Systematic Analysis of Transcriptomic Profile of the Effects of Low Dose Atropine Treatment on Scleral Fibroblasts using Next-Generation Sequencing and Bioinformatics.

This study has two novel findings: it is not only the first to deduct potential genes involved in scleral growth repression upon atropine instillation from a prevention point of view, but also the first to demonstrate that only slight changes in scleral gene expression were found after atropine treatment as side effects and safety reasons of the eye drops are of concern. The sclera determines the final ocular shape and size, constituting of scleral fibroblasts as the principal cell type and the major regulator of extracellular matrix. The aim of our study was to identify differentially expressed genes and microRNA regulations in atropine-treated scleral fibroblasts that are potentially involved in preventing the onset of excessive ocular growth using next-generation sequencing and bioinformatics approaches. Differentially expressed genes were functionally enriched in anti-remodeling effects, comprising of structural changes of extracellular matrix and metabolic pathways involving cell differentiation. Significant canonical pathways were correlated to inhibition of melatonin degradation, which was compatible with our clinical practice as atropine eye drops are instilled at night. Validation of the dysregulated genes with previous eye growth-related arrays and through microRNA-mRNA interaction predictions revealed the association of hsa-miR-2682-5p-KCNJ5 and hsa-miR-2682-5p-PRLR with scleral anti-remodeling and circadian rhythmicity. Our findings present new insights into understanding the anti-myopic effects of atropine, which may assist in prevention of myopia development.

Deduction of Novel Genes Potentially Involved in the Effects of Very Low Dose Atropine (0.003%) Treatment on Corneal Epithelial Cells Using Next-Generation Sequencing and Bioinformatics Approaches.

Background and Objectives: Atropine is a nonselective muscarinic antagonist which has been used to prevent worsening of myopia in children. Different concentrations of atropine were used for myopia, ranging from 0.01% to 1.0%. However, there are still potential toxicity of different doses of atropine to the cornea. Here, we present a study of investigating novel genes potentially involved in the effects of very low dose atropine treatment (0.003%) on corneal epithelial cells using next-generation sequencing (NGS) and bioinformatics approaches. Materials and Methods: Human corneal epithelial cells were treated with 0.003% atropine, cultured until confluence, and RNA extracted for differential expression profiling of mRNA and microRNA (miRNA) between control and atropine-treated corneal epithelial cells. The functional enrichment analysis for differentially expressed genes was performed using two bioinformatics databases, including Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity® Pathway Analysis (IPA). In addition, potential miRNA-mRNA interactions involved in atropine-treated corneal epithelial cells were predicted and validated using different miRNA target prediction databases. Results: Our results showed 0.003% atropine might suppress the apoptosis of corneal epithelial cells, potentially through Ras and protein kinase A signaling pathways. We also validated the possible miRNA regulations by using TargetScan and miRDB databases. Hsa-miR-651-3p-EPHA7, hsa-miR-3148-TMEM108 and hsa-miR-874-5p-TBX6 were validated as possible miRNA regulations involved in corneal epithelial cells treated with 0.003% atropine. Conclusions: These findings may contribute novel insights into therapeutic strategies for treating cornea with 0.003% atropine.

Proteomic analysis of retinal pigment epithelium cells after exposure to UVA radiation.

BACKGROUND: Age-related macular degeneration (AMD) is the primary cause of blindness and severe vision loss in developed countries and is responsible for 8.7% of blindness globally. Ultraviolet radiation can induce DNA breakdown, produce reactive oxygen species, and has been implicated as a risk factor for AMD. This study investigated the effects of UVA radiation on Human retinal pigment epithelial cell (ARPE-19) growth and protein expression. METHODS: ARPE-19 cells were irradiated with a UVA lamp at different doses (5, 10, 20, 30 and 40 J/cm2) from 10 cm. Cell viability was determined by MTT assay. Visual inspection was first achieved with inverted light microscopy and then the DeadEnd™ Fluorometric TUNEL System was used to observe nuclear DNA fragmentation. Flow cytometry based-Annexin V-FITC/PI double-staining was used to further quantify cellular viability. Mitochondrial membrane potential was assessed with JC-1 staining. 2D electrophoresis maps of exposed cells were compared to nonexposed cells and gel images analyzed with PDQuest 2-D Analysis Software. Spots with greater than a 1.5-fold difference were selected for LC-MS/MS analysis and some confirmed by western blot. We further investigated whether caspase activation, apoptotic-related mitochondrial proteins, and regulators of ER stress sensors were involved in UVA-induced apoptosis. RESULTS: We detected 29 differentially expressed proteins (9 up-regulated and 20 down-regulated) in the exposed cells. Some of these proteins such as CALR, GRP78, NPM, Hsp27, PDI, ATP synthase subunit alpha, PRDX1, and GAPDH are associated with anti-proliferation, induction of apoptosis, and oxidative-stress protection. We also detected altered protein expression levels among caspases (caspase 3 and 9) and in the mitochondrial (cytosolic cytochrome C, AIF, Mcl-1, Bcl-2, Bcl-xl, Bax, Bad, and p-Bad) and ER stress-related (p-PERK, p-eIF2α, ATF4 and CHOP) apoptotic pathways. CONCLUSIONS: UVA irradiation suppressed the proliferation of ARPE-19 cells in a dose-dependent manner, caused quantitative loses in transmembrane potential (ΔΨm), and induced both early and late apoptosis.

