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To investigate the tissue distribution and solubilities of various αA-crystallin truncation products in the cataractous ICR/f rat model.
Rat lenses from precataractous (21-day) and postcataractous (100-day) ICR/f rats were sectioned and applied to a matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) target plate. Mass spectrometry images were collected to obtain a macromolecular profile of the abundant lens proteins. Separately, age-matched lenses were extracted into water-soluble (WS) and water-insoluble/urea-soluble (WI-US) fractions and subjected to MALDI-TOF mass spectrometry to correlate the protein solubilities with the imaging data. Protein identities were assigned by using a top-down proteomics approach on a high-resolution mass spectrometer.
Ten novel αA-crystallin truncation products were identified, along with six previously known αA-crystallin truncation products. Nearly all truncations exhibited nuclear localization, with larger truncated products displaying a ringlike localization that progressed outward toward the extranuclear, cortical region. The distributions were similar in both ages with the only significant difference being the amount of tissue area encompassed by a particular species with increasing age. Almost all nuclear products fractionated into the WI-US fraction, whereas the five largest extranuclear species exhibited mixed solubility.
A successful methodology for the sectioning and imaging of pre- and postcataractous ICR/f rat lenses has been established. Data collected from these analyses indicate that there are multiple αA-crystallin truncation products present in both pre- and postcataractous rats. Furthermore, these species have defined lenticular localizations and unique solubilities that may be a consequence of lens development and protein function within the lens environment.
The ocular lens is an intriguing tissue, in part because of its atypical development and differentiation. The lens is formed by primary fiber cells derived from the ectoderm during early embryonic development that eventually become the lenticular nucleus.1 Subsequently, mature fiber cells (derived from a monolayer of epithelial cells present on the anterior surface of the lens) elongate to wrap around this nucleus, forming concentric layers.1 As a requirement for light transmission, the mature fiber cell is programmed to degrade all cellular organelles, rendering it incapable of further protein synthesis.2 Because of this, the lens retains all proteins that were synthesized before terminal fiber cell differentiation. This phenomenon is remarkable, as the lens must maintain transparency to function while possessing very high protein concentrations (greater than 320 mg/mL in humans1), which are necessary to refract and focus incoming light.
The crystallins are the most abundant class of proteins in the human lens3 and are subdivided into three major groups, the α-, β-, and γ-crystallins, each having a particular function. The α-crystallins, comprising nearly half of the total soluble lens proteome,4 are ~20-kDa proteins that form large molecular mass aggregates ranging upward from 800 kDa when complexed with substrates.4,5 They are small heat shock proteins possessing a chaperone-like function for binding and sequestration of denatured protein substrates.4,6 There are two subtypes of the α-crystallins, the αA and αB, with ~60% sequence homology, that can form hetero-oligomers within the lens environment at a 3:1 ratio.1,7 These large complexes help to maintain the solubility of denatured lens proteins to prevent aggregation, a hallmark of cataract formation.
We studied cataract progression by using a whole-tissue approach involving matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry imaging (MSI). This technique permits the detection and analysis of ionizable macromolecules within a tissue section by mass spectrometry, while retaining tissue distribution and localization.8 It is similar to classic immunohistochemistry, but is enhanced because MALDI-TOF MSI is not limited by antibody availability or specificity and simultaneously produces a chemical profile of tens to hundreds of ionizable protein species present across the tissue section during a single experiment.9 MALDI-TOF MSI typically allows for the detection of ionizable proteins within the range of 1,000 to 50,000 Da,10 which is amenable to lens tissue studies because of the small average monomeric protein molecular masses of the abundant crystallin proteins (~20 kDa).1
In the present study, we used tissue sections from both pre- (21 days of age) and post-cataractous (100 days of age) ICR/f rat lenses (Fig. 1). This animal model of age-related cataract disease is derived from the Wistar rat.11 Cataracts form in the ICR/f rat by ~10 weeks of age, with development beginning in the cortical region of the lens. As development continues, the opacification extends into the nuclear region, eventually producing a fully opaque lens cataract.12 Although the exact cause is not fully understood, the development of the cataract in the ICR/f rat may result from an early oxidative insult derived from abnormalities in lipid metabolism.13 As a result, changes within the lens microenvironment14–17 are semipermanent, as the lens is incapable of synthesizing or degrading proteins. This early insult renders these animals susceptible to cataract formation as time progresses and the repair–maintenance mechanisms are depleted.
To our knowledge, this study is the first to use MSI to investigate a small rodent lens, as well as the first comparison of MSI data from pre- and postcataractous lenses obtained from a laboratory model of cataracts. By combining the MSI imaging data with subsequent MALDI-TOF MS analysis of water-soluble (WS) and water-insoluble/urea-soluble (WI-US) ICR/f lens protein extracts, we have observed specific properties at pre- and postcataract lens stages. In the ICR/f rat lens, the αA-crystallin protein and its numerous truncation products are segregated by spatial distribution (nuclear versus extranuclear) and their intrinsic solubilities. These truncations may not be the primary cause of the cataract, as these features were detected in both the early and late stages of development in the ICR/f lens.
All reagents used in the study were of the highest quality available. High-performance liquid chromatography (HPLC)–grade solvents, including acetonitrile (ACN), ethanol (EtOH), and methanol (MeOH), as well as pure formic acid (99%; FA), were purchased from Fisher Scientific (Pittsburgh, PA). Sinapinic acid (SA) was purchased from Sigma-Aldrich (St. Louis, MO), and the water was purified (Milli-Q purification system; Millipore, Billerica, MA). All animal procedures were performed with the approval of and within the guidelines set forth by the University of Alabama at Birmingham Institutional Animal Care and Use Committee and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Frozen intact rat eyes (−80°C) were thawed, and lenses were dissected with the aid of a dissecting microscope (EZ4-D; Leica, Bannockburn, IL). The lenses were carefully extracted by corneal incision around the periphery of the eye. Care was taken to ensure the proper orientation of the lens, with the anterior side facing upward. The lenses were washed with deionized water to remove any contaminants and refrozen in a cryostat (CM3050-S; Leica, Bannockburn, IL) at −20°C, before sectioning. They were attached to the center of the cryostat chuck by layering optimal cutting temperature (OCT) medium. The lenses were sectioned into 20-μm slices, and thaw-mounted onto a methanol-layered, gold-coated MALDI plate (Applied Biosystems, Foster City, CA). Multiple sections were applied to each MALDI plate. After being placed on the plate, the sections were allowed to air-dry and then were washed with sequential submersion in 70% EtOH and 95% EtOH for 60 seconds per solution to remove lipids and salts and fix the proteins. The processed plates were then placed in a vacuum desiccator for at least 15 minutes before matrix deposition.
Matrix was applied with an acoustic reagent multispotter device (Portrait 630 Spotter; Labcyte, Sunnyvale, CA) that is capable of delivering 170 pL of matrix solution per spot. The matrix (15 mg/mL SA in 50:40:10 ACN:H2O:FA solution) was applied with a 400-μm offset-interleaving technique to achieve a final center-to-center spot distance of 200 μm across the entire tissue section. The spotter was operated in "fly-by" mode, which enables reapplication of the matrix at each spot for a total of 40 cycles (1 droplet/cycle), to ensure matrix saturation on the tissue.
