea recently has been assessed in vivo using an ocular response analyzer, revealing a greater maximal deformation of the MFS cornea compared to control, which in turn indicates decreased resistance to bending.39 This evidence supports the proposal that elastic fibers have a pivotal role in providing the cornea with mechanical strength/elastic recoil, and maintaining corneal curvature.
By using SBF SEM to quantify elastic fibers, we are limited to analyzing an approximately 30 × 30 μm area of tissue throughout the stroma, with only 1 cornea from each group being analyzed due to the technical difficulty and time consuming nature of the technique. It is likely that the elastic fiber content varies slightly between each Fbn1+/− cornea given the nature of the disease, ultimately resulting in a variable ratio of elastic fiber loss and fiber disorganization. The issue is complicated further by the fact that fibrillin-2 may compensate for the lack of fibrillin-1 proteins, as shown in mouse ciliary zonule.40 Nevertheless, we were able to show important alterations in the cornea of the mgΔloxPneo Fbn1+/− mice, indicating that the mutant fibrillin-1 exerts a dominant-negative effect on the microfibrils. In comparison to human,8 elastic fibers are more densely populated in the mouse cornea, where they may be responsible for accommodating the increase in corneal curvature.
In the mouse cornea, elastic fibers are present throughout the entire stroma, whereas in human cornea they are highly concentrated in an approximately 10 μm region of stroma immediately anterior to Descemet's membrane, with concentration decreasing rapidly in the anterior direction.8 In accord with the studies of Hanlon et al.,9 our 3D volume rendering showed the presence of elastic fibers running in many directions parallel to the surface of the cornea and often bifurcating, similar to findings observed in the central human cornea.8 Comparisons of 3D reconstructions above Descemet's membrane indicated that elastic fibers in the Fbn1+/− stroma were structurally different, appearing less ordered than the WT. Similarly, histologic examination of the lens capsule in human MFS patients revealed qualitative and quantitative differences, with significantly less fibrillin-1–containing microfibrils in addition to the presence of misshapen, irregular, and fragmented bundles.41
To our knowledge, this is the first study to demonstrate a reduction in elastic fiber density and microfibril disorganization in the cornea of a mouse model for MFS. In MFS, it is thought that the mutant protein that is produced has a dominant-negative effect over the normal protein, causing adverse activity on the deposition, stability, or function of the protein encoded by the normal allele of the Fbn1 gene.42 Therefore, it is likely that the disorganized elastic fibers that are laid down in the mutant mouse cornea either contain the mutant protein, or their deposition has been diminished as the mutant protein has attempted to assemble with normal fibrillin-1 monomers, resulting in a structurally altered elastic system. A reduction in number and disorganization of elastic fibers would ultimately result in a biomechanically less responsive cornea, as the tissue relies more heavily on the collagen to deform reversibly under stress following the pull of extraocular muscles and changes in intraocular pressure.
The reduced number/disorganization of elastic fibers in Fbn1+/− did not seem to have an adverse effect on overall structure of the cornea. Elastic fibers were similar in appearance to those previously reported in mouse cornea, with some fibers seen in close association with keratocytes (data not shown), suggesting a possible mechano-transductional role in the stroma.9 The slight reduction in mean fiber diameter in Fbn1+/− cornea may be caused by less individual microfibrils making up the bundles. There currently are no reported structural changes associated with MFS.
Glycosaminoglycans side chains of the proteoglycan protein core provide hydration and swelling pressure to tissues, enabling them to withstand compression, in addition to mediating collagen diameter and IFS. Therefore, it was thought that differences in proteoglycan quantity/distribution may contribute to the differences in cornea thickness. No differences in proteoglycans were evident throughout the stroma of WT and Fbn1+/− cornea. Electron dense filaments passing into an elastic fiber space suggest an association between CS/DS GAGs and fibrillin. The CS/DS proteoglycan decorin has been shown to be localized to fibrillin microfibrils in the skin.43 It has been suggested that CS/DS containing proteoglycans associate with fibrillin and contribute to microfibril organisation.44 Furthermore, fibrillin-1 and MAGP-1 (microfibril-associated glycoprotein-1) were shown to interact with decorin in cultured fetal bovine chondrocytes.45 In the rat eye, CS GAGs are associated with fibrillin-rich ciliary zonules.46 To our knowledge, this is the first study to indicate that GAGs colocalize with fibrillin in the cornea. It is likely that proteoglycans have an important role in the assembly and maintenance of elastic fibers.
X-ray scattering revealed that IFS was lowest at the central region of all specimens, increasing at the periphery, as observed previously in human24,47 and mouse23 corneas. Interfibrillar Bragg spacing was on average approximately 8 nm higher in the central region of Fbn1+/− corneas, an unexpected result given that these corneas were significantly thinner than the WT's. Given that the corneas were fixed, the specific arrangement of collagen fibrils may have been altered.48 Center-to-center fibril spacing in the cornea is governed by several factors, mainly stromal hydration49 and proteoglycan composition/abundance.50 Thinner corneas accompanied by higher average IFS in the center indicates that Fbn1+/− samples consist of fewer lamellae compared to WT's. This has similarities to keratoconic cornea, where x-ray scattering showed normal fibril spacings, indicating that the thin cornea in keratoconus is a result of reduced collagen quantity.51 Interestingly, earlier work by Patey et al.52 discovered a significant increase in collagen diameter and IFS in keratoconus as the disease progresses in severity, particularly in the middle and central stroma, leading to Pouliquen53 stating that this is the reason for stromal thinning and ectasia, since there are fewer lamellae. Therefore, findings from this current study showing increased IFS and thinner corneas in fibrillin-1–deficient animals are consistent with findings observed in keratoconus, suggesting that similar biochemical mechanisms may be active. As well as having a structural role, microfibrils forming elastic fibers are known to have a regulatory role in the extracellular matrix by binding cytokines (e.g., bone morphogenetic proteins and TGF-β).54 It may be possible that elastic fibers have a role in collagen biosynthesis, which could be negatively impacted by alterations in fibrillin-1 content.
