[Show abstract][Hide abstract] ABSTRACT: Purpose
The purpose of this study is to evaluate the functional and morphological changes in subretinal xenografts of human retinal progenitor cells (hRPCs) in B6 mice treated with Cyclosporin A (CsA; 210 mg/l in drinking water).
The hRPCs from human fetal eyes were isolated and expanded for transplantation. These cells, with green fluorescent protein (GFP) at 11 passages, were transplanted into the subretinal space in B6 mice. A combination of invasive and noninvasive approaches was used to analyze the structural and functional consequences of the subretinal injection of the hRPCs. The process of change was monitored using spectral domain optical coherence tomography (SDOCT), histology, and electroretinography (ERG) at 3 days, 1 week, and 3 weeks after transplantation. Cell counts were used to evaluate the survival rate with a confocal microscope. ERGs were performed to evaluate the physiologic changes, and the structural changes were evaluated using SDOCT and histological examination.
The results of the histological examination showed that the hRPCs gained a better survival rate in the mice treated with CsA. The SDOCT showed that the bleb size of the retinal detachment was significantly decreased, and the retinal reattachment was nearly complete by 3 weeks. The ERG response amplitudes in the CsA group were less decreased after the injection, when compared with the control group, in the dark-adapted and light-adapted conditions. However, the cone-mediated function in both groups was less affected by the transplantation after 3 weeks than the rod-mediated function.
Although significant functional and structural recovery was observed after the subretinal injection of the hRPCs, the effectiveness of CsA in xenotransplantation may be a novel and potential approach for increasing retinal progenitor cell survival.
[Show abstract][Hide abstract] ABSTRACT: To compare the power of the 4 intraocular pressure (IOP) measures, that is, peak, mean, range, and SD, over a 24-hour period in predicting IOP variations in order to determine which measure of IOP fluctuation correlates best with actual office-hour readings in glaucoma patients and healthy subjects.
For this prospective study, 25 subjects with untreated primary open-angle glaucoma and 33 healthy individuals were hospitalized for 24 hours. Measurements of the subjects' IOP for both eyes were recorded with a Goldmann applanation tonometer every 3 hours in the sitting position during the daytime (9 AM to 9 PM) and with a TonoPen in both the sitting and supine positions for 24 hours. Only 1 eye was selected randomly per subject for the final analysis. The strength of association between the estimated values and the actual 24-hour IOP data in habitual body positions was analyzed using the coefficient of determination (R). The differences were calculated. The percentage of subjects with estimated IOP values falling within the cutoff values from the 24-hour data were assessed.
The peak IOP was captured outside office hours in 57% of the young subjects, 75% in the elderly control group, and 52% of the glaucoma patients. The estimation of the strength of association for the mean IOP and peak IOP showed strong to moderate correlations (R range from 0.29 to 0.95) compared with the estimation of range and SD of IOP fluctuation, which demonstrated weak to moderate relationships (R range from 0.001 to 0.69). The percentage of significant cases mostly corresponded with the correlation.
With the combination of sitting and supine position readings during office hours, the study provides promising results in estimating the mean and peak IOP in glaucoma patients and healthy subjects; however, it showed little advantage in range and SD of IOP fluctuation.
No preview · Article · Apr 2014 · Journal of glaucoma
[Show abstract][Hide abstract] ABSTRACT: Objective:
To investigate the morphological and functional changes of the laser-induced choroidal neovascularization (CNV) in rhesus monkeys.
Experimental study. Eight adult rhesus monkeys weighted 4 to 7 kg were used in this study. CNVs were induced with small high-energy laser spots at short pulse duration by an argon green laser. Eyes were monitored weekly by color fundus photography, fluorescence fundus angiography (FFA) , and optical coherence tomography (OCT) . Fluorescein leaking intensities of grade 4 CNV lesions were analyzed by the method of ANOVA for repeated measures. Electroretinogram (ERG) was performed before laser photocoagulation and 56 days after laser photocoagulation and the data were analyzed with paired t-test.
(1) FFA revealed that the mean intensities of grade 4 CNV lesions were 89.44 ± 26.28, 97.56 ± 26.47, 110.22 ± 29.76, 100.26 ± 29.24, 91.77 ± 28.11, 77.76 ± 24.85 and 63.23 ± 22.34 on day 14, day 21, day 28, day 35, day 42, day 49, and day 56 respectively and the differences were statistically significant (F = 39.715, P < 0.01) . The differences between any time-point and its previous time-point were also statistically significant (t14-21 = 4.824, P < 0.01; t21-28 = 5.225, P < 0.01; t28-35 = 7.378, P < 0.01;t35-42 = 2.954, P < 0.05; t42-49 = 5.386, P < 0.01; t49-56 = 6.138, P < 0.01). (2) OCT images showed retinal edema, subretinal fluid and hyper-reflective lesions of CNVs in the laser sites and histopathology showed that fibrovascular tissues together with proliferating retinal pigment epithelium cells were seen in the laser sites. (3) ERG data revealed that implicit time of dark-adapted b wave (t = 4.23, P < 0.01) increased while the amplitudes of dark-adapted a wave (t = 6.35, P < 0.01) , dark-adapted b wave (t = 3.12, P < 0.01) and light-adapted b wave (t = 3.93, P < 0.01) decreased 56 days after laser photocoagulation compared with those before laser photocoagulation.
The laser-induced CNV in non-human primate model shows continuous leakage on FFA examination and reduced amplitudes as well as increased implicit time on ERG examination, suggesting that laser-induced CNV primate model not only could be used to study the pathogenesis of CNV formation but also could be used for screening of drug effectiveness.
