The application of advanced imaging techniques in glaucoma

Imaging technologies, especially optical coherence tomography (OCT), have an important role in glaucoma diagnosis and monitoring. This review aims to critically appraise recent developments in imaging of the optic nerve head (ONH), retinal nerve fiber layer (RNFL), and macula in glaucoma. The review focuses on imaging of the ONH, retina, and associated structures, identifying five broad themes; 1) imaging of the RNFL, ONH and macula; 2) OCT angiography (OCTA); 3) structure function analysis; 4) novel methods of retinal imaging (beyond OCT and OCTA); and 5) artificial intelligence (AI). The use of imaging for glaucoma diagnosis and progression analysis is discussed. Measurements of RNFL, macular, and ONH have shown similar ability to detect glaucoma, though the majority of OCT diagnostic ability studies are limited by case-control design. Macular and ONH parameters such as Bruch’s membrane opening-minimum rim width (BMO-MRW) may be more useful in eyes with unusual optic disc appearance or high myopia, though the limitations of normative reference databases should be appreciated. Imaging should not replace perimetry, particularly for monitoring progression. Devices are likely to be developed that test structure and function concurrently, with results integrated using Bayesian statistical approaches.</p

Homology model of L6 protein highlighting the substitutions that suppress the defect in RbgA.

<p>Homology model of L6 protein of <i>B. subtilis</i> is shown as a surface representation in grey. The residues that are altered in L6 are highlighted in red with the corresponding amino acid labeled.</p

Suppressor mutations in ribosomal protein L6.

<p>Suppressor mutations in ribosomal protein L6.</p

Composition of in vitro matured particles.

<p>Protein occupancy in 44/45S and 70S samples normalized to that of L20. Samples are colored in pairs with the 44/45S intermediate in a lighter shade than the 70S particle. Samples from strain 1043 (RbgA-F6A) are on the left (blue, green), those from strain 1055 (RbgA-F6A, L6-RC3) are on the right (orange, red). Semitransparent dots signify unique peptide measurements. The median value is denoted with a larger opaque marker. Protein L6 is highlighted in red. Proteins significantly depleted are colored orange.</p

Mutations in L6 protein affect subunit joining/interaction.

<p>Ribosome profiles of strains expressing mutated L6 protein [representative strain RB1123 (<i>rplF</i>-R70P)]. The X-axis indicates the direction of the profiles from the bottom of the gradient (25%) to the top of the gradient (10%). The Y-axis depicts absorbance at 260 nm, which is equivalent for both plots depicted. Dashed lines indicate the migration of the 70S, 50S and the 30S complexes in the gradient.</p

Proposed model for the role of RbgA in promoting late-stage large ribosome subunit assembly.

<p>A late assembly intermediate (LAI_50-1) can proceed via two different pathways. Pathway 1 posits that RbgA binds prior to L6 (LAI_50-2) while pathway 2 indicates L6 binds prior to RbgA (LAI_50-3). When bound together (LAI_50-4), RbgA facilitates proper interaction between L6 and the maturing ribosome, which triggers the incorporation of late ribosomal proteins. Once proper incorporation occurs, RbgA leaves the complex. The role of GTP hydrolysis in the assembly process is discussed in the text.</p

Analysis of ribosome assembly in a L6 suppressor strains.

<p>The ribosome profiles of <i>rbgA</i>-F6A suppressor strains show an accumulation of a novel 44S complex. Ribosome profiles were analyzed from RB247 (wild-type cells), RB1043 (RbgA-F6A mutant), RB1051 (<i>rbgA</i>-F6A, <i>rplF</i>-R70P), RB1055 (<i>rbgA</i>-F6A, <i>rplF</i>-R3C), RB1057 (<i>rbgA</i>-F6A, <i>rplF</i>-H66L), RB1063 (<i>rbgA</i>-F6A, <i>rplF</i>-G5C), RB1065 (<i>rbgA</i>-F6A, <i>rplF</i>-G5S) and RB1068 (<i>rbgA</i>-F6A, <i>rplF</i>-T68R). Profiles were generated by sucrose density gradient centrifugation. Dashed lines indicate the migration of the 70S, 44S and the 30S subunits in the gradient.</p

<i>In vitro</i> maturation of large subunit intermediates.

<p><b>(A) <i>in vitro</i> maturation of 44S intermediate from RB1055.</b> Ribosome profile from cell lysate of strain RB1055 expressing mutated L6 protein (R3C) and RbgA-F6A protein after incubation at 0°C (blue) and 37°C (red) for 60 minutes. <b>(B) </b><b><i>in vitro</i></b><b> maturation of 45S intermediate from RB1043.</b> Ribosome profiles from cell lysate of strain RB1043 expressing RbgA-F6A protein and wild-type L6 protein after incubation at 0°C (blue) or 37°C (red) for 60 minutes. The X-axis indicates the direction of the profiles from the bottom of the gradient (43%) to the top of the gradient (18%). The Y-axis depicts absorbance at 260 nm, which is equivalent for both plots depicted. Dashed lines indicate the migration of the 70S, 50S, 44S and the 30S complexes in the gradient.</p

Interaction between L6 protein and the 50S ribosomal subunit.

<p><b>A</b>. Crystal structure of 50S subunit from <i>E. coli</i> (PDB ID:2AW4) with the position of L6 indicated in blue. The location of late binding ribosomal proteins that are missing or highly reduced in the 45S particle are also highlighted (L16 (green), L28 (yellow) and L36 (cyan)} or highly reduced {L27 (orange), L33a (purple), L35 (red). <b>B</b>. L6 (blue) binding region including helix 97 (colored magenta) is shown in a magnified view and the residues mutated in suppressor strains are colored in red at the N terminal of L6 protein.</p

Whole cell protein abundance.

<p>A heat map depicting the normalized ribosomal protein abundance in various RbgA perturbation strains. The rows correspond to strains induced RbgA expression (RB301:1 mM), RbgA depletion (RB301:6 µM), RbgA-F6A (RB1043), and RbgA-F6A with suppressor mutations (RB1051:R70P, RB1055:RC3, RB1057:H66L, RB1068:T68R). To facilitate comparison between conditions, each protein's abundance is normalized to that of L20 and then to the protein's level in strain RB301:1 mM. Protein abundance is colored from white (0.3) to red (1.5). Proteins depleted from the 44/45S particles are labeled in orange. Protein L6 is highlighted in red.</p