It is probable that the elastic fibers carry out functions that are not apparent from ultrastructural observation, such as cell signaling and mechanotransduction in the extracellular matrix. Fibrillin is thought to have an important role in controlling TGF-β activation and signaling in the extracellular matrix, with studies showing dysregulation of TGF-β activity contributing to the pathogenesis of MFS, with excessive levels of active TGF-β in the matrix.55–57 Transforming growth factor–β is involved in many processes in the cornea, including keratocyte activation, myofibroblast transformation and proliferation, and wound healing.58
Overall, it is likely that elastic fibers have a multifunctional role in the cornea. This study, along with our previous study demonstrating the presence of elastic fibers in human cornea,8 should encourage more MFS–related research in the cornea. Further work is needed to elucidate the precise roles of elastic fibers in the cornea, as this is relevant for MFS pathogenesis, corneal biomechanics, development, and potentially other areas, such as extracellular matrix homeostasis.
The authors thank Diamond Light Source for time on beamline I22 under proposal SM12810 and Olga Shebanova for her assistance with the beamline.
Supported by a Programme Grant MR/K000837/1 from the Medical Research Council (KMM), an MRC studentship (TLW), and a Grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - 2011/01477-0). Vision Science Bioimaging Labs are supported by Cardiff University School of Optometry and Visions Sciences and collaborative research projects.
Disclosure: T.L. White, None; P. Lewis, None; S. Hayes, None; J. Fergusson, None; J. Bell, None; L. Farinha, None; N.S. White, None; L.V. Pereira, None; K.M. Meek, None
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Baccaranicontri M, Vincenzi D, Cicchetti F, Mori G, Pasqualironchetti I. Immunocytochemical localization of proteoglycans within normal elastin fibers. Eur J Cell Biol. 1990; 53: 305–312.
Kielty CM, Whittaker SP, Shuttleworth CA. Fibrillin: evidence that chondroitin sulphate proteoglycans are components of microfibrils and associate with newly synthesised monomers. FEBS Lett. 1996; 386: 169–173.
Trask BC, Trask TM, Broekelmann T, Mechame RP. The microfibrillar proteins MAGP-1 and fibrillin-1 form a ternary complex with the chondroitin sulfate proteoglycan decorin. Mol Biol Cell. 2000; 11: 1499–1507.
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Povazay B, Hermann B, Unterhuber A, et al. Three-dimensional optical coherence tomography at 1050 nm versus 800 nm in retinal pathologies: enhanced performance and choroidal penetration in cataract patients. J Biomed Optics. 2007; 12: 041211.
Copyright 2017 The Authors
Fibrillin Microfibrils Keep the Cornea in Shape
Protective Role for CD1d-Reactive Invariant Natural Killer T Cells in Cauterization-Induced Corneal Inflammation
Deletion of the Chemokine Receptor CCR1 Prolongs Corneal Allograft Survival
Aquaporin-3-Dependent Cell Migration and Proliferation during Corneal Re-epithelialization
CD8 T Cells Mediate Transient Herpes Stromal Keratitis in CD4-Deficient Mice
Alpha-internexin as a Corneal Autoantigen in Spontaneous Autoimmune Keratitis Mouse Model
From Other Journals
Latent Sensitization in a Mouse Model of Ocular Neuropathic Pain
Universal Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells for Cellularization of a Corneal Scaffold
Corneal Cross-Linking with Riboflavin and UV-A in the Mouse Cornea in Vivo: Morphological, Biochemical, and Physiological Analysis
PTEN Inhibition Facilitates Diabetic Corneal Epithelial Regeneration by Reactivating Akt Signaling Pathway
Pathological-Corneas Layer Segmentation and Thickness Measurement in OCT Images
Eye Anatomy and Disorders


text:
Tag "Argentina"
Climate Change & Global Warming
Canada and Argentina to peer review each other's fossil fuel subsidies
June 15, 2018 0 Comments	49 views
G7 and G20 hosts commit to a process they hope will help them phase out taxpayer support for coal, oil and gas Canada and Argentina have announced they will conduct peer reviews of their public finance of fossil fuels.
"Phasing out inefficient fossil fuel subsidies is an important step in the transition to a low-carbon economy," said the statement.
Canada and Argentina are the hosts of this year's G7 and G20 respectively.
Canada, which is a member of both the G7 and G20, has committed to working to phase out inefficient fossil fuel subsidies every year since 2009.
This month, observers told Climate Home News that Canada's pledge to spend $3.5bn to nationalise the Kinder Morgan oil pipeline counted as a huge new subsidy.
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"As a G20 country, Argentina reaffirms its commitment to rationalise and phase out, over the medium term, inefficient fossil fuel subsidies that encourage wasteful consumption, recognizing the need to support the poor, and we will endeavour to make further progress in moving forward on this commitment," said Juan José Aranguren, Argentina's minister of energy and mining.
The announcement did not set a deadline for the review to be delivered.
"Canada's partnership with Argentina in this peer review demonstrates our commitment to ambitious climate and energy policies," said Jim Carr, Canada's minister of natural resources.
Echo Energy Announces Gas Discovery Onshore Argentina
May 18, 2018 0 Comments	49 views
Latin America focused upstream oil and gas company, Echo Energy plc, has announced a gas discovery at the ELM-1004 exploration well, which is located at the company's Fracción C asset onshore Argentina.
The well, which was drilled into the Lower Tobífera formation to a total measured depth of 5,774 feet, encountered over 130 feet of gas shows through the Upper Tobífera. "We are very excited to have confirmed the presence of a substantial gas column in this first well of our four well exploration program and look forward to the arrival of the completion rig and subsequent testing in June," Fiona MacAulay, Echo CEO, said in a company statement.
"Meanwhile our attention turns to ELA-1 where we expect to spud the well within the next few days," MacAulay added.
Echo announced the commencement of drilling on ELM-1004 on May 9.
