Probability of Agent Advancement.

<p>The sample agent’s probability of DR progression over time.</p

Training, Test, and Simulated Data.

<p>DM – Diabetes Mellitus.</p><p>NPDR - non proliferative diabetic retinopathy.</p><p>PDR - proliferative diabetic retinopathy.</p><p>BMI - body mass index.</p><p>A1c = Hemoglobin A1c.</p><p>Values reported as average ± standard deviation, or number (percent).</p><p>P value represents comparison of Test Data to Simulated Data.</p

Statechart model for Diabetic Retinopathy.

<p>DR: Diabetic Retinopathy NPDR: Non-proliferative Diabetic Retinopathy PDR: Proliferative Diabetic Retinopathy PRP: Pan-retinal Photocoagulation.</p

Size-exclusion gel permeation chromatography (GPC) analysis of lens proteins from αB-R120G knock-in mice.

<p>Lens proteins were separated into water-soluble and insoluble fractions, and then the water-soluble fractions were analyzed by GPC. <i>A, B)</i> Three-month-old lenses. <i>C, D)</i> Eight-month-old lenses. <i>A)</i> Refractive index profile of the water-soluble proteins in wild-type <i>(black)</i>, heterozygous mutant <i>(green)</i>, and homozygous mutant <i>(red)</i> lenses. <i>B)</i> Right-angle light scattering in proteins eluting from the size exclusion chromatography columns for wild-type <i>(black)</i>, heterozygous mutant <i>(green)</i>, and homozygous mutant <i>(red)</i> lens proteins. <i>C)</i> Refractive index profile of the water-soluble proteins from wild-type <i>(black)</i>, heterozygous mutant <i>(green)</i>, and homozygous mutant <i>(red)</i> lenses. <i>D)</i> Right-angle light scattering in proteins eluting from the size exclusion chromatography columns from wild-type <i>(black)</i>, heterozygous mutant <i>(green)</i>, and homozygous mutant <i>(red)</i> lens proteins. Note that while the y-axis of the light scattering intensity is lower in 8-month-old lens proteins <i>(D)</i>, it is spread out over 5 ml, compared with 3 ml in the 3-month-old lens proteins <i>(B)</i>. Thus, the area under the light scattering curve is greater at 8 months. Note also that the higher molecular weight for α-crystallin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017671#pone-0017671-t003" target="_blank">Table 3</a>) calculated by the software shows that the light scattering intensity with respect to the refractive index signal is higher in the 8-month-old than in the 3-month-old lens proteins.</p

Eye abnormalities in αB-R120G knock-in mice.

<p>WT, wild-type; Het, heterozygous; Homo, homozygous.</p

Gene-targeting strategy and genotype analysis.

<p><i>A)</i> Diagram illustrating the gene-targeting strategy used to produce αB-R120G gene knock-in mice. The numbered rectangles represent exons and the starred exon 3 indicates the mutated exon. Restriction sites relevant to Southern blot analysis are shown together with the size of the restriction fragments. Lox P sites are represented as closed triangles. The 3′ probe that was used for Southern blot analysis is shown below the mutated allele. Neo^r represents the neomycin cassette, which was excised by homologous recombination after introduction of the αB-R120G knock-in plasmid and the Turbo-Cre plasmid. Bold arrows indicate the PCR primers (pcr1 and pcr2) used to detect the wild-type and mutated alleles. <i>B)</i> Genomic DNA from ES clones was sequenced to verify the A to G mutation in the mouse αB-crystallin gene. <i>C)</i> Southern blot of <i>Sac</i>I digests showing clones 7, 24, 37, and a wild-type clone with no insertion. Of these, clone 24 demonstrated insertion of the plasmid containing neomycin <i>(mutant)</i>. The lower band (9.0 kb) represents the correctly targeted ES clone containing the insertion. The native αB-crystallin gene (11.5 kb) was present in each positive clone. <i>D)</i> PCR screening of mouse genomic tail DNA confirmed recombination of the Lox P sites showing that neomycin was deleted. At the 5′ end, a sense flanking primer (pcr1) was paired with an antisense αB-crystallin gene intronic primer (pcr2). These primers amplified a 768-bp band from the wild-type (WT) αB-crystallin gene compared with an 860-bp band from neomycin-deleted knock-in chromosomes. Both the 768- and 860-bp bands were amplified in heterozygous mice. Absence of the 768-bp band and detection of only the 860-bp band indicated homozygosity for the αB-R120G mutation.</p

Eye and lens phenotypes of αB-R120G knock-in mice.