Mass spectrometric analyses were performed in the linear, positive mode at a 20-kV accelerating potential on a time-of-flight mass spectrometer (Autoflex II Linear; Bruker Daltonik, Bremen, Germany) equipped with a laser (Smartbeam; Bruker Daltonik) capable of operating at a repetition rate of 200 Hz. The laser beam size was set to medium, estimated to be approximately 150 μm in diameter, at a laser power of 72%. A linear, external calibration was applied to the instrument before data collection, with a protein mixture of insulin (M+H+ = 5,734), cytochrome C (M+H+ = 12,361), myoglobin (M+H+ = 16,952), and trypsinogen (M+H+ = 23,982). Mass spectral data sets were acquired over lens sections with a raster step size of 200 μm obtaining 200 laser shots per spectrum (flexImaging software; Bruker Daltonik). After data acquisition, molecular images were reconstituted by the software. The data were smoothed with a Gaussian filter (width 2 m/z, four cycles), and baseline subtraction was applied by using the TopHat algorithm and normalized to total ion current with the software. Extracted peaks were plotted ±0.05% mass-to-charge units, to generate an image.
ICR/f rat eye samples were obtained at 21 and 100 days of age. The lenses were dissected from frozen intact eyes and washed with deionized water. To collect the WS proteins, the lenses were homogenized with 50 mM Tris-HCl buffer (pH 7.4) containing protease inhibitors (Complete Protease Inhibitor Cocktail; Roche, Basel, Switzerland) using a Teflon homogenizer in a 1.5 mL microcentrifuge tube. The samples were centrifuged at 18,000g at 4°C for 15 minutes, and the supernatant saved. This step was repeated to remove as much of the WS proteins as possible. Finally, the pellets were homogenized in a similar Tris-HCl buffer supplemented with 6 M urea, to obtain the WI-US proteins.
Samples were quantified by using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL), and read on a plate reader at 590 nm, to obtain the concentration. Protein (12 μg) from each sample was washed and concentrated with a C4-column (ZipTip; Millipore) eluted with 10 μL of 60% ACN and 0.1% TFA and dried in a centrifuge (SpeedVac; Agilent, San Diego, CA). Each sample was resuspended into 5 μL 50% ACN and 10% FA. An aliquot (1 μL) of the sample solution was mixed 1:1 with 15 mg/mL SA in 50% ACN and 10% FA and spotted onto a MALDI target plate (Bruker Daltonik). Spectra from these samples were collected on the same instrument as the tissue samples collecting data at a repetition rate of 100 Hz for a total of 2000 shots. The data were smoothed and baseline subtracted.
Protein separations were performed with an HPLC system (1100 Series; Agilent Technologies). ICR/f lens homogenates were loaded onto a 5-μm C5 column (250 × 2.0 mm ID; Jupiter 300; Phenomenex, Torrance, CA) pre-equilibrated with 5% ACN and 0.1% FA (buffer A) at a flow rate of 150 μL/min. Lens homogenates were eluted with a linear gradient of 2% to 50% buffer B (95% ACN/ 0.1% FA) over 40 minutes. The column eluate was collected with an automated fraction collector (Agilent Technologies) at 1-minute intervals into sterile 96-well plates (Eppendorf twin.tec, Hauppauge, NY). After each separation, the samples were dried in a centrifuge (SpeedVac; Agilent). The samples were resuspended in 30 μL of 50% ACN and 0.1% FA.
Individual fractions were directly injected into a 7-T mass spectrometer (LTQ FT-ICR; Thermo Scientific, Waltham, MA) via a chip-based infusion system (TriVersa NanoMate; Advion, Ithaca, NY). With each sample, a new spray nozzle was used to limit cross-contamination. High-resolution mass scans (m/z 200–2000) were performed in the FT-ICR cell with a resolution of 200,000 at m/z 400. Full scans were processed to deconvolute >2+ charge states to M+H+ (Xtract software package within the Xcalibur software; Thermo Scientific), with the setting of S/N, 2; fit factor, 44%; remainder, 25%; averaging table and a maximum charge state, 30. Individual charge states were manually isolated in the LTQ and fragmented by using CID (collision-induced dissociation) with N2 gas at 20% maximum intensity. Fragmentation spectra were collected as FT-ICR scans and analyzed (Xtract software; Thermo Scientific). De-isotoped MH+ precursor ions and fragment ions were submitted to an online engine (MS-Tag; http://prospector.ucsf.edu/prospector/html/instruct/tagman.htm/ provided in the public domain by the University of California San Francisco) and searched against the UniProt Rattus norvegicus database to obtain protein identities from the MS/MS spectra (www.uniprot.org/ The UniProt Consortium).18
Figure 1A shows a precataractous (21-day old), visibly transparent ICR/f rat lens, and Figure 1B shows a cataractous (100-day old) ICR/f rat lens in which there is a dense opacification throughout the entirety of the lens. Lenses such as these were used to evaluate changes in protein distribution and solubility during cataract development. Figure 2 shows the sectioned lens tissues and selected m/z signals that were obtained from 21-day (Figs. 2A–D) and 100-day (Figs. 2E–H) ICR/f rat lenses. In the optical images (Figs. 2A, A,2E),2E), there are obvious regions that are segmented by slight color changes (from white to translucent against the gold background of the MALDI target plate) that appear at various radii from the center of the tissue. These regions represent different stages of lens development, as the nuclear center was developed during embryogenesis with subsequent addition of lens fiber cells.19 From this, it is plausible to predict regional differences in protein signals, as these regions were formed at different points during lens development. We confirmed this by extracting images from the MSI experiment as indicated in Figures 2B–D and and2F–H.2F–H. For this purpose and for reasons discussed later, two of the major protein species present in the MSI experiment were chosen for analysis. The first protein is the N-terminally acetylated, full-length αA-crystallin (residues 1-173) protein, at an average mass of 19,835 Da ± 0.06% (Figs. 2B, B,2D,2D, D,2F,2F, F,2H,2H, blue). The second protein is an N-terminally acetylated αA-crystallin truncation product (residues 1-53), at an average mass of 6413 Da ± 0.11% (Fig. 2C, C,2D,2D, D,2G,2G, G,2H,2H, orange). Although other MSI studies have localized low-molecular-weight αA-crystallin truncation products (1-40, 1-50, and 1-58) to the nuclear core of other species,20–22 this is study is the first to identify the 1-53 truncation product as a major protein present in the lens.
The distribution of the full-length αA-crystallin in these rat lenses is in agreement with previous lens MSI studies, where it was found to associate only in the outermost cortical regions, whereas other posttranslationally modified forms (phosphorylated and truncated) were found to localize to subcortical regions deeper within the lens tissue.20–22 Despite the full-length αA-crystallin being present in lenses of both ages, the key distinguishing factor is the approximate 30% reduction in its cross-sectional area in the 100-day ICR/f lens (compare Fig. 2B with with2F).2F). Grey et al.22 recently showed a reduction in the full-length αA-crystallin abundance in progressively aged human lenses. In the rat lens, the αA-crystallin truncation product specifically localizes to the nuclear region, encompassing the whitest regions present in the optical tissue sections (Figs. 2C, C,2G).2G). Surprisingly, there appears to be no difference in the cross-sectional area of this product between the two different lenses, given their different ages and stages of cataract development.
The MSI data indicate three broad classes of radial protein distributions, which are depicted in Figure 3. Nuclear distribution was defined by the boundaries of the 1-53 αA-crystallin truncation product present in all lenses analyzed (dashed lines). The second boundary is defined as the nuclear ring that displays a ring-like distribution immediately outside the nuclear region that grows with age. The final class of lens protein distributions identified is the cortical distribution, in which the proteins fall beyond the nuclear ring and also display a ringlike presence, with the outermost region being defined by actively growing–dividing epithelial cells. As previously mentioned, these different distributions may represent different regions of the lens throughout the periodic stages of development. The complete MSI data are presented in Figure 4. Here, all the discernible peaks correlating to the αA-crystallin and its truncation products are shown (the protein identities are discussed later). Most of the distribution profiles are preserved between the different-aged sections with the most obvious difference being both the diffusion of particular species in the 100-day section (bottom) versus the 21-day section (top) and the reduced cross-sectional area of both full-length αA-crystallin species (the normal protein and the A-chain).