The well is situated approximately 2.5 miles north east of the existing Estancia La Maggie gas collection facilities.
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All comments are subject to editorial review.
Off-topic, inappropriate or insulting comments will be removed.
World Travel & Tourism Council Formally Joins Forces with the UNFCCC
April 30, 2018 0 Comments	64 views
Buenos Aires Playa is an urban beach on the Río de la Plata mostly used in the summer in Buenos Aires.
By Gobierno de la Ciudad de Buenos Aires from Ciudad Autónoma de Buenos Aires, Argentina – Buenos Aires Playa, CC BY 2.0, Link Guest essay by Eric Worrall The World Travel and Tourism Council meeting in Buenos Aires has announced a formal partnership with the UNFCCC to jointly tackle climate damage caused by global tourism.
The World Travel and Tourism Council (WTTC) and the United Nations Framework Convention on Climate Change (UN Climate Change) have agreed a common agenda for climate action in tourism.
Agreed at the WTTC Global Summit in Buenos Aires, Argentina last week, the partnership backs goals set by the Paris Agreement to maintain temperature levels at 2 degrees above pre-industrial levels, and the economic importance of tourism to the world's economy (10% of GDP and one in 10 jobs), the Common Agenda sets out a framework for the two organisations to recognise and address the linkages between tourism and climate change.
Announcing the agreement, Patricia Espinosa, Executive Secretary of UN Climate Change advised "this is the first time the tourism sector has actively engaged on a global level with the UN Climate agenda.
"We recognise that tourism has a huge role to play in addressing climate change.
While climate change itself poses significant risks to some tourism destinations, in many of the most high-risk areas, tourism can provide opportunities for communities to build resilience to its impacts.
At the same time, as a fast-growing sector, tourism has a responsibility to ensure this growth is sustainable and sits within the parameters set by the Paris Agreement.
… Read more: https://www.ausleisure.com.au/news/wttc-and-un-climate-change-in-new-partnership-to-tackle-global-warming/ The WTTC press release is available here Sadly this partnership may add to the moral burden of climate scientists, who already accumulate substantial air miles attending conferences in exotic holiday destinations like Paris, Cancun and Davos.
Personal inspections of environmentally endangered eco-tourist hotspots will likely be required, maybe even a rota of permanent monitoring in the most sensitive locations, to ensure places of special ecological significance are properly preserved.
More Polar Scientists Stuck in the Ice
March 13, 2018 0 Comments	61 views
Guest essay by Eric Worrall h/t Breitbart / James Delingpole – US scientists appealed to Argentina for help when the US icebreaker Laurence M Gould failed to break through the ice.
American scientists rescued from icebound Antarctic island
March 12, 2018 A group of American scientists was rescued from an island off Antarctica's coast after ice prevented a U.S. Antarctic Program research vessel from reaching them.
The four U.S. scientists and a support staff member conducting research on Antarctica's Joinville Island were airlifted by helicopter Sunday from an icebreaker ship dispatched by Argentina, said the National Science Foundation, which funds and manages the Antarctic program.
… Read more: https://www.yahoo.com/gma/american-scientists-rescued-icebound-antarctic-island-205205965–abc-news-topstories.html No doubt in a few decades helicopter rescues of scientists stuck in the global warming will be a thing of the past.
Having said that, perhaps the USA should consider upgrading its ice breaker fleet, just in case the ice melt takes a little longer than anticipated.
WUWT readers may remember this story from last year, where Chris Turney, leader of the ill fated "ship of fools" Spirit of Mawson expedition that go stuck in Antarctic sea ice said: "Penguins Don't Migrate, they're dying!" and of course blamed the dreaded "climate change" as the reason.
: +0.20 C (about 0.36 degrees Fahrenheit) above 30-year average for February.
Northern Hemisphere: +0.24 C (about 0.43 degrees Fahrenheit) above 30-year average for February.
CREDIT NASA/Kirk Knobelspiesse Clouds play a prominent role in moderating Earth's climate, but their role is still poorly… Guest essay by Eric Worrall Michael Bloomberg joins Leonardo DiCaprio and the Angry Birds character "Red" as a UN appointed climate envoy.
Brazil fights emissions cap for shipping, citing cost concerns
March 8, 2018 0 Comments	59 views
Ahead of a key meeting at the International Maritime Organisation in April, Brazil is leading objections to setting tough climate targets for the sector Brazil is seeking to water down prospective climate targets for the shipping sector, documents seen by Climate Home News show.
The group deleted sections proposing to cap greenhouse gas emissions from shipping at 2008 levels and reduce them "significantly" by 2050, arguing only relative measures of carbon intensity should be used.
An overarching vision to achieve a zero carbon sector by 2075 was replaced with the vaguer "no later than in the second half of this century".
It added that no policy measures "are expected to be implemented" before 2023, ignoring suggestions for action that could be taken sooner, such as speed restrictions.
The strategy should "give a signal to the private sector that the transition is inevitable," he said.
In a comment article first published in Brazilian business paper Valor Econômico and translated into English for CHN, researchers from the Institute for Climate and Society warned Brazil had become a laggard on this issue.
Chiarelli rejected the suggestion – raised by Influence Map research published last October – that Vale had excessive influence over Brazil's position.
"Vale has not captured the Brazilian delegation," he said.
A broader group of nearly 40 countries has signed up to the Tony de Brum Declaration, named after a Marshall Islands statesman, which is less specific but emphasises the urgency of action.
These include South American states Chile, Peru and Colombia, which face some of the same challenges as Brazil and have previously aligned with its IMO positions.
Emefcy Group, RWL Water form new company Fluence
July 27, 2017 0 Comments	151 views
Emefcy Group, RWL Water form new company Fluence.
Emefcy Group and RWL Water have completed their previously announced definitive merger agreement to form Fluence Corporation.
The agreement, which was initially announced in May this year, brings together latest technologies and delivery platforms to optimise treatment solutions for the water cycle.
Fluence's products will address all segments of the water value chain, from desalination to wastewater treatment and waste-to-energy.
