Rapid assessment methods in eye care: An overview

Reliable information is required for the planning and management of eye care services. While classical research methods provide reliable estimates, they are prohibitively expensive and resource intensive. Rapid assessment (RA) methods are indispensable tools in situations where data are needed quickly and where time- or cost-related factors prohibit the use of classical epidemiological surveys. These methods have been developed and field tested, and can be applied across almost the entire gamut of health care. The 1990s witnessed the emergence of RA methods in eye care for cataract, onchocerciasis, and trachoma and, more recently, the main causes of avoidable blindness and visual impairment. The important features of RA methods include the use of local resources, simplified sampling methodology, and a simple examination protocol/data collection method that can be performed by locally available personnel. The analysis is quick and easy to interpret. The entire process is inexpensive, so the survey may be repeated once every 5-10 years to assess the changing trends in disease burden. RA survey methods are typically linked with an intervention. This article provides an overview of the RA methods commonly used in eye care, and emphasizes the selection of appropriate methods based on the local need and context.     

Selective Laser Trabeculoplasty Complicated by Cystoid Macular Edema: Report of Two Cases

 Purpose: Selective laser trabeculoplasty, a relatively novel treatment for open angle glaucoma, is frequently associated with mild post-operative intraocular inflammation. Methods: We report two uncommon cases of cystoid macular edema within a few weeks of routine selective laser trabeculoplasty. Results: Visual acuities and macular thicknesses of the two cases returned to baseline after medical treatment, but in one case, the cystoid macular edema persisted for months. Conclusion: Cystoid macular edema after selective laser trabeculoplasty is fortunately a rare complication, but it might be more common in patients with predisposing factors, and it can be resistant to treatment.     

Outcome of partial internal biliary diversion for intractable pruritus in children with cholestatic liver disease

Purpose

Children with cholestatic disorders have undergone liver transplantation for intractable pruritus unresponsive to medical therapy even in the absence of liver failure. Biliary diversion procedures interrupt the entero-hepatic circulation of bile acids allowing them to be excreted in the feces thereby lowering the total bile acid pool. We evaluated the outcome of partial internal biliary diversion (PIBD) in children with intractable pruritus from inherited cholestatic disorders.

Methods

The records of children who underwent PIBD over a 4-year period were reviewed for etiology of liver disease, demographic data, preoperative and postoperative biochemical profile and improvement of pruritus. Standard statistical methods were used for analysis.

Results

Of the 12 children, 10 had progressive familial intrahepatic cholestasis (PFIC) and 2 had Alagille syndrome (AS). PIBD was done using an isolated jejunal loop as a conduit from gall bladder to mid ascending colon. Median period of follow up was 30 months. Pruritus resolved in nine children with significant reduction of serum bile acids (P < 0.02).

Conclusion

To our knowledge, this is the largest reported series of children with PIBD. PIBD is a safe, well-tolerated and effective alternative to liver transplant in children with PFIC and AS who have intractable pruritus in the absence of synthetic liver failure.     

