Evaluation of ganglion cell-inner plexiform layer thickness in the diagnosis of preperimetric glaucoma and comparison to retinal nerve fiber layer.

Purpose: The aim of this study was to evaluate the diagnostic ability of optic nerve head (ONH), RNFL, and GC-IPL parameters in differentiating eyes with PPG from normals. Methods: This was a retrospective, cross-sectional, observational study. We studied 73 eyes of 41 patients and compared them to 65 eyes of 34 normal persons. Each patient underwent detailed ocular examination, standard automated perimetry, GC-IPL, ONH, and RNFL analysis. PPG was defined as eyes with normal visual field results and one or more localized RNFL defects that were associated with a glaucomatous disc appearance (e.g., notching or thinning of neuroretinal rim) and IOP more than 21 mm Hg. Diagnostic abilities of GC-IPL, ONH, and RNFL parameters were computed using area under receiver-operating curve (AUROC), sensitivity and specificity, and likelihood ratios (LRs). Results: All GC-IPL parameters differed significantly from normal. The ONH, RNFL, and GC-IPL parameters with best area under curves (AUCs) to differentiate PPG were vertical cup to disc ratio (0.76), inferior quadrant RNFL thickness (0.79), and inferotemporal quadrant GC-IPL thickness (0.73), respectively. Similarly, best LRs were found for clock hour 5, 6, and 12 thicknesses among RNFL; inferior sector and inferotemporal sector thicknesses among GC-IPL parameters. Conclusion: Diagnostic abilities of GC-IPL parameters were comparable to RNFL parameters in differentiating PPG patients from normals. The likelihood of ruling in a disease was greater with GC-IPL parameters.

Linear discriminant score for differentiating early primary open angle glaucoma from glaucoma suspects.

PURPOSE: To determine the diagnostic accuracy of a linear discriminant function (LDF) based on macular ganglion cell complex (GCC), optic nerve head (ONH) and retinal nerve fibre layer (RNFL) for differentiating early primary open-angle glaucoma (POAG) from glaucoma suspects. METHODS: In this cross-sectional study, data from consecutive 127 glaucoma suspects and 74 early POAG eyes were analysed. Each patient underwent detailed ocular examination, standard automated perimetry, GCC and ONH and RNFL analysis. After adjusting for age, gender and signal strength using the analysis of covariance; Benjamin-Hochberg multiple testing correction was performed to detect truly significant parameters to calculate the LDF. Subsequently, diagnostic accuracy of GCC and ONH and RNFL were determined. The obtained LDF score was evaluated for diagnostic accuracy in another test set of 32 suspect and 19 glaucomatous eyes. Data were analysed with the R-3.2.1 (R Core Team 2015), analysis of variance, t-test, Chi-square test and receiver operating curve. RESULTS: Among all GCC parameters, infero temporal had the best discriminating power and average RNFL thickness and vertical CDR among ONH and RNFL parameters. LDF scores for GCC had AUROC of 0.809 for a cut-off value 0.07, while scores for ONH and RNFL had AUROC of 0.903 for a cut-off value - 0.24. Analysis on combined parametric space resulted in avg RNFL thickness, vertical CDR, min GCC + IPL and superior GCC + IPL as key parameters. LDF scores obtained had AUROC of 0.924 for a cut-off value 0.1. The LDF was applied to a test set with an accuracy of 84.31%. CONCLUSION: The LDF had a better accuracy than individual GCC and ONH and RNFL parameters and can be used for diagnosis of glaucoma.

Individual differences in the effect of social defeat on anhedonia and histone acetylation in the rat hippocampus.

Major depression is a growing problem worldwide with variation in symptoms and response to treatment. Individual differences in response to stress may contribute to such observed individual variation in behavior and pathology. Therefore, we investigated depressive-like behavior following exposure to repeated social defeat in a rat model of individual differences in response to novelty. Rats are known to exhibit either high locomotor activity and sustained exploration (high responders, HR) or low activity with minimal exploration (low responders, LR) in a novel environment. We measured anhedonia using the sucrose preference test in HR and LR rats following exposure to social defeat stress or in basal, non-defeated conditions. We then compared histone acetylation in the hippocampus in HR and LR defeat and non-defeated rats and measured mRNA levels of histone deacetylases (HDAC) 3, 4, 5, and Creb binding protein (CBP). We found that basally, HR rats consumed more sucrose solution than LR rats, but reduced consumption after exposure to defeat. LR rats' preference was unaffected by social defeat. We found that HR rats had higher levels of histone acetylation on H3K14 and H2B than LR rats in non-stress conditions. Following defeat, this acetylation pattern changed differentially, with HR rats decreasing acetylation of H3K14 and H2B and LR's increasing acetylation of H3K14. Acetylation on histone H4 decreased following defeat with no individual variation. Basal differences in CBP expression levels may underlie the observed acetylation pattern; however we found no significant effects of defeat in levels of HDACs 3, 4, 5 in the hippocampus.