Although Figures 2D and and2H2H indicate a clear partition of the αA-crystallin and its truncation product within the tissue, they provide no information about their solubility characteristics. This parameter is very important, because it is the basis on which the development and progression of the age-related cataract is posited. By fractionating whole lens homogenates into WS and WI-US fractions and using standard MALDI-TOF MS analysis, we were able to correlate the MSI signals with their intrinsic solubilities. As indicated in Figure 5A, the full-length αA-crystallin was found to be highly enriched within the WS lens fraction in both 21-day (dashed black line) and 100-day (dashed gray line) lens protein extracts, although the corresponding WI-US proteins (solid black and solid gray lines) have minimal intensities. This information indicates that the full-length αA-crystallin is freely soluble when present in both the pre- and postcataractous lens tissue. Given the functions of the full-length αA-crystallin, these data were not surprising. However, the observed WI-US distribution of the 1-53 amino acid residue truncation of the αA-crystallin (Fig. 5B) was of great interest. Combining this discovery with its tissue distribution, it becomes apparent that the nuclear regions of both lenses contain protein/peptide species that are present in a WI state well before the development of a visible nuclear cataract.
In the 21-day ICR/f lens, the tissue was transparent (Fig. 1A) and contained limited light-refracting precipitates, although its 100-day counterpart (Fig. 1B) was fully opaque. The MSI and solubility data alone did not reveal a significant difference between the two lenses. By observing the mass spectral profiles of the two ages (Fig. 6), one can see a level of uniformity between the ages in the WS and the WI-US profiles. Figure 6A shows the WS profile comparison of the 21-day (dashed black line) and the 100-day (dashed gray line) ICR/f rat lens extracts. With few exceptions, nearly all the signals that were present in the 21-day lens were also present in the 100-day lens, indicating that the most abundant WS signals did not correlate directly with the development of the cataract. For the most abundant WI-US samples (Fig. 6B), there were peaks that were common to both, but new species (asterisks) began to appear in the 100-day ICR/f rat lens (solid gray line). These signals may correspond to various truncation products, such as the 1-53 αA-crystallin C-terminal truncation discussed earlier, that result from proteolytic events occurring in the ICR/f rat lens.15,17 Although these data show an increase in WI-US truncation products over time, again, they demonstrate that some protein species do not directly correlate with cataract development, as they were present in the pre- as well as postcataractous lenses.
Having identified multiple peaks by MSI that are fractionated by tissue localization and/or discrete solubility characteristics, it was then necessary to determine each peak's identity. MSI is a useful tool in providing information on the distribution and localization of the most abundant ionizable protein species within a tissue section; however, it lacks the capability of providing the exact identity of those peaks. The observed species were identified by analyzing the m/z signals detected in the previous MALDI-TOF experiments (both the MSI and solubility profiling) with a top-down directed mass spectrometry approach, as outlined in Figure 7. By analyzing the accurate mass of the abundant protein species on a hybrid linear quadrupole ion trap (LTQ) 7-T Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS), identification could be made in silico, based on mass matching of the N-terminally acetylated αA-crystallin protein and its possible truncation products (Figs. 7A, A,7B).7B). When the FT-ICR mass spectrometer detects a multiply charged ion (the [M+5H]5+ molecular ion), a deconvoluted, accurate monoisotopic mass can be assigned (Fig. 7B, 6409.26 Da). Comparatively, the peak that corresponds to this value in the MSI data was within 0.11% of this mass, indicating a very close association of these peaks based on both the mass and the abundance. The mass accuracy of the FT-ICR data alone are sufficient to identify this peak as the N-terminally acetylated αA-crystallin truncation product (residues 1-53), based on the calculated mass of 6409.19 Da (~14 ppm mass accuracy). However, top-down proteomics provides a partial amino acid sequence tag to confirm a protein assignment. In this case, a specific m/z value (precursor ion series, Fig. 7A) can be isolated in the ion trap and subjected to CID via inert nitrogen gas yielding fragment ions. These fragment ions, which are indicative of sequential cleavage of amino acid residues, are then transferred into the FT-ICR MS for m/z detection (Fig. 7C). This permitted accurate mass analysis of the fragment ions for submission to an online MS-Tag search engine to validate the previously associated protein identification.
With this approach, 18 different species of the αA-crystallin were identified (Table 1). Of these 18 species, only 6 have been identified in the ICR/f rat,17 another was found in a similar MSI study22 (residues 1-101), two were variations of the full-length αA-crystallin, and the remaining nine were novel truncation products. Most of these products were C-terminally truncated forms exhibiting specific tissue localization and solubility characteristics as summarized in Table 1. In general, the smaller the truncation product, the more likely that it was localized toward the nuclear region of the lens as well as a tendency to be WI-US. All these identified αA-crystallin truncation products were found in both ages, regardless of cataract status. The most notable difference was a dispersion and expansion of all truncation products larger than 151 residues with age. Each of these species exhibited a nuclear ring organization at the precataractous stage, only to become more diffuse and larger in surface area at the postcataractous stage. Also observed was the limited distribution of the full-length αA-crystallin protein to the outermost regions in the postcataractous sections.
Cataract is often produced by a dysregulation of the folding of proteins in the lens that condense to form large, light-scattering precipitates. This may seem to be a simplistic view of cataract development, but the fact that lens proteins remain in place and contribute to transparency throughout the life of an organism adds a new level of complication. The ability of these proteins to survive in a dynamic environment with stressors that include oxidation,23 deamidation,24 and truncations,25 as well as various chemical adducts with glutathione,26 sugars, and other proteins/peptides,23,27 while maintaining solubility is a significant departure from normal biochemistry. In this MALDI-TOF imaging study, αA-crystallin, the predominant lens crystallin, was distributed in different regions of the lens tissue according to its level of truncation. As a general rule, truncation products involving more than 10 C-terminal residues were found to colocalize near/within the nuclear region, the region that was formed during embryogenesis. Studies on αA-crystallin have shown that in aged lenses, multiple C-terminal truncations are present and that these products are altered in their chaperone capacities,28 solubilities,29 and the rate of subunit exchange within the complexes.30 These altered capacities may dictate their co-localization to specific regions in the lens. If that notion is true, the various functions of the αA-crystallin would be associated with its localization and the particular functional requirements necessary for that region. Overall, such compartmentalization may help to establish the necessary chaperone functions, indices of refraction, and overall lens health.
Our MSI data indicate that specific proteins co-localize in the nucleus of the lens. In 1998, Sweeney and Truscott31 described a nuclear barrier in aged human lenses that isolated the nucleus. This barrier is a product of lens development that occurs with age, and its formation may be the result of various biochemical events including the compaction of fiber cells,32 membranes decorated with denatured proteins,33 changes in membrane lipid composition,33,34 and truncations occurring on the aquaporin water channels.35 Studies have shown that water and glutathione, a major lens antioxidant, are reduced in their ability to diffuse into the nucleus which may prevent the delivery of nutrients and antioxidants to the nuclear core,31,36 resulting in an altered proteomic profile that is specific to this region. Although the initial development of this nuclear barrier was studied in human lenses, lenses taken from Wistar rats (the ICR/f's parental strain) have shown that the nuclear core has similar exclusion properties. In two studies,37,38 the MP20 protein, an abundant lens membrane protein, was found to redistribute from the cytoplasm to the cellular membrane in maturing fiber cells, by an unknown mechanism. This action was hypothesized to be the necessary impediment to dye accumulation in the nuclear core of the lens by a physical barrier.38 In support of this, our study indicated αA-crystallin truncation products in the lenses of the ICR/f rat (as noted in Fig. 4) were also found to tightly surround the nucleus and thus may be involved in the development, function, and impermeable nature of this barrier.