Modulation of frustration in folding by sequence permutation

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray–scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.Highly denatured states of globular proteins resemble statistical random coils when examined with low-resolution techniques such as X-ray scattering (1) and hydrodynamic analyses (2). However, a higher-resolution view provided by experimental models (3–6) and simulations (7) shows that the conformational ensemble is biased toward low-contact-order (CO) structures, e.g., α-helices, β-turns, and β-hairpins, which form and melt in less than a few microseconds. During folding, these nascent structures presumably coalesce into higher-order assemblies of ever-increasing free energy until reaching the transition-state ensemble (TSE) that leads to the native conformation. From another perspective, this assembly process mediates a global collapse of the chain in an unfavorable solvent (8). Landscape theory (9) posits that, in the simplest scenario, native-like substructures appear and lead without pause to the TSE and the native conformation in an apparent two-state fashion. However, simulations have found that topological frustration, e.g., the premature formation of a substructure that impedes access to the productive TSE, can lead to the accumulation of intermediates (I) that must unfold to some extent to traverse the folding reaction coordinate successfully (8, 10, 11). Experimental and computational studies on the folding of the α-subunit of Trp synthase (12, 13), the chemotaxis response regulator Y (CheY) (10, 14), a pair of apo-flavodoxins (8, 15, 16), and tandem titan domains (17) revealed frustration in the form of off-pathway intermediates (I_OFF). Thus, as-yet unexplored aspects of sequence and structure can add complexity to folding reactions.The observed inverse relationship between CO and folding rate constant (18) implies that elements of secondary structure that are near in sequence and near in space will associate preferentially over those that are distant in sequence. However, if such low-CO substructures are not involved in the productive TSE, they could serve as sources of frustration. A case in point is CheY, a member of the very common flavodoxin-fold family with its classic α/β/α sandwich architecture. The (β/α)5 motif displays the α1 and α5 helices on one face of the parallel β-sheet and the α2, α3, and α4 helices on the opposing face (Fig. 1 A and B). The proposed kinetic folding mechanism (Fig. 1C) (14) involves two parallel folding channels defined by the cis and trans isomers of the prolyl peptide bond between K109/P110. The unfolded proteins (U) in both the major trans (U_t) (90%) and minor cis (U_c) (10%) channels sample an off-pathway submillisecond intermediate (I_BP), I_BPt and I_BPc, respectively, before the rate-limiting isomerization reaction in the I_BPt→I_BPc step. I_BPc unfolds to the U_c state before accessing the productive TSE leading to the native conformation (N) in the U_c →N_c step. Further complicating the mechanism is an on-pathway intermediate, I_ON, between U_c and N_c that has been observed by equilibrium NMR measurements (19) and in Gō-model simulations (10) but not by circular dichroism (CD) or flow fluorescence (FL) experiments.Open in a separate windowFig. 1.(A) Topology diagram of CheY. The N-terminal folding subdomain is highlighted in yellow, and the C-terminal folding subdomain is highlighted in blue. The effects of each permutation on the continuity of cluster 1 (blue), and cluster 2 (red) are shown. (B) Clusters of ILV residues are superimposed on the crystal structure of CheY (Protein Data Bank ID code: 3CHY). Cluster 1 (blue) has a lower CO and resides on the α2/α3/α4 side of the central β-sheet. The larger cluster 2 (red) contains high-CO contacts and resides on the α1/α5 side of the β-sheet. (C) The folding mechanism of WT CheY. The major pathway is highlighted in red. The U_c →N_c step, involving the on-pathway intermediate, is designated by the triple dots.Mutational analysis (20, 21) has revealed a nucleation-condensation folding mechanism for CheY in which the N-terminal subdomain [residues 1–70, (βα)1–2β3] serves as the nucleus for the subsequent condensation of the C-terminal subdomain [residues 70–129, α3(βα)4–5] (Fig. 1A). However, native-centric simulations identified contacts between the N- and C-terminal subdomains, centered around (βα)3–4, early in folding that are incompatible with access to the productive TSE and that lead to frustration in the folding mechanism (10). Another perspective is provided by the branched chain aliphatic side chains (BASiC) hypothesis, which supposes that large clusters of isoleucine, leucine, and valine (ILV) side chains serve as cores of stability in folding intermediates (11, 14). Both these clusters have been shown to have a high contact density (22). CheY has two ILV clusters, each serving to fuse the surface helices to each other and to the central β-sheet (Fig. 1B). The smaller cluster (cluster 1) contains 10 side chains and primarily links α2(βα)3β4 on one face of the β-sheet; the larger cluster (cluster 2) contains 15 side chains and links the β-strands to α1 and α5. The sequence spanned by the smaller cluster agrees closely with the (βα)3–4 segment identified as the source of frustration in the simulations and, importantly, involves only low-CO contacts. If cluster 1 were to form early and, by sequestering β3, impede the development of the productive TSE in the N-terminal subdomain, (βα)1–2β3, the BASiC hypothesis would provide an alternative explanation for frustration in the folding of CheY.Permutations in the sequence of CheY provide a means to compare the subdomain model and the ILV cluster model as explanations for the frustration in folding detected by simulations and experiments. By fusing the natural N and C termini with a short linker peptide (Gly-Ala-Gly) and inserting new termini in the loops after β2, β3, and β4, it is possible to cleave within the N-terminal subdomain (Cpβ2), between the subdomains (Cpβ3), and within the C-terminal subdomain (Cpβ4). Related to the ILV clusters, Cpβ2 cleaves cluster 1 and leaves cluster 2 largely intact, Cpβ3 cleaves both clusters, and Cpβ4 cleaves only cluster 2 (Fig. 1A). Our simulations and experiments on these permutants show that aspects of both models describe the relationships between sequence, structure, and frustration in the folding of CheY. The results also show that frustration can be modulated by sequence permutations that can bias the initial stages of folding toward the productive TSE and away from kinetic traps.