The comprehensive distributed water services firm will provide local and sustainable treatment and reuse solutions to businesses and communities across the globe.
Fluence CEO Henry Charrabe said: "The combination of complementary decentralised and packaged water and wastewater treatment solutions is paving the way for Fluence to emerge as the leading global provider of innovative water and wastewater solutions serving municipal, industrial and commercial markets.
"In pairing RWL Water's proven execution capabilities and standardised solutions with Emefcy's breakthrough technologies, Fluence operates at the forefront of the movement that is solving the world's most pressing water challenges." The company will continue to serve and support all existing RWL Water and Emefcy customers and will bring the latter's wastewater treatment technology to new clients across the globe.
Fluence executive chairman Richard Irving said: "The merger will substantially accelerate Emefcy's deployment in China and other key markets while making significant operational and distribution gains." The operating and regional entities of Fluence are located around the globe, including the US, China, Australia, Mexico, Argentina, Brazil, Italy, France, Israel and Dubai.
Could Argentinian Politics Beat the Vaca Muerta?
Argentina's Vaca Muerta is one of the few places in the world outside of the United States developing unconventional oil and gas.
"But because of government intervention in the energy market, the implementation of price controls, as well as some rather dubious government maneuvering within the energy sector, investment plummeted, and as a consequence, the production of natural gas plummeted to the point where Argentina now imports approximately one-quarter of the natural gas it consumes," Jones explained.
So, Jones said, the government froze energy prices.
That clearly reduced the incentive for many energy companies to invest." As such, Cuenca said, "The first step of the development of the Vaca Muerta will be to restore energy self-sufficiency or at least energy autonomy to Argentina, as well as allow Argentina to return to being a natural gas exporter." 12 View Full Article Later gas price incentives by the government – boosting the price to $7.50 per million British Thermal Units (MMBtu), compared to U.S. prices closer to $3 per MM Btu – expire in 2021.
U.S. cost efficiencies, business environment and labor costs are likely to keep its wells more economic than those in Argentina.
Part of what's pushing up the per-well cost is the powerful labor unions in Argentina.
Eventually, the unions and the provisional government came together to sign the Vaca Muerta agreement in January.
It's not a regulatory [challenge] purely – it's not a government issue – it's more about the unions allowing operations to be more efficient," Cuenca said.
The election of Mauricio Market is a pivotal part of the new attitude toward maintaining the energy business in Argentina.
"I think a lot of people will be holding back from pushing forward with investment and other commitments in Argentina until they have further confirmation that the Market government is here to stay." 12 View Full Article

redpajama_set_name: RedPajamaCommonCrawl


text:
Sorkhakan (, also Romanized as Sorkhakān and Sorkhekān; also known as Sorkhagān, Sorkhegān, Sorkh Kūh, and Surkh Kān) is a village in Negar Rural District, in the Central District of Bardsir County, Kerman Province, Iran. At the 2006 census, its population was 338, in 80 families.

References

Populated places in Bardsir County

redpajama_set_name: RedPajamaWikipedia


AUBURN HILLS, Mich / TOKYO – Chrysler LLC and Nissan Motor Co., Ltd., today announced two new agreements for the supply of products between both companies. In January, Nissan agreed to supply Chrysler with a new car based on the Nissan Versa sedan for limited distribution in South America on an Original Equipment Manufacture (OEM) basis in 2009.
Nissan will manufacture an all-new, fuel-efficient small car based on a unique Chrysler concept and design. This new segment entry for Chrysler will be sold in North America, Europe and other global markets in 2010, and manufactured at Nissan's Oppama Plant in Japan.
Chrysler will manufacture a full-size pickup for Nissan. Based on a Nissan unique design, this truck will be manufactured at Chrysler's Saltillo (Mexico) Assembly Plant. In order to accommodate this product, Chrysler will shift volume from Mexico to its U.S.-based assembly plants that produce pickup trucks. Sales of the pickup in North America will start in 2011.
This latest OEM supply agreement extends a long standing product exchange relationship between the two corporations, with Nissan affiliate JATCO already supplying Chrysler with transmissions since 2004.
"In January, we said we would continue to look for additional OEM opportunities with Chrysler," said Carlos Tavares, Executive Vice President, Nissan Motor Company. "This latest agreement builds on Nissan's proven track record to deliver win-win product exchanges with multiple manufacturers around the world," continued Tavares. Since the signing of the first OEM in January, the two companies have also agreed to maintain an open dialogue to explore further product-sharing opportunities.


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Braces are the most common type of orthodontic treatment. Braces work to gradually straighten your teeth by using metal brackets that are connected by a wire. These brackets and wires work together to exert a constant, gentle pressure on the teeth. Invisalign works in a similar way, but rather than using metal brackets and wires, Invisalign uses clear plastic aligner trays to move your teeth. Both braces and Invisalign are effective for achieving a straight, healthy smile. Dr. Duke will help you determine which type of orthodontic treatment is best for your smile. We always consider your personal preferences and individual dental needs when planning dental and orthodontic treatment.
To learn more about orthodontics and how we can improve your smile, we welcome you to call 801-295-7171 or visit us at Dental Designs of Bountiful today. We are dedicated to helping you achieve the healthy, attractive smile you deserve!
Invisalign® is a revolutionary new way to achieve the smile of your dreams. Rather than using metal brackets and wires to straighten your teeth, Invisalign involves wearing a series of smooth, clear plastic aligners. The aligners are custom made to fit your smile and guide your teeth to their ideal positions. Approximately every two weeks, you will visit Dental Designs of Bountiful to switch to a new set of aligners to continue adjusting the alignment of your teeth. Invisalign aligners are removable, allowing you to brush, floss, and eat as usual. Most patients also find Invisalign aligners to be exceptionally comfortable.
We welcome you to contact us today at 801-295-7171 to discover whether Invisalign in Bountiful, Utah, is right for you!