Investigating the solubility of lens proteins by the use of mass spectrometry (Figs. 5, ,6)6) showed the signals that were common between the cataractous states in both the WS and WI-US fractions. Associating the solubilities of the full-length and truncated αA-crystallin with the MSI data showed a trend for these products to be distributed in different regions of the lens on the basis of their solubility. The WI-US truncation products were found in the nuclear region, whereas the WS full-length αA-crystallin was exclusively in the extranuclear regions. The function of these truncation products and their localization to the nuclear core encourages the development of future hypotheses relating to the function of the N terminus of the αA-crystallin and its relation to multimerization and subunit exchange in its chaperone activities.39,40 As previously stated, the preeminent difference between the sections was the amount of surface area covered by a particular species. The general trend was an increasing amount of αA-truncation products in the 100-day old ICR/f lens section. This process may be a result of fiber cell alterations and subsequent liquefaction that occurs as a result of cataract development in this laboratory animal model.41 Another observation was the age-related, limited distribution of the full-length αA-crystallin proteins to the outermost periphery of the lens section. This distribution difference may be the result of fiber cell degeneration,41 whereby the cellular contents of cortical fibers, including the αA-crystallin, are no longer contained within a single fiber cell. They are free to interact with other proteins (either folded or unfolded) within the lens cortex, to form cataractous aggregates that remain undetectable by the technology used in the present study.
That WI-US proteins are already present in the nuclear region of young precataractous ICR/f lenses demonstrates the importance of obtaining a more comprehensive understanding of the development of the cataract. The data in this study indicate that WI-US proteins may be a result of normal lens biology in this animal model and not necessarily the direct cause of cataract development. Although it is necessary to evaluate the αA-crystallin truncations discussed herein in the Wistar rat, it is important to note that many of the observed truncation products were also found in the 21-day ICR/f rat lens nucleus. A lens at this age has no opacification,12 and therefore we conclude that the presence of these species indicates they may not be directly responsible for cataract development in this model. Other events (such as water dysregulation,12 ionic imbalances,15) may initiate cataract formation in the ICR/f lens that leads to the localization changes observed in Figure 4. Therefore, understanding what causes the switch from normal lens protein aging to cataractogenesis is an important question for lens biology, a question that can begin to be answered by studying the truncation products found in other animal models.
The experimental techniques used in this study have added to this understanding, but present limitations. For example, the particular matrix and solvent conditions used in the MSI experiments dictate which signals are measured across the tissue, and only a subset of all expressed proteins are detected in a single MSI analysis. Furthermore, large-molecular-mass aggregates of crystallin proteins, known to correlate with cataract formation, cannot be detected by the current MALDI-TOF instrumentation. Therefore, it is likely that other proteins and protein complexes that were not detected in this study are involved in cataractogenesis. Future work will focus on the identification, tissue distribution, and characterization of solubilities of these and other lens proteins. This work is a beginning in a new and exciting area of study of lens cataracts. With continuing advances in the technology, we will be able to look more rigorously at tissue sections, to understand proteomic changes that occur during cataractogenesis that were previously limited by the specificity and availability of antibodies.
Supported by Grant FY2007-08-84 from the EyeSight Foundation of Alabama; P50 AT00477 from The National Center for Complementary and Alternative Medicine and the NIH Office of Dietary Supplements to the Purdue University-University of Alabama (UAB) at Birmingham Botanicals Center for Age-related Disease (Connie M. Weaver, PI); and EY13462 from the National Institutes of Health (NIH) (KLS). Funding for the purchase of the mass spectrometers at the University of Alabama at Birmingham came from NIH/NCRR (National Center for Research Resources) Shared Instrumentation Grants S10 RR13975-01 and RR17261-01 (SB). Funds for the operation of the UAB Biomedical FT-ICR MS Laboratory were provided in part by the supporters of the UAB Comprehensive Cancer Center and the Department of Biochemistry and Molecular Genetics.
Disclosure: D.R. Stella, None; K.A. Floyd, None; A.C. Grey, None; M.B. Renfrow, None; K.L. Schey, None; S. Barnes, None
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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For sale $7,500
...4 wheel disc brakes and 4 speed manual transmission. Very rare fiberglass full body rail buggy. Economical 4 cal vw engine that starts, runs and sounds great. Don't let ...
Oklahoma City OK, United States1 month ago on spreadmyad.com
For sale $2,300
I have A street legal dune buggy that I bought at texas it runs and has its oklahoma plates probly epired havent checked but do not have the title ,becuase when I changed the texas title to oklahoma the tag agency wanted me to take it to them to inspected and they would send it to me by mail ...
California 20619, MD, United States1 month ago on craigslist.org
For sale $32,500
...registered as spcn (special construction) with sb100 smog exemption meaning it is 100% California street legal with no smog required ever. This process took 11 months to complete!! The buggy itself is a Funco chromoly chassis fitted with a turbocharged 2.5L Subaru sti engine and Mendeola md4S transmission. It has upgraded Weddle gears with a more streetable 3rd and ...
Holtsville NY, United States23 hours ago on ebay.com
For sale Auction
2005 Custom Built street legal show winner 1st place 2005 Monster mash Dune buggy nys title 2005 Custom with vin number assigned by dmv all new components in 05 fully powder coated removable 3 Piece Body Kit (dutz fasteners) adjustable dragster wing Chassis xtc motorsports $ seat Beachcomber Chassis New from torsion assy, Steering Box, ghia Disc Brakes, 2 Section ...
Tempe 85287, AZ, United States1 month ago on ebay.com
For sale $19,000
...2 seat busa pre-runner buggy $19,000 Light weight, 2 seat fully loaded street legal 1300cc Hayabusa pre-runner buggy is in perfect condition and has all the comforts of a full size car. This 2010 car has been in storage and barely broken-in. Title is free & clear with current registration. This vehicle is light weight, nimble and ...
Helix 97835, OR, United States2 weeks ago on craigslist.org
For sale $5,975
1962 vw dune buggy Street legal Paid registration clean title Roll cage and tow bar included 1600 dual port Runs and ready to go back seat new electrical wires New paint new upholstery bug motor Call show contact info for more information
Berlin 21811, MD, United States3 weeks ago on ebay.com
For sale $5,500
Dune Buggy vw Empy Dune Buggy 1973 Karmann Gia Frame Street Legal vin # ... Miles The Buggy Runs Well and id being sold as a project car I have owned this buggy for 5 years and am selling it to free up money for some new toys. It has been garage kept since I have owned it.
Coinjock 27923, NC, United States3 weeks ago on ebay.com
For sale Auction
...I installed heavy duty axles and cv joints last year. This vehicle is totally street legal and inspected in nc, with all lights, turn signals etc. I just replaced the ...
Follow this search and create an email alert for Joyner buggies street legal
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subset: commoncrawl


text:
The average rating for a hearing aid center in the Sanger area is 4.78 out of 5 stars. Here's what one of our users said about Physicians Hearing Services at CCENT, Fresno, CA. "Professional experience and care. They took the time to explain what was best for me and why. Lots of follow up care to make sure I had a good result with my hearing aids. " - John B.
