A screen for regeneration-associated silencer regulatory elements in zebrafish.

Regeneration involves gene expression changes explained in part by context-dependent recruitment of transcriptional activators to distal enhancers. Silencers that engage repressive transcriptional complexes are less studied than enhancers and more technically challenging to validate, but they potentially have profound biological importance for regeneration. Here, we identified candidate silencers through a screening process that examined the ability of DNA sequences to limit injury-induced gene expression in larval zebrafish after fin amputation. A short sequence (s1) on chromosome 5 near several genes that reduce expression during adult fin regeneration could suppress promoter activity in stable transgenic lines and diminish nearby gene expression in knockin lines. High-resolution analysis of chromatin organization identified physical associations of s1 with gene promoters occurring preferentially during fin regeneration, and genomic deletion of s1 elevated the expression of these genes after fin amputation. Our study provides methods to identify "tissue regeneration silencer elements" (TRSEs) with the potential to reduce unnecessary or deleterious gene expression during regeneration.

Modulation of red blood cell oxygen affinity with a novel allosteric modifier of hemoglobin is additive to the Bohr effect.

PURPOSE: The Bohr effect describes hemoglobin's affinity for oxygen dependent on solution pH. Within pH range 6.0-8.5, hemoglobin's oxygen affinity decreases with decreasing pH. This results in increased oxygen delivery to metabolically active, acidic tissues and improved oxygen uptake in basic regions including lung tissue. Myo-Inositol tripyrophosphate (ITPP) translocates the erythrocyte membrane and allosterically modifies hemoglobin (Hb). We tested the hypothesis that ITPP does not abrogate the Bohr effect. METHODS: Experiments were conducted to determine the effect of increasing concentrations of ITPP on P50 with varying pH. We incubated 10 mL red blood cells at 37 °C for 1 h with ITPP concentrations from 0 to 240 mM. The Clark oxygen electrode (Hemox-Analyzer; TCS Scientific, New Hope, PA) determined oxygen affinity of each sample, in triplicate, using buffers pH 6.8, 7.4, and 7.6. A mixed linear regression model with fixed effects for ITPP concentration and pH was used. RESULTS: Increasing ITPP concentration and decreasing pH increased P50 (p < 0.0001 for ITPP concentration, p < 0.0001 for pH). ITPP modulated increased P50 in normal pH (7.4) and acidic condition pH (6.8); with no effect at alkaline pH (7.6). CONCLUSION: The Bohr effect is conserved, with ITPP augmenting the decreased oxygen affinity seen with tissue acidosis, while not affecting oxygen affinity in conditions similar to a pulmonary microenvironment.

Astigmatic axis is related to the level of spherical ametropia.

PURPOSE: Against-the-rule (ATR) astigmatism has been shown to be a risk factor for subsequent myopia development. In this study, we evaluated the relationship between astigmatic axis and the level of spherical ametropia in both myopes and hypermetropes. METHODS: Astigmatic axes were analyzed in two distinct cohorts. First, 53 high myopes from families that were recruited for linkage analysis were compared with an age-matched control group derived from family members. Second, cross-sectional data were analyzed for 90,884 subjects attending 19 optometric practices in the north of England. Initially, the relationship between astigmatic axis and cylinder power and between axis and sphere power were analyzed in 21- to 40-year olds and 21- to 30-year olds, respectively, to control for the effects of age. Multivariate logistic regression analysis was then performed using data for all compound astigmats in the cohort to examine the effect of sphere power, cylinder power, age, and sex on the odds of subjects having either ATR or with-the-rule (WTR) astigmatism. RESULTS: In the genetic study cohort, there was an excess of WTR astigmats in the high myopes compared with controls, but this only reached significance for the right eye. In the much larger optometric practice sample, the association of WTR astigmatism with high myopia was highly significant. A parallel increase in WTR astigmatism was also found for high hypermetropes. In addition, the odds of having WTR astigmatism were increased if subjects were young or had a high cylinder power. ATR astigmatism occurred more often with increasing age and in subjects with lower spherical ametropia. Indeed, for 21- to 30-year-old subjects with low myopia (> or =-2.00 DS in the least minus meridian), ATR occurred more often than WTR astigmatism. CONCLUSION: Astigmatic axis was found to be related to the level of ametropia, with both a higher spherical component or higher cylinder power increasing the odds of astigmatism being WTR. Low ametropes, particularly myopes, were more likely to have axes ATR.