Stepwise candidate drug screening for myopia control by using zebrafish, mouse, and Golden Syrian Hamster myopia models.

BACKGROUND: We developed a preclinical protocol for the screening of candidate drugs able to control myopia and prevent its progression. The protocol uses zebrafish, C57BL/6 mice, and golden Syrian hamster models of myopia. METHODS: A morpholino (MO) targeting the zebrafish lumican gene (zlum) was injected into single-cell zebrafish embryos, causing excessive expansion of the sclera. A library of 640 compounds with 2 matrix metalloproteinase (MMP) inhibitors (marimastat and batimastat), which have the potential to modulate scleral remodelling, was screened to identify candidates for mitigating scleral diameter expansion in zlum-MO-injected embryos. The myopia-prevention ability of compounds discovered to have superior potency to inhibit scleral expansion was validated over 4 weeks in 4-week-old C57BL/6 mice and 3-week-old golden Syrian hamsters with form-deprivation myopia (FDM). Changes in the refractive error and axial length were investigated. Scleral thickness, morphology of collagen fibrils in the posterior sclera, messenger RNA (mRNA) expressions, and protein levels of transforming growth factor-ß2 (TGF-ß2), tissue inhibitor of metalloproteinase-2 (TIMP-2), MMP-2, MMP-7, MMP-9, and collagen, type I, alpha 1 (collagen Iα1) were investigated in C57BL/6 mice, and MMP-2, MMP-9, and MMP activity assays were conducted in these mice. FINDINGS: In the zebrafish experiment, atropine, marimastat, batimastat, doxycycline, and minocycline were the drugs that most effectively reduced expansion of scleral equatorial diameter. After 28-day treatment in diffuser-wearing mice and 21-day treatment in lid-sutured hamsters, myopic shift and axial elongation were significantly mitigated by eye drops containing 1% atropine, 50 µM marimastat, 5 µM batimastat, or 200 µM doxycycline. MMP-2 mRNA expression in mouse sclera was lower after treatment with atropine, marimastat, batimastat, or doxycycline. The protein levels and activity of MMP-2 and MMP-7 were significantly reduced after treatment with atropine, marimastat, batimastat, doxycycline, and minocycline. Furthermore, scleral thickness and collagen fibril diameter were not lower after treatment with atropine, marimastat, batimastat, or doxycycline than those of occluded eyes. INTERPRETATION: Stepwise drug screening in a range of models from zlum-MO-injected zebrafish to rodent FDM models identified effective compounds for preclinical myopia control or prevention. On the basis of the 640 compounds that were screened, MMP inhibitors may offer alternatives for clinical trials. FUNDING: This research was supported by grants from Taiwan's Ministry of Science and Technology and Ministry of Health and Welfare.

Scopoletin 8-Hydroxylase-Mediated Fraxetin Production Is Crucial for Iron Mobilization.

Iron (Fe) is an essential mineral nutrient and an important factor for the composition of natural plant communities. Low Fe availability in aerated soils with neutral or alkaline pH has led to the evolution of elaborate mechanisms that extract Fe from the soil solution. In Arabidopsis (Arabidopsis thaliana), Fe is acquired by an orchestrated strategy that comprises mobilization, chelation, and reduction of Fe3+ prior to its uptake. Here, we show that At3g12900, previously annotated as scopoletin 8-hydroxylase (S8H), participates in Fe acquisition by mediating the biosynthesis of fraxetin (7,8-dihydroxy-6-methoxycoumarin), a coumarin derived from the scopoletin pathway. S8H is highly induced in roots of Fe-deficient plants both at the transcript and protein levels. Mutants defective in the expression of S8H showed increased sensitivity to growth on pH 7.0 media supplemented with an immobile source of Fe and reduced secretion of fraxetin. Transgenic lines overexpressing S8H exhibited an opposite phenotype. Homozygous s8h mutants grown on media with immobilized Fe accumulated significantly more scopolin, the storage form of scopoletin, supporting the designated function of S8H in scopoletin hydroxylation. Fraxetin exhibited Fe-reducing properties in vitro with higher rates being observed at neutral relative to acidic pH. Supplementing the media containing immobile Fe with fraxetin partially rescued the s8h mutants. In natural Arabidopsis accessions differing in their performance on media containing immobilized Fe, the amount of secreted fraxetin was highly correlated with growth and Fe and chlorophyll content, indicating that fraxetin secretion is a decisive factor for calcicole-calcifuge behavior (i.e. the ability/inability to thrive on alkaline soils) of plants.

iTRAQ protein profile analysis of Arabidopsis roots reveals new aspects critical for iron homeostasis.

Iron (Fe) deficiency is a major constraint for plant growth and affects the quality of edible plant parts. To investigate the mechanisms underlying Fe homeostasis in plants, Fe deficiency-induced changes in the protein profile of Arabidopsis (Arabidopsis thaliana) roots were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) differential liquid chromatography-tandem mass spectrometry on a LTQ-Orbitrap with high-energy collision dissociation. A total of 4,454 proteins were identified with a false discovery rate of less than 1.1%, and 2,882 were reliably quantified. A subset of 101 proteins was differentially expressed upon Fe deficiency. The changes in protein profiles upon Fe deficiency show low congruency with previously reported alterations in transcript levels, indicating posttranscriptional changes, and provide complementary information on Fe deficiency-induced processes. The abundance of proteins involved in the synthesis/regeneration of S-adenosylmethionine, the phenylpropanoid pathway, the response to oxidative stress, and respiration was highly increased by Fe deficiency. Using Fe-responsive proteins as bait, genome-wide fishing for partners with predictable or confirmed interologs revealed that RNA processing and ribonucleoprotein complex assembly may represent critical processes that contribute to the regulation of root responses to Fe deficiency, possibly by biasing translation efficiency.