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» Call (661) 750-6008


text:
Tags » Born Standing Up
Ten Lessons from Steve Martin
I am a big fan of stand-up comedy. I have enormous respect for the men and women—whether professionals or amateurs—who get up on stage and do their best to make us laugh. 1,053 more words
Tricking Yourself Into Creativity
I am ashamed to admit that it is almost November, and aside from necessary reading for work, I've only managed to read three books this year. 526 more words
Be So Good...
I've loved Steve Martin for ages. I recently rewatched "LA Story." So genius! My sister recommended I read his book, "Born Standing Up." I have yet to finish it, but even the first few chapters are amazing. 383 more words
Born Standing Up by Steve Martin
This was much more of a reserved read than I thought it was going to be. I think given the fact that it is written by and about Steve Martin, it would have been much more of a humorous look into stand up but it was actually a very deep account of his journey from Disneyland magic tricks to being one of the biggest names in the industry. 114 more words


text:
Is it all about brand recognition?
Garrett Harris 8 p.m., May 23
Posted April 6, 2000
Tucked away obscurely in two shady canyons behind the San Gabriel Valley community of Monrovia is one of the most beautiful parks in the Los Angeles area: Monrovia Canyon Park. Adding spice to the rich ...


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
32 year old man | Gardena, CA, USA | Seeking women 25-35 within 20 miles
Active within 24 hours
Laugh tell it hurts, smile untell your mouth is sore, and never stop moving forward.
Hello all. My name is Michael. Me. Im straight forward, but down for earth, Loyal to my friends and family, Comfortable with myself, Goofy at times, Serious when I need to be. I enjoy my job. I have list goals that never stops changing or growing. Im done the single thing and it was fun. But now Im ready to find someone I can get to know and build a life with. I am very much a t shirt, jeans and flip flops kind of guy. But I do like to get dressed up when the time calls for it. Music. I like all kinds. Lately its been alternative rock, inde rock , R@B and always anything the classic rock like the Beatles. I've been to Philippines, Canada, Mexico, 10 of the 50 states. I love road trips. Vegas, San Diego, long drive or rides up the coast.
Camping , Coffee and conversation , Cooking , Dining out , Fishing/Hunting , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Nightclubs/Dancing , Video games
Auto racing / Motorcross, Billiards / Pool, Cycling, Martial arts, Other types of exercise, Swimming, Walking / Hiking, Weights / Machines
I exercise 3-4 times per week.
I like Dogs, Other
Some other viewpoint
The beach in a worm sunny day, Malibu cannon riding my bike, the roman shop down the street, my dads house, bj restaurants, any Starbucks.
FOOD, sunny days, cars, beach, beer, my bike, and take a naps.
hiking, fishing, driving fast, watching movies, games, and working out.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
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|5'11" (180cms)
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text:
There are few things more exciting than a fresh passport stamp in a new country, but all those international adventures can leave your documents looking worse for wear. Keep your precious passport protected--and add an extra shot of style to your travel accessories--with one of these handmade passport covers. We've rounded up a range of styles from preppy stripes to vintage maps, and all of 'em are under 20 bucks. Bon voyage!
According to the Associated Press, Arkansas police have destroyed the 2006 offense report for Josh Duggar, which detailed how the then-14-year-old had molested five girls, including his sisters, on the orders of Judge Stacy Zimmerman. "As far as the Springdale Police Department is concerned, this report doesn't exist," Springdale's police representative Scott Lewis told AP.
Happy Couchiversary!
But still no cancellation yet.
Netflix confirmed today that "A Very Murray Christmas" will be streaming this December.


text:
"ROAWWWRRR!" South Florida Wrestling Photo Outtakes
Tuesday, February 15, 2011 | 4 years ago
In this week's feature, staff writer Francisco Alvarado takes a closer look at South Florida's local independent wrestling leagues. Young wrestlers participate to gain experience and exposure that may ultimately lead to the WWE.
Our cover for "ROAWWWRRR!" features wrestlers Joey Saint and Brian "The Beast" Brody. Here at the photos from that photo shoot. For more outtakes, check out the behind-the-scenes video.
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Closer Look: Klima in Miami Beach4 days ago
Miami Dolphins Cheerleaders 2015 Final Auditions at Miniaci…19 days ago
Marco Rubio Announces Presidential Bid at Freedom Tower1 month ago
Miami Open 2015 at Tennis Center at Crandon Park2 months ago
Scenes From Spring Break 2015 on South Beach2 months ago
FIA Formula E Miami ePrix 2015 in Downtown Miami2 months ago


text:
Shopping outside the US? International Shipping is now available!
Outfitting your vehicle with Pure halogen headlights by Putco not only sheds new light on the road but offers you the opportunity to customize your ride a bit, with several white bulb options available as well as blue and yellow bulbs. Putco, long known and respected for quality and innovation, has been manufacturing and distributing original and licensed aftermarket accessories for SUVs, trucks and cars for over 45 years.
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text:
Wrong, most will indeed post with ot without a vid card. Just guessing or wanting something to be true wont make it happen.
Most systems don't post unless there is a video card running.
Jump to content
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|Windows 10 Technical Preview in Microsoft Beta (Windows 10)
Posted 01 January 2013 - 01:19
Check digikey, match up the farad rating and be sure that it's voltage rating is equal or higher and,if possible, look for a low ESR one. The spacing looks cramped so be sure to check the data sheet to be sure it'll fit in the existing footprint.
Posted 01 January 2013 - 02:09
Pretty much this - this is true for all capacitors. As a matter of fact - the voltage rating doesn't really have a maximum. Although in my experience, the higher the voltage, the bigger the cap - so this does matter within reason
That's normal with old PCs; best to get a new one
My vista laptop sucks as well mehh thats why i use the ipad more
Posted 02 January 2013 - 02:46
Wrong, most will indeed post with ot without a vid card. Just guessing or wanting something to be true wont make it happen.
Posted 02 January 2013 - 07:14
Tried that.
There might be an upset in RAM or RAM slot. Replace the RAM in another slot and try.
Posted 07 January 2013 - 10:18


text:
This empirical study analyzes the long run behavior of provincial house prices in South Africa based on the club convergence and clustering procedure of Phillips and Sul. Using quarterly data covering the period of 1976Q2–2012Q4, 1974Q1–2012Q4 and 1977Q3–2012Q4 for the large, medium, and small middle segments of the housing market, respectively, we test the law of one price across nine provinces. The empirical findings suggest that the nine provinces do not form a homogeneous convergence club. Unlike the small middle segment, which consists of two convergence clubs of seven and two provinces, the large and medium middle segments have three convergence clubs corresponding to three segmented independent local markets. Possible intuitive explanations for the existence of such clubs are discussed and resulting policy implications provided.
New firm formation is an important determinant of regional development. Entrepreneurial decisions on location choice are affected by numerous factors. Given the importance of local regional characteristics, this study focuses on the dynamics behind the regional formation of new firms in Turkey using balanced panel data from 1997 to 2006. An initial set of analyses underline the ongoing persistence of the unequal distribution of new firms. Among various factors, results obtained from different panel models suggest the importance of local demand, provincial business cycles, human capital development, and financial availabilities to explain the regional differences of new firm formation in Turkey. These results, which are robust to the inclusion of spatial links, also confirm that the proposed channels are common for manufacturing, services, and trade-based production.