Long before Amazon announced the company would be entering the grocery business, the independent supermarket industry has been utilizing – and succeeding in – e-commerce to better serve communities and differentiate themselves from competitors.
There's a good reason for grocers to be online too, with nearly one fifth of all grocery spending – a total of $100 billion – expected to come from online shoppers by 2025. However, online shopping won't replace trips to the store any time soon, according to a new Gallup poll. Think of it as just one more opportunity for independents to deliver high quality service and exceed the expectations of the consumers they know best.
It's not just the large chains who are exceling in the space either. Broulim's Fresh Foods, an independent grocer with 10 stores that has served Idaho and Western Wyoming for the past 90 years, recently entered the online space. It paid off big, too.
The supermarket launched Broulim's Online, powered by Rosie, in August 2015 to provide online access to groceries acquired through click and collect at the store or by delivery. Customers can shop online and have their food items delivered six days a week, and Broulim's promises all in-store sale prices will be found online and only the best produce will be brought to shoppers' front door.
But that wasn't enough – the supermarket was eager to get consumers excited about buying online. So, in May 2016, the store hired a film crew to visit the homes of five Broulim's Online customers to capture their experience. In total, they produced four 15-second customer testimonial spots and a four-minute interview highlight video.
From there, they executed a two week long digital campaign that included Facebook engagement and traffic ads, YouTube pre-roll ads, and email marketing campaigns. The hard work paid off with big results and Broulim's witnessed a significant increase in awareness of the online service, user signups, and order flow.
The campaign even gained nearly 150,000 online impressions, over 1,100 click throughs on Facebook and YouTube, and over 850 email subscribers opened marketing emails. As a result, the store saw a combined online revenue increase of 21 percent from August to September in two different locations. They also collectively had over 100 new users sign up for the online service, representing a 91 percent increase over the previous month and the most single signups in a single month in six months. Consequently, the new users were effectively converted with 126 first-time orders.
But Broulim's didn't just increase their service or grow the customer base – they gained recognition as a leader in the grocery industry, besting more than 300 entries to be named this year's "Outstanding Marketer" – one of the highest honors of NGA's Creative Choice contest – at The 2017 NGA Show.
The annual Creative Choice Awards contest, presented by Kellogg's and Unilever, honors and recognizes the best marketing and merchandising programs throughout the industry that both boost sales and build customer loyalty. And if you're planning to put on a creative program, campaign, or event like Broulim's we want to know about it! With 11 different submission categories, from public service to grand opening and digital marketing, there's something for everyone.


text:
module Data.Aeson.Validation.Internal.Context
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import Data.Aeson.Validation.Internal.Prelude

-- Breadcrumbs into a JSON object in reverse order.
data Context
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redpajama_set_name: RedPajamaGithub


text:
Michigan Association of Railroad Passengers
Amtrak Status
MARP Vision
Bylaws and Resolutions
Annual Meeting Minutes – November 13, 2010 – Durand
Durand Union Station, Durand
Those Signing In: Hugh Gurney, Clark Charnetski, John Langdon, Don Monteith, Tim Fischer, Dietrich Bergmann, George Schlaepfer, Harry Gow, Steve T. Sobel, John DeLora, Mary Myers, Daniel Myers, Julien Wolfe, Pat Post, Richard F. Pekarek, Ren Farley, Dan Platz, Larry Swartz, Jim Wallington, Larry Sobczak, Steve Vagnozzi, Doug Wilson, Jim Hinkins, Dave Randall, John Guidinger, Marilyn Guidinger, Kay Chase, Robert Tischbein, Rich Vavra-Musser, Robert Patterson, Diane Patterson, Andrew Kent, Tim Hoeffner, Norma Ward, Kathleen Newell, Kaz Fujita
Also Present: Douglas DeLora, Martha K. Wolfe, Scott Hercik, Jane Hinkins, Phyllis Bergmann, Joe Moran, Pat Norris
Call to Order: The meeting to order at 10:18 a.m. All present introduced themselves.
Treasurer's Report: Treasurer Randall reported that as of October 31, 2010, MARP had $3,375.00 in its checking account and the Pratt Fund was valued at $14,205.95. Langdon moved approval. The motion was seconded by Monteith and approved by the members present.
Secretary's Report: After review, Tischbein moved approval of the minutes of the October 23, 2010 meeting. The motion was seconded by Langdon and approved by the members present.
Bonding Bill: Langdon called attention to the fact that a bill is now before the Michigan Senate that would authorize use of state building authority bonding as the state match in rail projects. He urged all present to write a letter to their own state senator urging his or her support. Langdon has a sample letter he will share.
Chairman's Annual Report: Tischbein reviewed the Chairman's Annual Report, which had been handed to all present in printed form. In response, DeLora noted that MARP's website had seen an increase in hits from 30,000 annually to 100,000 annually. NARP is looking at this as a model.
Gow expressed thanks for MARP's response to the Detroit River International Crossing project and for its successful efforts to extend Amtrak Thruway Service to Sault Ste. Marie.
One member pointed out a problem he noted recently when his bus arrived late at the Kalamazoo depot. Not a human being was in sight. The member was being picked up, but wondered what a stranger on that bus would have done. Wallington noted that this issue would be on the agenda for meetings with Amtrak and Indian Trails.
Outreach: Tischbein thanked all who had helped with Outreach over the past year, including Gurney, Newell, Langdon, Sobel, Charnetski, Hinkins, Fischer, Chase, Valderas, Frezell, Wallington, DeLora, Pekarek and Swenson.
Guest Speaker: Tim Hoeffner, MDOT Intermodal Policy Administrator, reported progress on a number of fronts. MDOT has received a $3.2 million grant to study the High Speed Rail Project. This is a joint project with Indiana and Illinois and will include an Environmental Review. The process will take 18-21 months. FRA is anxious to see this study move quickly.
A total of $151 million was awarded for the Dearborn-Kalamazoo sector. MDOT had requested $241 million. Hoeffner plans to meet face to face with Norfolk Southern officials next week to discuss the project. Many in the passenger rail community are truly excited about the possibility of passenger trains running on their own rails.
