A Novel BEST1 Mutation in Autosomal Recessive Bestrophinopathy

Citation: Lee CS, Jun I, Choi S, et al. A novel BEST1 mutation in autosomal recessive bestrophinopathy. Invest Ophthalmol Vis Sci. 2015;56:8141-8150. DOI:10.1167/iovs.15-18168 PURPOSE. To describe the clinical characteristics associated with a newly identified mutant of autosomal recessive bestrophinopathy (ARB) and confirm the associated physiological functional defects. METHODS. Two patients with ARB from one family underwent a full ophthalmic examination, including dilated fundus examination, fundus photography, fluorescein angiography, fundus autofluorescence imaging, spectral-domain optical coherence tomography (OCT), electroretinography (ERG), and electrooculography (EOG). Subsequently, genetic analysis for bestrophin-1 (BEST1) mutations was conducted through direct Sanger sequencing. The effect of ARB-associated mutations of BEST1 on the cellular localization was determined by in vitro experiments. Whole-cell patch clamping was conducted to measure the chloride conductance of wild-type BEST1 and the identified BEST1 mutants in transfected HEK293T cells. RESULTS. Two related patients (66-year-old brother and 52-year-old sister) presented with reduced visual acuity and bilateral symmetrical subretinal deposits of hyperautofluorescent materials in the posterior pole. Spectral-domain OCT showed macular thinning with submacular fluid. The female patient had a concomitant macular edema associated with branched retinal vein occlusion in the left eye, which responded well to intravitreal bevacizumab injections. Genetic analysis demonstrated that both patients were compound heterozygous for one novel (Leu40Pro) and one previously identified (Ala195Val) BEST1 variant. HEK293T cells transfected with the identified BEST1 mutant showed significantly small currents compared to those transfected with the wild-type gene, whereas cells cotransfected with mutant and wild-type BEST1 showed good chloride conductance. Cellular localization of BEST1 was well conserved to the plasma membrane in the mutants. CONCLUSIONS. We have identified and described the phenotype and in vitro functional aspects of a new BEST1 mutation causing ARB. Clinically suspected ARB cases warrant genetic confirmation to confirm the diagnosis

RNA-Guided Genome Editing in Drosophila with the Purified Cas9 Protein

We report a method for generating Drosophila germline mutants effectively via injection of the complex of the purified Cas9 protein, tracrRNA, and gene-specific crRNAs, which may reduce delayed mutations because of the transient activity of the Cas9 protein, combined with the simple mutation detection in GO founders by the T7E1 assay.

Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines: less involvement of metallothionein

BACKGROUND: Heptaplatin is a new platinum derivative with anticancer activity against various cancer cell lines, including cisplatin-resistant cancer cell lines (Cancer Chemother Pharmacol 1995; 35: 441). METHODS: Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines has been investigated in connection with metallothionein (MT). Cytotoxicity was determined by an MTT assay. MT mRNA, was determined by RT-PCR assay. Transfection study was carried out to examine the function of MT. RESULTS: Of various gastric cancer cell lines, SNU-638 and SNU-601 showed the highest and lowest levels of MT mRNA, respectively, showing 80-fold difference. The IC(50 )values of SNU-638 to cisplatin, carboplatin and heptaplatin were 11.2-fold, 5.1-fold and 2.0-fold greater than those of SNU-601, respectively. Heptaplatin was more effective against cisplatin-resistant and MT-transfected gastric cancer sublines than cisplatin or carboplatin was. In addition, heptaplatin attenuated cadmium, but not zinc, induction of MT. CONCLUSION: These results indicate that molecular mechanisms of heptaplatin effective against cisplatin-resistant gastric cancer sublines is at least in part due to the less involvement of MT in heptaplatin resistance as well as its attenuation of MT induction

Effect of mucilage from yam on activation of lymphocytic immune cells

The immunostimulating activities of mucilage fraction from yam were investigated. The proliferation of BSA-primed lymph node cells was enhanced between 4.1- to 10.9-fold compare to control, when cultured with 1 to 25 µg/mL of yam-mucilage fraction. It showed strong immunopotentiating activity than ginseng extract and as remarkable as Bifidobacterium adolescentis M101-4 known as a positive immunostimulator. Mitogenicity to lymph node cells was fully induced by concanavalin A and lipopolysaccharide. The proliferation of splenocytes and Peyer's patch cells was enhanced between 5.0- to 14.1-fold and 2.4- to 6.4-fold, respectively, when cultured with 1 to 25 µg/mL of yam-mucilage fraction. It enhanced the production of cytokines such as tumor necrosis factor-α and IL-6 in the culture of RAW 264.7 macrophage cells. In the culture of lipopolysaccharide-stimulated RAW 264.7 cells, production of cytokines was as similar as compared to controls. In unstimulated RAW 264.7 cells, both tumor necrosis factor-α and IL-6 production were enhanced between 15.6- to 60.1-fold and 2.3- to 9.1-fold, respectively. Mucilage fraction from yam is expected to be a safe immunopotentiator to maintain the host immunity and develop a physiologically functional food

Topical application of epidermal growth factor accelerates wound healing by myofibroblast proliferation and collagen synthesis in rat

Recombinant human epidermal growth factor (rhEGF) stimulates the proliferation and migration of epithelial cells in human cell culture systems and animal models of partial-thickness skin wounds. This study investigated the effect of a topical rhEGF ointment on the rate of wound healing and skin re-epithelialization in a rat full thickness wound model, and verified whether or not the rhEGF treatment affected both myofibroblast proliferation and collagen synthesis in the dermis. When rhEGF (10 µg/g ointment) was applied topically twice a day for 14 days, there was significantly enhanced wound closure from the 5th to the 12th day compared with the control (ointment base treatment) group. A histological examination at the postoperative 7th day revealed that the rhEGF treatment increased the number of proliferating nuclear antigen immunoreactive cells in the epidermis layer. In addition, the immunoreactive area of alpha-smooth muscle actin and the expression of prolyl 4-hydroxylase were significantly higher than those of the control group. Overall, a topical treatment of rhEGF ointment promotes wound healing by increasing the rate of epidermal proliferation and accelerating the level of wound contraction related to myofibroblast proliferation and collagen deposition

A case of hemocholecyst associated with hemobilia following radiofrequency ablation therapy for hepatocellular carcinoma

Radiofrequency ablation (RFA) is performed as an alternative to surgical resection for primary or secondary liver malignancies. Although RFA can be performed safely in most patients, early and late complications related to mechanical or thermal damage occur in 8-9.5% cases. Hemocholecyst, which refers to hemorrhage of the gallbladder, has been reported with primary gallbladder disease or as a secondary event associated with hemobilia. Hemobilia, defined as hemorrhage in the biliary tract and most commonly associated with accidental or iatrogenic trauma, is a rare complication of RFA. Here we report a case of hemocholecyst associated with hemobilia after RFA for hepatocellular carcinoma that was successfully managed by laparoscopic cholecystectomy