<p>Eyes were dilated in non-anesthetized mice and examined using a slit lamp. <i>A)</i> Slit lamp micrographs of 5-week-old mice. <i>Left panel</i>, wild-type mice with clear lenses. <i>Middle panel</i>, heterozygous αB-R120G knock-in mice displaying low-grade opacities in the nuclear and posterior regions of the lens. <i>Right panel</i>, homozygous αB-R120G knock-in mice with prominent opacity in the lens nucleus (stage 3) and the small eye phenotype. The dotted line shows the outline of the eye. <i>B)</i> Lens opacity and corneal abnormality in 10-month-old αB-R120G mouse eyes. <i>Top left</i>, wild-type. <i>Top right</i>, left eye. <i>Bottom left</i>, right eye of an αB-R120G heterozygous mutant mouse. <i>Bottom right</i>, eye of homozygous mutant mouse showing complete (stage 4) opacity. <i>C)</i> Corneal abnormality in αB-R120G heterozygous mutant mouse eye. The wild type eye was normal. <i>D)</i> Immunoblot analysis of lens water-soluble and water-insoluble proteins using an antibody specific for αB-crystallin. <i>E)</i> Histological analysis of αB-R120G heterozygous lenses. Lens sections were stained with hematoxylin and eosin (H&E). <i>Top panel</i>, wild-type lens with normal epithelial and fiber cell morphology. <i>Middle panel</i>, an αB-R120G heterozygous lens with a multilayered region of cells. <i>Lower panel</i>, a higher magnification image of the multilayerd region shown in the rectangle of the <i>middle panel</i>. <i>F, G)</i> Immunofluorescence analysis of major intrinsic protein (MIP) expression in wild-type and αB-R120G heterozygous knock-in lenses. <i>F)</i> Mid-sagittal lens sections stained with anti-MIP (AQP0) to visualize fiber cell membranes. Equatorial region of the lens. Sections of wild-type <i>(left panel)</i> and homozygous <i>(right panel)</i> lenses are shown. Visualization of fiber cell membranes at the onset of differentiation (cell elongation) region. <i>G)</i>. Cross-sections of anterior cortical fibers of the lens. Sections of wild-type <i>(left panel)</i> and heterozygous <i>(right panel)</i> lenses are shown.</p

Molecular weight (M_w) and hydrodynamic radius (R_h) of crystallins in αB-R120G mice.

<p>*Proteins from completely opaque 9-month-old homozygous mutant lenses were largely water-insoluble. The estimated molecular weight of the α-crystallin fraction was >10×10⁶. Most of the lens proteins did not go through the column filter (0.2-µm). The concentration of protein that went through the column was very low (0.128 mg/ml). Insufficient soluble protein made it difficult to accurately evaluate the R_h value from viscosity measurements.</p><p>**Protein concentration of the homozygous lens proteins was too low for an accurate determination of R_h. Data for two independent sets of 4-month-old mutant lenses (group 1 and group 2) demonstrate minor differences between litters.</p><p>WT, wild-type; Het, heterozygous; Homo, homozygous.</p

Histopathology and αB-crystallin insolubility in αB-R120G knock-in mice.

<p><i>A)</i> Additional staining of 10-month-old αB-crystallin R120G homozygous mouse TA with Congo red shows multiple congophilic inclusions (<i>red; open arrows</i>). Cytochrome oxidase staining (<i>brown</i>) is absent or cleared from the central region of scattered myofibers (<i>starred fibers</i>). Immunofluorescence using an antibody against ubiquitinated proteins (<i>red</i>) highlights large ubiquitinated aggregates within scattered fibers. Blue staining is nuclear DNA. Scale bar is 50 µm. <i>B)</i> Triple immunofluorescence staining of TA muscle from 10-month-old wild-type or αB-R120G homozygous mutant mice using anti-αB-crystallin (<i>green</i>), anti-desmin (<i>red</i>), or anti-ubiquitin (<i>red in bottom right panel only</i>), and nuclear DNA (<i>blue</i>). Open arrowhead shows αB-crystallin and desmin co-aggregate, while closed arrowhead denotes isolated desmin inclusion. Individual myofibers are outlined in white. Scale bar is 50 µm. <i>C)</i> Immunoblot using αB-crystallin antibody of total, detergent-soluble, or detergent-insoluble proteins from TA muscle lysates of 4.5-month-old wild-type (<i>WT</i>), αB-R120G heterozygous (<i>het</i>), or homozygous (<i>homo</i>) mutant knock-in mice. Histopathology was performed on at least two mice per age.</p

Statistical analysis of data in Table 1.

<p>Asterisks denote statistical significance with p<0.05.</p><p>WT, wild-type; Het, heterozygous; Homo, homozygous.</p