Plans for the West Detroit connector are moving along and should see fruition in the near future.
Thanksgiving trains on the proposed Ann Arbor-Detroit commuter line were never scheduled. Money was a problem as well as issues with the seating in the refurbished coaches. Bids for leasing locomotives have been awarded and the successful bidder has painted one of their locomotives in the MITRAIN color scheme.
WALLY communities must now decide on the location of stations in their respective communities.
At this point in time, MDOT has no clear direction as to where the new governor stands on the various rail projects, though he appears to support both WALLY and the Ann Arbor-Detroit commuter service. MDOT will continue to take an incremental approach to these projects.
Michigan residents are voting by taking the train. Ridership in October was up 20% on the Wolverine line and 39% on the Blue Water line.
In response to Vavra-Musser's query about reaction to Ohio and Wisconsin opting out of high speed rail, Hoeffner noted that MDOT had been talking to Talgo, who recently located a facility in Wisconsin. MDOT is particularly interested in bringing maintenance of all Midwest trains to a facility in Waterford.
Hoeffner conceded that Michigan may have to pick up the costs of the Wolverine under PRIIA. He is not comfortable with the capital costs that are being quoted.
Wallington noted that more cars on the Blue Water were bringing more riders. Hoeffner added that MDOT's efforts to rebuild relations with CN after a wreck in Charlotte several years ago were also paying off.
In response to a question from Robert Patterson as to how the $150 million grant will be broken down, Hoeffner stated that FRA is quite flexible. Norfolk Southern has expressed a willingness to take less than the appraised value for the Dearborn-Kalamazoo line, and that can be applied as part of the 20% state match. Some of the money will have to go to maintenance and not acquisition.
Hoeffner feels that only minor upgrades are needed to permit 110 mph speeds, though Norfolk Southern wants to hold at 90 mph. Norfolk Southern will continue operating freights on the line after acquisition.
In response to a question from Langdon, Hoeffner indicated he didn't know whether the new Amtrak station in Grand Rapids would be staffed. It could be that Indian Trails or Greyhound would handle ticket sales.
Robert Patterson expressed concern that increased speeds and more frequencies would adversely affect communities along the Chicago-Detroit line. If the Pere Marquette switches to the Amtrak line north of New Buffalo, that would mean even more rail traffic through that city. Has New Buffalo expressed concerns about more trains passing through their community? Hoeffner responded that signal technology can forestall the need for grade separations. MDOT can also work with local communities to improve local traffic flow. Amtrak trains will pass a grade crossing in 30-40 seconds, a wait that most motorists can tolerate.
In response to a concern that the East Lansing station closes before the Amtrak train is scheduled in the evening, Hoeffner noted that he needed to talk to Michigan State about that.
According to Hoeffner, the hope for a station track at Battle Creek is still on the books. Funding was shifted to West Detroit, a higher priority. The ultimate solution at Battle Creek is a flyover.
Responding to a question from Kent, Hoeffner stated that federal grants and the Comprehensive Transportation Fund will cover capital expenses. Revenue from ridership should cover routine maintenance. We must work to grow the business to cover fixed costs.
As to the Michigan Owl, the decision to run the train over the Labor Day weekend was made too late to really promote it. MDOT is not in a position to fund that train at this time.
MDOT pays for service under Amtrak's second option, where Michigan pays an agreed upon fixed cost for service during the year. If ridership is up, Amtrak keeps any additional amount that it takes in. MDOT feels that this is the better option because it gives Amtrak an incentive to do a better job. Because ridership is up, Hoeffner anticipates that the basic contract will be slightly lower in 2011. Amtrak may receive additional amounts for special trains in the Ann Arbor-Detroit line.
MDOT is working with Amtrak on increased Business Class seating. At this point, a whole car is not needed. MDOT does not see Quiet Cars on Michigan lines at the present time. Hoeffner has asked his operations staff to look into the feasibility of connecting additional cars en route on some trains.
Responding to a question from Tischbein, Hoeffner stated that MDOT has taken no position on the proposed abandonment of a rail line serving Ontonagon. The community is looking for a buyer for the plant that is closing there.
In conclusion, Hoeffner stated that he can't predict what the new governor will do as far a rail passenger service. He seems to have an urban agenda, supporting high speed and commuter rail.
Lunch: At this point, a break was taken for a delicious catered luncheon.
Thanks to Retiring Executive Committee Members: Gurney paid tribute to those going off the Executive Committee at this time, including Tim Fischer, Steve Sobel and Andrew Kent. He also recognized the contributions of those who had left the committee earlier, including Mike Whims and Michael Frezell. A Certificate of Appreciation was given to Clark Charnetski for his thirty-five years of service to the Michigan rail passenger.
Tribute to John DeLora: Bergmann then offered tribute MARP's founder, John DeLora, who has served the cause of the Michigan rail passenger for thirty-seven years.
DeLora called a small number together for the first meeting in November, 1973. Two months later, in January, 1974, the first newsletter came out. It has been published continuously ever since, under several names, The Michigan Mainliner; The Wolverine Passenger: and now The Michigan Passenger. The organization was incorporated in January, 1975, with John DeLora, John Guidinger and Craig Harris as Incorporators.
From 1977-1982, there was a subsidiary organization, The Michigan Passenger Foundation, whose aim was to assist passengers in those early Amtrak days. Bergmann illustrated highlights in DeLora's leadership right up to October, 2010, when he was present in Jackson for the announcement of the $150 million high speed rail grant.
An engraved plaque was presented in recognition of DeLora's "40 years at the throttle".
Scott Hercik then added to the list of DeLora's accomplishments, followed by Harry Gow, who thanked DeLora for his work on the legislation and his support for VIA. Unfortunately, VIA is currently building a new Windsor station in Walkerville, which will make connections to Detroit very difficult. Gow presented DeLora with a VIA thermos to take on his trips.