Free tissue transfer for trophic ulcer complicating leprosy.

Plantar ulceration is the commonest disability in leprosy and occurs in about 10 to 20% of leprosy patients. Various loco-regional flaps have been described for reconstruction of trophic ulcers; however, very large defects are not amenable to local flaps and free flaps form one of the important treatment options. We present a case of a post Hansen's trophic ulcer over the forefoot managed using a radial artery forearm free flap. Debridement of the osteomyelitic bone, removal of the bony prominences, coverage by a well-vascularised tissue, end-to-side arterial anastomosis, use of anterior tibial as the recipient vessel and good postoperative compliance in foot care on the part of the patient gave us good results.

Core histone acetylation is regulated by linker histone stoichiometry in vivo.

We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo. Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of [(3)H]acetate into all four core histones. Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with [(3)H]acetyl-CoA. Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells. In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells. Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease. The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo.

Dynamic binding of histone H1 to chromatin in living cells.

The linker histone H1 is believed to be involved in chromatin organization by stabilizing higher-order chromatin structure. Histone H1 is generally viewed as a repressor of transcription as it prevents the access of transcription factors and chromatin remodelling complexes to DNA. Determining the binding properties of histone H1 to chromatin in vivo is central to understanding how it exerts these functions. We have used photobleaching techniques to measure the dynamic binding of histone H1-GFP to unperturbed chromatin in living cells. Here we show that almost the entire population of H1-GFP is bound to chromatin at any one time; however, H1-GFP is exchanged continuously between chromatin regions. The residence time of H1-GFP on chromatin between exchange events is several minutes in both euchromatin and heterochromatin. In addition to the mobile fraction, we detected a kinetically distinct, less mobile fraction. After hyperacetylation of core histones, the residence time of H1-GFP is reduced, suggesting a higher rate of exchange upon chromatin remodelling. These results support a model in which linker histones bind dynamically to chromatin in a stop-and-go mode.

Effects of H1 histone variant overexpression on chromatin structure.

The importance of histone H1 heterogeneity and total H1 stoichiometry in chromatin has been enigmatic. Here we report a detailed characterization of the chromatin structure of cells overexpressing either H1(0) or H1c. Nucleosome spacing was found to change during cell cycle progression, and overexpression of either variant in exponentially growing cells results in a 15-base pair increase in nucleosome repeat length. H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication. Overexpression of H1(0) and, to a lesser extent, H1c results in a decreased rate of digestion of chromatin by micrococcal nuclease. Using green fluorescent protein-tagged H1 variants, we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H1(0) variant. Overexpression of H1(0) results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels. Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H1(0) protein could independently give rise to this unique mononucleosome species. These results in part explain the differential effects of H1(0) and H1c in regulating chromatin structure and function.

Overproduction of histone H1 variants in vivo increases basal and induced activity of the mouse mammary tumor virus promoter.

BALB/c 3T3 cell lines containing integrated copies of the MMTV promoter driving a reporter gene were constructed. Expression vectors in which either of two H1 variants, H10 or H1c, were under control of an inducible promoter were introduced into these lines. Surprisingly, overproduction of either variant resulted in a dramatic increase in basal and hormone-induced expression from the MMTV promoter. H1 overproduction also slowed the loss of MMTV promoter activity associated with prolonged hormone treatment. Transiently transfected MMTV reporter genes, which do not adopt a phased nucleosomal arrangement, do not display increased activity upon H1 overproduction. Thus the effects observed for stable constructs most likely represents a direct effect of H1 on a chromatin-mediated process specific to the nucleosomal structure of the integrated constructs. Induction of increased levels of acetylated core histones by treatment with trichostatin A also potentiated MMTV activity and this effect was additive to that caused by H1 overproduction. However, the effects of TSA treatment, in control or H1-overproducing cells, were eliminated by inhibiting protein synthesis. TSA treatment does not necessarily potentiate MMTV promoter activity by increasing core histone acetylation within the MMTV promoter but perhaps by altering the synthesis of an unlinked transcriptional regulator.

Differential effect of H1 variant overproduction on gene expression is due to differences in the central globular domain.

The in vivo overproduction of two mouse histone H1 variants in homologous mouse fibroblasts has opposite effects on gene expression. Overproduction of H1(0) results in repression of transcript levels of all polymerase II genes tested. In contrast, overproduction of H1c results in elevated levels of transcripts. We created a series of chimeric H1 genes in which the regions encoding the three structural domains common to this family of these proteins were systematically switched. Overexpression of these genes in vivo resulted in the accumulation of large amounts of the chimeric H1 in chromatin. Analysis of the effects of overproduction of these proteins revealed that the differential effect of H1 variant overproduction on gene expression is due to differences in the central globular domain.