Malignant choroidal melanoma with suspected vitreous seeding - A case report.

Retinal perforation with vitreous seeding is an uncommon condition of treatment-naïve choroidal malignant melanoma. We reported a 52-year-old male who had a pigmented tumor protruding from choroid of his right eye. He had only black shadow sensation for 4 months then a rapid deterioration of vision. Fundus examination showed vitreous haze with many pigmented materials. B-scan ultrasonography revealed a mass with low internal reflectivity and vitreous opacity. The eyeball was enucleated and a stage IIIA melanoma was confirmed by the pathologist. No local recurrences or metastases were found during 31-month follow-up. Although vitreous seeding may indicate rapid tumor growth, early enucleation may insure a better prognosis.

Bilateral uveitis associated with concurrent administration of rifabutin and nelfinavir.

Rifabutin-associated uveitis has been recognized as a dosage-dependent side effect. Previous studies have reported that clarithromycin or fluconazole may elevate concentrations of rifabutin through inhibition of metabolism through the cytochrome P-450 pathway. Nelfinavir is a protease inhibitor widely used in the treatment of human immunodeficiency virus (HIV) infection. The interactions between protease inhibitors and rifabutin have not been reported in clinical practice. Therefore, we present a case of bilateral uveitis associated with coadministration of rifabutin and nelfinavir. Uveitis did not subside until discontinuation of rifabutin. To our knowledge, this is the first report of uveitis with concurrent administration of rifabutin and nelfinavir. Our finding reminds us that rifabutin dosage should be reduced when it is administered with protease inhibitors.

Strategy for stable and high-level expression of recombinant trehalose synthase in Escherichia coli.

Trehalose is a nonreducing disaccharide and has a wide range of applications in food and biorelated industry. This sugar can be synthesized from maltose in one step by trehalose synthase. In this study, we attempted to overproduce trehalose synthase from Picrophilus torridus (PTTS), a thermoacidophilic archaea, in Escherichia coli . However, overproduction of PTTS was hampered when the T7 promoter-driven PTTS gene (PT7-PTTS) on a multicopy plasmid was employed in E. coli . The factors limiting PTTS production were identified in a systematic way, including the codon bias, plasmid instability, a redundant gene copy, a high basal level of PTTS, and metabolic burden resulting from the mutlicopy plasmid DNA and antibiotics. To overcome these difficulties, an E. coli strain was developed with insertion of PT7-PTTS into the chromosome and enhanced expression of genomic argU tRNA and ileX tRNA genes. Without the selective pressure, the constructed producer strain was able to produce a stable and high-level production of recombinant PTTS. Overall, we proposed a simple and effective method to address the issue that is most commonly raised in overproduction of heterologous proteins by E. coli .

Heat shock induces apoptosis through reactive oxygen species involving mitochondrial and death receptor pathways in corneal cells.

Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Here, we are the first to investigate the biological effect of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. Our results indicate that heat shock exhibits effective cell proliferation inhibition by inducing apoptosis. Heat shock triggers the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activity. In addition, heat shock triggered the death receptor apoptotic pathway indicated by a change in Fas ligand expression, resulting in caspase-8 activity. Furthermore, we also found that generation of reactive oxygen species (ROS) is a critical mediator in heat shock-induced apoptosis. In addition, the antioxidant vitamin C significantly decreased heat shock-mediated apoptosis. Taken together, these findings suggest a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells.

Occult plastic intravitreal foreign body retained for 30 years: a case report.

A 41-year-old man had suffered trauma to the right eye 30 years ago. In March 1996, he underwent trabeculectomy and peripheral iridectomy under the diagnosis of open angle glaucoma in the right eye. Autoperimetry at that time revealed visual field constriction. In addition, ocular examination showed that the cup/disc ratio of his right eye was increased. Cataract was diagnosed in September 2002 and cataract extraction was performed on October 22, 2002. Aplastic intravitreal foreign body was detected during the operation. However, preoperative B-scan ultrasonography had failed to detect an intraocular foreign body (IOFB), and the previous fluorescein angiography had shown only retinal pigment epithelium changes. This case reminded us that we should be alert to an occult IOFB in the event of ocular trauma, even if none had been detected during prior imaging examinations.

Polymerase chain reaction identification of a hymenopteran insect in the cornea: a case report.

The type of corneal injuries associated with insect encounters is related to the composition of the foreign body. However, previous reports on corneal foreign bodies as insects were rarely based on scientific evidence. Here, we report on a 49-year-old male who was stung in his left eye by an unknown insect. Emergent keratotomy was performed to remove the embedded corneal foreign body. The removed foreign body was observed under light microscopy, and a fragment of insect was suspected. The sample was sent for molecular analysis. The polymerase chain reaction product was sequenced, subjected to a BLAST search, and identified as an ichneumonoid member of the insect order Hymenoptera.