Larry Swartz of America By Rail praised DeLora for his achievements. Michigan's rail network feeds passengers to America By Rail and helps make the business a success. As a token of thanks, Swartz presented DeLora with a $1,000 gift certificate.
Others speaking in praise of DeLora included Phyllis Bergmann, Clark Charnetski, Norma Ward, and George Schlaepfer, one of the signers of the original charter.
DeLora acknowledged all who had spoken and made presentations and pledged to continue his work in support of passenger rail service on the national level.
Adjournment: The meeting adjourned to enjoy a festive cake in honor of the occasion.
Respectfully submitted, Accepted:
Hugh D. Gurney Robert Tischbein
Secretary Chair
Previous PostEditorial: Keep light-rail opportunity on track for city, regionNext PostMeeting agenda – January 15, 2011 – Williamston
November/December 2019 edition of OnTrack January 12, 2020
MARP meets in Kalamazoo on Nov.16, 2019 November 5, 2019
September/October 2019 edition of OnTrack October 14, 2019
August 2019 edition of OnTrack August 18, 2019
Register now for MARP's 2019 Annual Meeting! August 10, 2019
July 2019 edition of OnTrack July 25, 2019
June 2019 edition of OnTrack June 24, 2019
MARP meets in Sanford on June 22 June 10, 2019
May 2019 edition of OnTrack May 9, 2019
March/April 2019 edition of OnTrack March 30, 2019
Learn about 30-year transportation plan at MARP's April meeting March 27, 2019
February 2019 edition of OnTrack February 22, 2019
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text:
"The Outsider" Recap: "Que Viene El Coco"
by Gena Radcliffe January 26, 2020 January 26, 2020
Holly picks up one thread after another, & begins to get a full, chilling picture of what the team might be up against.
Warning: don't read until you've seen the episode!
The recap of the premiere episode of The Outsider mentioned Stephen King's fascination with twins and doppelgangers, exploring it in several books to varying degrees of creepiness. Perhaps his most well-known take on it is The Dark Half, a novel that is eerily meta in terms of King's feelings about his writing alter ego, Richard Bachman. Like Thad Beaumont, the protagonist, King "retired" Bachman for a while, after using Bachman's name to write "Rage," a short story about a school shooting that he would eventually regret publishing, even refusing new printings of it. In The Dark Half, Beaumont's alter ego, George Stark, not happy about his retirement, comes to life as a rotting, murderous version of Beaumont, killing those he believed gave Beaumont the idea to let him go. The novel ends on an ambiguous note, never really saying either way if Stark is real, or an alternate personality of Beaumont's.
Though traditional doppelganger folklore doesn't specifically say as much, pop culture has determined that they're always manifestations of our darkest impulses and desires. Your doppelganger isn't likely to be out there feeding orphans and helping old ladies cross the street (unless you yourself are a doppelganger — or did I just blow your freaking mind??). That idea is chilling enough on its own, but the latest episode of The Outsider, "Que Viene El Coco," gives it an extra malevolent, breathtakingly cruel twist.
If you're enjoying Cynthia Erivo as Holly Gibney, you're in luck, as fully 75% of the episode is devoted to her. The show is still dancing around what exactly is the nature of her "quirkiness," but, to be fair, it's not entirely relevant. Despite what her social limitations might be, Holly is out there literally walking the beat on this case, while Ralph's (Ben Mendelsohn) research so far has been reserved largely to his computer and watching security camera footage.
Still in Dayton, she learns more about Heath Hofstetter (Martin Bradford), the nurse who worked at Terry's father's nursing home and was later convicted of the murder of two little girls. Although she has to endure a blast of pepper spray to the eyes first, Holly hears from a co-worker of Heath's (Suehyla El-Attar) a story all too similar to Terry's — irrefutable DNA evidence despite a solid alibi that puts him nowhere near the scene of the crime.
Holly is out there literally walking the beat on this case, while Ralph's research so far has been reserved largely to his computer and watching security camera footage.
In exchange for a casual date with Andy Katcavage (Derek Cecil), a flirty former detective who worked the Hofstetter case, Holly learns that, like Frankie Peterson's family, the murdered little girls' mother and grandfather both died shortly thereafter, as did Hofstetter's mother and brother. "It's like a plague," the detective notes. A plague that, perhaps, starts with scratching someone, as we learn Heath Hofstetter did, when Terry bumped into him at the nursing home. And, as Ralph discovers, Terry (or who he thinks is Terry) did to Claude Bolton (Paddy Considine) the night of Frankie Peterson's murder.
Holly follows a new thread to New York City, where Heath visited a few months before the murders and had a fling with Maria Caneles (Diany Rodriguez), a bartender at his hotel. Astonishingly, Maria is also in jail for the murder of a child, under the same circumstances as Heath and Terry — DNA match despite a solid alibi, tragedy and death touching the family members of victim and accused killer alike, etc. Maria denies ever spending time alone with Heath, though, according to flashbacks, he spent time with someone who looks like her, and who scratched his back during sex.
"Que Viene El Coco" fills the audience with the same kind of dread as King's The Stand, in that there's some sort of unknowable force that we can't see on a path of single-minded, locust-like destruction. Sometimes it uses people for its tools, sometimes it uses people for its prey, but sooner or later everyone is touched by it, and everyone pays for it one way or another. Though it's assumed that Claude will soon be inexplicably spotted in two different places at the same time, with the blood of another dead child on his hands, Jack (Marc Menchaca) seems to be preparing himself to be some sort of tool for this mysterious being's use as well. In one scene, he buys what appears to be the entire inventory of a hardware store, only to leave it all piled up in the middle of the woods next to the rotting remains of a deer carcass, as if it's an offering to whatever it was that left the burn on the back of his neck.