This paper analyzes the competitiveness of Indonesian wood-based products using revealed symmetric comparative advantage (RSCA), Spearman rank correlation (SRC), product mapping, and constant market share (CMS) analyses. RSCA results show that Indonesia had a comparative advantage in wood-based products, especially in Standard International Trade Classification (SITC) 245, 248, 251, 634, 635, 641, and 821. SRC results find that Indonesian wood-based products do not show a structural shift in comparative advantage during 1991–2010 while product mapping places Indonesia in group A (comparative advantage and a net exporter) in SITC 245, 634, and 635. CMS reveals that the increase in wood-based product export was caused by an increase in world exports, and commodity and market distribution effects. This study examines Indonesia's log export ban policies, which have been inconsistently implemented for the past several decades and adversely affect the country's global competitiveness, and recommends that Jakarta eliminate these policies and impose an appropriate export tax rate on logs.
The paper seeks to provide a theoretical foundation to the diverse empirical findings with regard to the role of liberalization in perpetuating gender inequality in the labor market. It studies the role of female labor supply decisions on gender wage inequality by considering the interrelation between liberalized policies, gender wage inequality, and female labor force participation. A three–sector Harris-Todaro general equilibrium model has been developed to examine the effects of liberalized trade, investment, and labor market policies on the gender wage gap and female labor force participation. There are two underlying assumptions: first, women are paid less because they are employed in the informal sectors; even within the informal sector, they are engaged in particularly low paying activities because of gender segregation. Second, female labor supply decisions or female labor force participation is endogenous, and depends on male and female wages, and the unemployment of male workers. The comparative static analysis indicates that while a reduction in import tariff may narrow the gender wage gap and raise female workforce participation, foreign capital inflow and labor market deregulation may aggravate gender wage inequality and lower female labor force participation.


text:
|Teaser image - Sam Gilbey's limited edition Ninja Scroll poster
|Fri, 9 Nov 2012
From Manga UK Press Release:
"It gives us great pleasure to provide a sneak preview of the stunning artwork created by the acclaimed British illustrator (of Scott Pilgrim and Call of Duty fame) Sam Gilbey...
The teaser image (attached) is a small section of the limited edition Ninja Scroll poster that will only be available to those attending the Ninja Scroll premiere on 16th November, and will be signed by Sam Gilbey too!
In his own words about the teaser image, Sam says that,
"I often include portraits of real people in my work and capture likenesses, but because Ninja Scroll is hand-drawn in a very specific way, I felt it was important to reinterpret the characters in my own style. Here's a sneak peek at a small detail, specifically Kagero, the beautiful ninja."
Ninja Scroll will premiere at the Hackney Picturehouse Cinema on Friday 16th November. This is a premium ticket event and includes admission to the movie, an advance copy of the standard edition Blu-ray and a limited edition, signed and numbered variant Ninja Scroll movie poster by Sam Gilbey. There will be only 300 tickets available for this event making the poster an instant collector's item and world exclusive.
Tickets can be booked online here or by calling the box office 0871 902 5734"
Related: Ninja Scroll, Manga Ent., Yoshiaki Kawajiri
|Reported by chaos — Comments: 13
Episodes 1-7 (Streaming on Crunchyroll) "Man cannot live by bread alone; he must have peanut butter." - James A. Garfield If the stereotypes of the.. Read more (0 comments)
"I can't dance, I can't talk. Only thing about me is the way I walk. I can't dance, I can't sing. I'm just standing here selling everything." -.. Read more
Quiet, unassuming fifteen-year-old Mikado Ryugamine moves from his safe but boring countryside home to Ikebukuro, Tokyo to start high school with his old friend Masaomi Kida.. Read more (0 comments)


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Free forum : Engage is an international Level 6 guild that resides in Illyfue [PVE] server of Forsaken World. Being one of the first few guilds to have conquered a Guild Base.
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Andy Biggs, Other Politicos Tied to Gilbert Religious Group Labeled as Anti-Gay "Hate Group" by Southern Poverty Law Center
The Southern Poverty Law Center rolled out with its new round of "hate group" labels, adding the Arizona chapter of the charity/religious advocacy group United Families International to the list for being "anti-gay."
Republican state Representative Andy Biggs is the former policy adviser to the group, and his wife Cindy Biggs is currently listed as the organization's secretary and treasurer on its website. Congressional candidate Matt Salmon's mother Nancy used to be the president.
According to Republican state Representative Cecil Ash's website, he and his wife have been affiliated with the group for "several years."
United Families International is one of 26 "active anti-gay groups" listed by the Southern Poverty Law Center, alongside Faithful Word Baptist Church -- the Tempe church with the pastor who says "the government should put [homosexuals] to death" -- and Fred Phelps' world-famous Westboro Baptist Church.
What exactly the Southern Poverty Law Center doesn't like about United Families International isn't mentioned, and we've got a call out to get that information.
According to the general "anti-gay" overview provided, it's pretty much described as a religious crusade against equal rights for gays and lesbians.
"For Christian Right leaders, the gay rights movement and its so-called 'homosexual agenda' are the prime culprits in the destruction of American society and culture," the description says. "In the words of Focus on the Family founder James Dobson, the battle against gay rights is essentially a 'second civil war' to put control of the U.S. government in the right hands, meaning those who reject gay rights."
Here are some interesting things from the United Families International "guide to family issues" about gays:
- "Pedophilia is widespread among the homosexual community."
- "Reputable studies and decades of successful treatment show that homosexual behavior can be changed."
- "Discrimination on the basis of gender or race is vastly different from discrimination on the basis of sexual practice."
- "It is not marriage, but women in marriage, that help to contain and channel the male sexual appetite."
- "In fact it is more compassionate to discourage homosexuality than to tolerate it."
There are also accompanying "fact" sheets for topics such as "the harms of pornography" and some opinions on abortion.
Still, some view the Southern Poverty Law Center as the political-correctness police. For example, it's gone after the "Men's Rights Movement" in its most recent publication -- a group eloquently described by a Business Insider reporter as "men who are rude about women and their sex lives."
Reason isn't happy about it either.
Either way, United Families International is on the list.
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This year I'm enjoying a first - coaching a girl's sport, basketball. A team made up of Notre Dame de Sion juniors in the CYO league, it's a rather unusual arrangement as most coaches do things I don't. Practice, for instance. They also use those white boards to diagram plays, make strategic substitutions and scout their opponents. That's not me, not us.
For its business success and long-standing commitment to the community, Rosewood Services earned the title of Business of the Year for 2012.
For several decades, local consultant Rick Ball has been helping business owners and investors make sound decisions about their futures. He founded Ball Consulting Group LTD, specializing in offering financial and management consulting.
Anyone who has worked with teenagers knows how saintly educators are. Teachers dedicate themselves to impacting the lives of their students. But one teacher in particular has earned special recognition for going above and beyond as a role model and servant leader in 2012.
VICTORIA - The St. John-Hudson High School had four players in double figures in a 58-25 victory over Victoria on Friday night.
GAINESVILLE, Fla. - Mike Rosario scored 18 points, Erik Murphy added 17 and No. 2 Florida bounced back from a humbling loss earlier in the week to thump Mississippi State 83-58 on Saturday.
The Claflin Lions Club is sponsoring basketball author and historian Steven Michael Farney on Monday, Feb. 18, at the Claflin Community Center, located at 409 A Street in Claflin.
MADISON, Wis. - Ben Brust hit a 3-pointer from just inside midcourt to tie the game at the end of regulation and added a tie-breaking 3 with less than 40 seconds left in overtime as Wisconsin beat No. 3 Michigan 65-62 on Saturday.
MANHATTAN - Kansas State is now clearly in the driver's seat in the Big 12.
NORMAN, Okla. - Buddy Hield came away with the steal, ran out to the other end of the court for a dunk that wouldn't count. It was still time for Oklahoma to celebrate, and Hield would get bounced around on fans' shoulders in a rare on-the-court postgame party at the Lloyd Noble Center.