Marc Menchaca & Hettienne Park in The Outsider (HBO)
We can also see the being doing its insidious work on Glory (Julianne Nicholson), who tries to contend with reporters disguising themselves as homeschool teachers, and threats against the lives of her daughters to "even the score" with quiet strength and dignity. You can see her will sagging a little, though, and this is all part of the nefarious plan, to wear her and her children down until either they hurt themselves, or someone hurts them. As Idylis Castro (Susanna Guzman), a visitor at the prison who overhears Holly's conversation with Maria, explains to Holly later, this is the nature of a creature who goes by many names in many cultures: El Coco, Black Annis, Baba Yaga, the Boogeyman. "The Grief Eater," Idylis calls it, who wreaks unimaginable horror and then dines on the pain it causes. "The suffering of the family is its dessert," Idylis says.
In short, Frankie Peterson is dead. Terry Maitland is dead. But it's still not finished yet. It's still hungry. If the mere idea of that doesn't send a chill up your spine, particularly when you think about someone you know who never seems to have any kind of luck but bad, I don't know what to tell you.
Ralph seems to still be grudging at best about the fantastical elements Holly is exploring, but can no longer deny that something he may never fully understand is going on when he's paid a visit by Merlin Cassidy (Jakob Gruntfest), the kid who originally stole the van later used in Frankie Peterson's kidnapping and murder. He admits to Ralph that he did see who drove the van away from where he left it in Dayton, but was too frightened initially to tell him. Merlin draws a figure whose facial features aren't quite formed — blurry, you might say, like Jessa Maitland's nighttime visitor. The visitor Ralph tells Jessa she doesn't need to worry about, because he isn't real. We tell our children these things to comfort them, or in the inverse, to make them behave, even though, as Idylis tells Holly, "It doesn't matter. It still takes what it wants."
Random Thoughts:
"Que Viene El Coco" is an information packed episode, so much so that I wonder how all of it is going to be stretched out over another six episodes.
Despite its tonal resemblance to True Detective, The Outsider overall seems far more interested in showcasing female characters and actresses, and "Que Viene El Coco" in particular is a goldmine of excellent performances, including Erivo, Rodriguez, and Guzman.
That being said, I do find it odd that Glory, who can't be older than her late 30s or early 40s, doesn't know the correct word for "blogger."
It's here that the adaptation is starting to divert somewhat from the novel (other than killing off Ralph and Jeannie's son rather than having him just be away at camp). I'm now no longer entirely sure how this is all going to play out, and it's weirdly exciting.
I mentioned it in last week's recap, but much of the effect of The Outsider would be minimized if not for the bone=chilling score by Danny Bensi and Saunder Jurriaans.
I love the closing shot of Holly continuing her research while soaking in the tub. We should all have that kind of work ethic.
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ben mendelsohncynthia erivoderek cecildiany rodriguezhbojakob gruntfestjulianne nicholsonmarc menchacamartin bradfordpaddy considinestephen kingsuehyla el-attarsusanna guzman
Gena Radcliffe
Gena Radcliffe is the co-host of the award-winning (not really) horror podcast Kill by Kill, and has also written for F This Movie, Anatomy of a Scream, and Grim magazine (although the Spool is her pride and joy). Her pitch graveyard and "pieces that don't really belong anywhere else" can be found at genaradcliffe.com, and you can see her slowly losing her mind at Twitter under @porcelain72.
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"The Outsider" Recap: "Fish in a Barrel" & "Roanoke"
HBO's adaptation of Stephen King's supernatural murder mystery is off to an eerie, understated start.
The Outsider: "Dark Uncle" Sends an Ominous Message
Ralph reluctantly takes on a partner as a colleague has a brush with the impossible.
"The Outsider" Recap: "Tigers and Bears"
The team figures out where to center their attack, as the penultimate episode of the season continues to struggle with pacing.
Next: Box Office Report: "Bad Boys for Life" Hangs On for a Second Week
Reading: "The Outsider" Recap: "Que Viene El Coco"


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we understand that you need factories
we understand that you need cars
we understand that you need electricity
we understand that you need new technologies
BUT AS YOU CANNOT GIVE THESE NECESSITIES UP, WE ASK THAT YOU AT LEAST PLANT A TREE
"ხე მაინც დარგე"
How dire is the situation?
Since 2015, Georgia has lost an area 341 times the size of a football pitch every month to wildfires. The threat of deforestation is huge, and if you're living in Georgia, you've probably seen some of its effects already. Hotter summers, less rainfall, decreased humidity and harsher sunlight – those are all tell-tale signs of a changing climate that scientists have attributed, at least to a certain degree, to what's now a rapid loss of trees and cover vegetation.
Green4Georgia is a non-profit organization, which is acting in order to preserve and proliferate local, endemic trees in Georgia. We plant seedlings, promote reforestation/afforestation, and advocate the green economy for the betterment of our environment. In order to achieve these goals, we are creating a platform to facilitate donations, organize events, and promote going green within the Georgian population and corporations.
We plant forests in the cities!
Forests are gradually disappearing in the cities. Forest that kept hundreds of tons of carbon and generated thousands of litres of oxygen. Forest that was abundant of birds and animals for several decades. Forest where unique trees and plants used to grow several decades ago. Forest that helped the population to survive from starvation and frost in the absence of electricity and natural gas.
Zugdidi Botanical Garden and Arboretum of Kolkhetian Flora
On June 24, 2021, a Memorandum of Understanding (MOU) was signed with the Management Center of Botanical Gardens and Zugdidi Municipality Town Hall. The cooperation aims to create an arboretum of Kolkhuri Flora and nursery in the territory of Zugdidi Botanical Garden. Herewith, it is planned to implement annul activities aimed at raising awareness of the society about eco education, environment, nature and biodiversity preservation issues.
Trees are important for the environment
Trees are natural purifiers. Every tree you see around you absorbs harmful pollutants to return clean and breathable oxygen. In urban centers plagued with pollutants like carbon monoxide and ozone and respiratory irritants like dust and soot, trees act as a buffer mechanism to soak in all these toxins. The role of tress as nature's detoxing agent cannot be overemphasized. With the world's growing dependence on fossil fuels and widespread global deforestation, trees provide a time-tested mechanism to reduce the many harmful effects of air pollution and climate change.
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