MANHATTAN – Tradition and heritage is a big part of what makes agriculture such an attractive way of life for so many Kansans. The lifeblood of our existence, the farms and ranches in Kansas, provide food, fuel and fiber for the world.
GREAT BEND - Lynda Marie Garrett, 60, died Feb. 8, 2013 at her home in Great Bend.
ELLINWOOD - William Henry "Bill" Kimpler, 89, died Dec. 8, 2012 in Oklahoma City.
TOPEKA - Lorenzo M. Lemuz, 88, Topeka, died Feb. 2, 2013 at Midland Hospice Care.
HUTCHINSON - Donald Orville "Don" Stafford, 99, died Feb. 8, 2013, at Elm Grove Estates, Hutchinson.
Page 1115 of 2261
About 100 Kansas high school students flocked to Barton Community College to get hands-on experience from Barton's art professionals and participate in a juried art exhibit on Thursday.
Great Bend police received reports of criminal damage to property at two locations on Saturday morning. Someone threw paint on the side of the house at 1208 Morphy St., and someone spray painted the Funtime Bingo building at 2805 Ninth St.
Great Bend police made a narcotics arrest at 1:38 a.m. Saturday in the 2200 block of Jackson Street, after a driver allegedly failed to use her turn signal.
A 20-year-old man told Great Bend police he was battered by someone carrying a knife, around 9:46 p.m. Friday in the area of 19th and Holland St. He did not know the subject. An aggravated battery case was taken.
Girl Scout Brownies in Troop 11142 recently conducted a sweet community service project at the Heartland Cancer Center in Great Bend. They made and delivered 50 chemo kits filled with journals of inspirational pictures and quotes, along with pens, toothbrushes, hand sanitizer, lotion, candy and mints. They also made special kits with toys for children.
S.R.C.A Dragstrip will host Thunder on the Plains on May 15-17 at 455 W. Barton County Rd.; 2½ miles west of Great Bend on the old Air Force base. The event is one of 45 scheduled across the country where drivers earn points leading to divisional and national championships as well as a chance at a share of the year-end purse of more than $519,000. Categories include Comp Eliminator, Super Stock, Stock Eliminator, Super Comp, Super Gas and Super Street.
Kansas Department of Wildlife, Parks and Tourism staff are hosting a series of informational meetings to hear public input on duck hunting zone boundaries in the Kansas Low Plains Early, Late and Southeast Zones.
Great Bend Fire/EMS
LOS ANGELES (AP) - Houston's strategy of intentionally fouling poor free-throw shooter DeAndre Jordan backfired in a big way.
MEMPHIS, Tenn. (AP) - First-year coach Steve Kerr knows a crucial moment in the playoffs, so he understands exactly the test his Golden State Warriors face trailing Memphis 2-1 in their Western Conference semifinal.
DETROIT- The weekend showdown between the Tigers and Royals lasted into Monday morning before Omar Infante's 10th-inning sacrifice fly decided the rubber match of their three-game set, sending Kansas City to a 2-1 win at Comerica Park.
Katherine Mary Heeke of Great Bend, graduated with a Bachelor of Arts Degree in Church Ministries during Southwestern Assemblies of God University's 107th commencement ceremony, held on May 1.
Fort Hays State University, which has seen the number of its graduates nearly triple over the past decade, will divide those graduates into two separate ceremonies this spring.
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Dear Diane Von Furstenberg: What's the Perfect Color for Spring?
Plus, her tips on looking chic and notes on wardrobe staples in this week's installment of WWDVFD?READ MORE »
Plus, her tips on looking chic and notes on wardrobe staples in this week's installment of WWDVFD?READ MORE »
The Cut has teamed up with Estée Lauder to offer you the chance to win a $500 gift card to a fine retailer of your choice. Ten runners-up will receive an Estée Lauder Pure Color Pops collection. Enter now!READ MORE »
Mad Men Understood Human Behavior Better Than Any Show on TV
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Can Lemon Water Really Help You Lose Weight?
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Can medication efficacy be predicted in schizophrenia patients?
This question is the focus of an international research consortium (CRESTAR), in collaboration with scientists from the CIMH. Among others the researchers aimed to elucidate whether medication efficacy in schizophrenia can be predicted by attributes that can be assessed prior to treatment initiation. First results indicate that medication will be less effective in patients who carry more risk genes for schizophrenia. The study has now been published in the scientific journal Molecular Psychiatry.
Schizophrenia is a major psychiatric disorder, often characterized by a chronic progression in symptoms. This in turn leads to pronounced psychosocial impairment and the requirement for permanent care.
At present, psychiatrists are unable to predict which medication will be effective in a given schizophrenia patient.
The EU-funded CRESTAR project (http://www.crestar-project.eu) aims to investigate this research topic. A particular focus of its research work is a highly potent antipsychotic medication, Clozapine, which has been available since decades. Clozapine however, is only used after at least two trials of other medications have proven to be unsuccessful, as in a small number of cases life-threatening side-effects arise. This approach leads to a delay in effective treatment and a worsening of long-term prognosis.
The CRESTAR researchers are now investigating: First, whether clear indicators for the development of these adverse effects can be identified at an earlier time-point; and second whether clozapine will be effective in a given individual patient. Identification of these factors would allow the safe and effective initiation of clozapine during the early stages of illness.
CRESTAR researchers used information concerning many thousands of genetic markers to show that antipsychotic medication in general will be less effective in patients with a higher genetic load for schizophrenia. This genetic load is already present at birth. Despite the existence of familial schizophrenia, most schizophrenia patients have no family history of the disease. Thus a genetic test to determine the genetic load would represent a first step towards individualized therapy.
Until present psychiatric genetic research has generated few results of direct relevance to treatment, and individual prediction is still not possible. The results of the present study are only a small step into this direction. According to the first author of this recent CRESTAR study, Josef Frank, this is likely to change in the future, since through the development of new biostatistical methods, an increasing amount of information is being obtained concerning genetic variation across the whole genome.
Identification of increased genetic risk scores for schizophrenia in treatment resistant patients.
Josef Frank, Maren Lang, Stephanie H Witt, Jana Strohmaier, Dan Rujescu, Sven Cichon, Franziska Degenhardt, Markus M Nöthen, David A Collier, Stephan Ripke, Dieter Naber & Marcella Rietschel. Molecular Psychiatry (2014). doi:10.1038/mp.2014.56
Contact at the CIMH:
Prof. Dr. Marcella Rietschel
Central Institute of Mental Health
Department of Genetic Epidemiology in Psychiatry
E-Mail: firstname.lastname@example.com
Sigrid Wolff | idw - Informationsdienst Wissenschaft
Further reports about: > CIMH > Identification > Molecular > Psychiatry > antipsychotic > clozapine > medication > schizophrenia
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Nanoengineers at the University of California, San Diego developed a gel filled with toxin-absorbing nanosponges that could lead to an effective treatment for skin and wound infections caused by MRSA (methicillin-resistant Staphylococcus aureus), an antibiotic-resistant bacteria. This "nanosponge-hydrogel" minimized the growth of skin lesions on mice infected with MRSA - without the use of antibiotics. The researchers recently published their findings online in Advanced Materials.
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Bluffton, SCUSA 29910
June 2, 2013
New to the area and looking to make friends and explore. I have one extended kayak/camping trip under my belt, but it was a few years ago and I am still a beginner. Looking forward to learning and meeting people!
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A WARRANT has been granted for the arrest of a Worksop restaurant owner.
Mohammed Abdul Kashem is facing 15 charges relating to the contravening of several food hygiene regulations at Monsoon, on Ryton Street.
The charges have been brought about by Bassetlaw Council's environmental health department. On Friday magistrates in Worksop issued a warrant to be granted for Kashem's arrest after he failed to appear in court.


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List all words that contain apyrase
2 words found.
List all words containing apyrase, sorted by length
Some random words: cajole twa bield hoise up clabber toil
This is not a dictionary, it's a word game wordfinder.
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First automatic answering machine patented in 1935.
Invention popular with Orthodox Jews who were forbidden to answer the phone on the Sabbath.
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Though the technology for recording telephone conversations was invented in the late 1800s, the first automatic answering machine was not patented until 1935. The Swiss inventor Willy Müller developed a three-foot-tall (nearly a metre tall) answering machine which became popular with Orthodox Jews who were forbidden to answer the phone on the Sabbath.
SOURCE: About.com
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Watch This BMW Execute the Worst Parking Job Ever
We'll admit that parking spots are getting smaller in most parking lots but this driver is parking her BMW like she's trying to fit a submarine into a kid's pool.
We'll admit that parking spots are getting smaller in most parking lots but this driver is parking her BMW like she's trying to fit a submarine into a kid's pool.
The fall season is underway and that means millions are traveling for Thanksgiving and gearing up for Christmas. Nearly 24 million passengers are expected to fly this Thanksgiving, an increase of about 150,000 passengers from last year.
Today is October First and usually that means its time to get ready for Halloween putting up decorations, finding the right costume, even hitting out to the haunted houses. Well what could be more scary then a haunted house maybe a real ghost perhaps a car?
Driving a car is always risky, but if you want to stay safe, you may want to avoid the nation's capital.
Five-year-old Evan loves 'Cars' so much that he literally wore out the wheels on his Lightning McQueen Power Wheel toy car. So, his dad Sean, an employee at the aftermarket tuning company FFTEC Motorsports, rebuilt the ride from the ground up, even adding a zippy 500 watt electric motor that's capable of outputting .66 horsepower.
A 22-year-old Virginia woman performed a remarkable feat last Saturday when she reportedly lifted a car off her father who was pinned beneath, then saved his life with CPR.
Over 50 Marine Poolees on hand to wash your car for any donation you choose this weekend in Amarillo.
If you've ever driven on the 405 freeway in Los Angeles during rush hour then the thought of your car transforming into a tank and crushing everyone in front of you crosses your mind once every 2.3 seconds.
An Ohio woman took the phrase "frivolous lawsuit" to a new level with her lawsuit over her towed car.
Michelle R. Mathis of Columbus, Ohio asked for $500 billion in damages from the city after they impounded her 2002 Saturn SC2.
Have you ever passed someone in a sports car, watched them rev the engine like a pompous jerk and find yourself thinking something maybe a bit off-color about them? Well, this video shows what we all wish would happen to that guy. In this case, the driver is behind the wheel of a Lamborghini, a yellow one. Yeah… a yellow Lamborghini. We know what you're thinking and we agree.
A recent study of accidents involving the gas pedals of vehicles yielded some age and gender-specific results.
A 40 year old Florida man was arrested for crushing a teen girls car in a spontaneous monster truck rally. That's right, I said SPONTANEOUS monster truck rally.


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I heard recently that Brisbane's "suncorp stadium" has been voted the best rugby league stadium in Australia. It certainly lives up to the name (especially for state of origin...
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Enogra Reservoir circuit
Kedron Brook Track
Los Angeles Tour: Silver Lake
I heard recently that Brisbane's "suncorp stadium" has been voted the best rugby league stadium in Australia. It certainly lives up to the name (especially for state of origin). You wont see a crowd quite like the Brisbane crowd.
Suncorp Stadium is fantastic for spectators. The stadium is small enough that you are close to all the action and yet big enough to attract the best of events. From ease of just getting to and from the Stadium to food outlets to personal facilities - it is ideal!
Undoubtedly the best place to watch rugby league in Australia. there is not a bad seat in the place. The train station is just down the road and the buses disembark under the stadium. Travel is free with your ticket so that's a bonus !!!
The beer is cold but far too expensive, same with the food ... but the...
Suncorp stadium, best in Australia.
Not one negative comment about it
Great view from the close stands, easy access to your seating.
Free public transport to the stadium, well organised and free flowing even when there are 40,000 people trying to get out after a game. Within 15 minutes you are out of the stadium and onto public transport.
The only thing I can say: If it's your first time in Australia go and see a rugby game. You won't regret it and it's a great experience!
A wonderful venue to view NRL football matches , trains run from the City of Brisbane from most areas surrounding Brisbane, good facility ,great views of the game ,and an overall pleasing experience for families as well as individuals.
I have been to SuncorpStadium many times and I find that the view of the ground is great from all seats no matter where you are. Also the public transport system that gets you there and gets you away is superb.
Suncorp Stadium is easily the best sports venue in Australia, and probably further, to watch any rectangle field game. Absolutely perfect views from any seat in the place.
Public transport sport access isn't the best and car parking is a mission and isn't cheap. But worth it to go see your team play.
For sporting events, this venue is one of the top in Australia, particularly for the NRL, where the fan base is quite strong. Soccer and Rugby are a close second, but still neither code has the fan base to bring consistent big crowds to games. State of Origin games over the past few years have been sold out as long...
Register now for free — and start getting automatic notification of new reviews, responding to traveler feedback, adding new photos to your listing and much more.
Hi there - we are travelling to Suncorp from Toowoomba for Foo Fighters on 24 Feb.
I would appreciate anyones advice on where to find SECURE parking & best form of travel to the stadium from the parking facility.
Many Thanks!
See all questions


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Portfolios distributed in Mizoram
Aizawl: Mizoram Chief Minister Lal Thanhawla on Monday allotted portfolios to his council of ministers who were sworn-in on Saturday.
The Chief Minister retained Political and Cabinet, Vigilance, General Administration Department, Secretariat Administration Department, Department of Personal and Administrative Reforms, Information and Public Relations, PWD, Power and Electricity and District Council Affairs.
R Lalzirliana retained Home and Rural Development and was also allocated Agriculture and Excise and Narcotics, Lalsawta got Finance.
Lalsawta was also allocated Finance, Planning and Programme Implementation, Taxation and Law and Judicial departments.
Former speaker R Romawia was allocated Higher and Technical Education, Art and Culture, Land Revenue and Settlement and Parliamentary Affairs.
H Rohluna was given School Education, Industries (including Geology and Mineral Resources) and Trade and Commerce.
Zodintluanga retained his earlier portfolios -- Urban Development and Poverty Alleviation and Sports and Youth Services while he also got Public Health Engineering which was earlier held by S Hiato, who was defeated.
PC Lalthanliana, earlier a Minister of State was elevated to the Cabinet and got Horticulture, Local Administration and Social Welfare.
Former Deputy Speaker John Rotluangliana got Food, Civil Supplies and Consumer Affairs, Transport and Tourism.
Among the Ministers of state - Lal Thanzara was allocated Health, Minor Irrigation and Information and Communication Technology while he would assist the Chief Minister in PWD and Power portfolios.
Lalrinmawia Ralte was allocated Environment and Forests, Cooperation, Labour, Employment and Industrial Training and Soil and Water Conservation.
C Ngunlianchunga got AH And Veterinary, Disaster Management and Printing and Stationery, while Dr BD Chakma got Sericulture and Fisheries.
In the new ministry, former ministers - JH Rothuama, PC Zoramsangliana, Lalrinliana Sailo and Nihar Kanti Chakma were not included.
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