No preview · Article · Mar 2014 · [Zhonghua yan ke za zhi] Chinese journal of ophthalmology
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the safety and efficacy of intravitreal injection of (99)Tc-MDP, a decay product of (99m)Tc-MDP, on the development of laser-induced choroidal neovascularization (CNV) in rhesus monkeys.
Experimental CNV was induced by argon laser with a small high-energy laser spot. Monkeys were given 50 μL of (99)Tc-MDP at a concentration of 0.005 μg/mL (n = 6) or 0.01 μg/mL (n = 6) by intravitreal injection once a week immediately after laser injury for a period of 56 days. Control animals were treated with the same volume of PBS (n = 6) in the same way. Eyes were monitored by ophthalmic examination, color fundus photography, fluorescence fundus angiography (FFA), optical coherence tomography (OCT) and histology. Incidences of grade 4 CNV lesions as well as the leakage areas of grade 4 CNVs on the late-phase of fluorescein angiograms were measured in a standardized, randomized and masked fashion fortnightly. The maximum widths and heights of grade 4 CNVs were also calculated by histology at the end of the experiment. Toxicity of (99)Tc-MDP on the retina was evaluated by electroretinogram (ERG) and histologic analysis.
(99)Tc-MDP reduced the incidences of grade 4 CNVs by 33.33 % and 39.40 % in the 0.005 μg/mL and 0.01 μg/mL groups, respectively, compared with the PBS group on day 28 (P < 0.05; n = 6). The leakage areas of grade 4 CNVs were smaller in the 0.005 μg/mL (0.7136 ± 0.0283 mm(2), p <0.01; n = 6) and 0.01 μg/mL (0.4351 ± 0.0349 mm(2), p < 0.01; n = 6) groups than those in the PBS control group (0.9373 ± 0.0455 mm(2); n = 6) in a dose-dependent manner on day 28. OCT and histology also showed that the sizes of CNVs were smaller in the (99)Tc-MDP treated groups than those in the PBS group. Although intravitreal injection of (99)Tc-MDP led to mild inflammatory reaction in the anterior chamber, histology and ERG findings demonstrated that (99)Tc-MDP did not cause any change in histological structure or function of the retina (p>0.05).
Intravitreal injection of (99)Tc-MDP can inhibit the development of laser-induced CNV without toxic effect on retina, suggesting that (99)Tc-MDP has therapeutic potential for CNV related diseases.
No preview · Article · Jan 2014 · Albrecht von Graæes Archiv für Ophthalmologie
[Show abstract][Hide abstract] ABSTRACT: Purpose:
We evaluated the effect of ginkgo biloba extract on visual field defect and contrast sensitivity in a Chinese cohort with normal tension glaucoma.
In this prospective, randomized, placebo-controlled crossover study, patients newly diagnosed with normal tension glaucoma, either in a tertiary glaucoma clinic (n = 5) or in a cohort undergoing routine general physical examinations in a primary care clinic (n = 30), underwent two 4-week phases of treatment, separated by a washout period of 8 weeks. Randomization determined whether ginkgo biloba extract (40 mg, 3 times per day) or placebo (identical-appearing tablets) was received first. Primary outcomes were change in contrast sensitivity and mean deviation on 24-2 SITA standard visual field testing, while secondary outcomes included IOP and self-reported adverse events.
A total of 35 patients with mean age 63.7 (6.5) years were randomized to the ginkgo biloba extract-placebo (n = 18) or the placebo-ginkgo biloba extract (n = 17) sequence. A total of 28 patients (80.0%, 14 in each group) who completed testing did not differ at baseline in age, sex, visual field mean deviation, contrast sensitivity, IOP, or blood pressure. Changes in visual field and contrast sensitivity did not differ by treatment received or sequence (P > 0.2 for all). Power to have detected a difference in mean defect as large as previously reported was 80%.
In contrast to some previous reports, ginkgo biloba extract treatment had no effect on mean defect or contrast sensitivity in this group of normal tension glaucoma patients. (http://www.chictr.org number, ChiCTR-TRC-08000724).
[Show abstract][Hide abstract] ABSTRACT: Background
The result of primary open-angle glaucoma is the loss of retinal ganglion cells. Transient receptor potential cation channel 6 is a pressure-related channel that may function in the survival of retinal ganglion cells. The purpose of this study was to evaluate the expression levels of the transient receptor potential cation channel 6 gene in patients with primary open-angle glaucoma. DesignRandomization study at Zhongshan Ophthalmic Center in China. Participants80 primary open-angle glaucoma patients and 75 cataract patients recruited from Zhongshan Ophthalmic Center. Methods
Total RNA was extracted from the leukocytes of the peripheral blood collected. The levels of transient receptor potential cation channel 6–messenger RNA were determined by real-time polymerase chain reaction. Related factors including age, intraocular pressure, optic cup-to-disc ratio and visual field defect were analysed accordingly. Main Outcome MeasuresClinical examination and the messenger RNA level. ResultsThe expression level of the transient receptor potential cation channel 6 gene in the leukocytes of primary open-angle glaucoma patients was two times higher when compared with control cataract patients. The gene expression level was also correlated with intraocular pressure and cup-to-disc ratio. Treatment with different anti-glaucoma drugs did not affect the gene expression. Conclusions
Increasing expression levels of the transient receptor potential cation channel 6 gene in the blood accompanies chronic elevation of intraocular pressure in primary open-angle glaucoma and may serve as a genetic biomarker for primary open-angle glaucoma.
No preview · Article · Nov 2013 · Clinical and Experimental Ophthalmology
[Show abstract][Hide abstract] ABSTRACT: Microorganisms contain a large number of biocatalysts, which are of great potential in industrial applications. However, the traditional cultural approaches can obtain only less than 1% of microorganisms. As a culture-independent method, metagenomics is an advanced solution by means of extracting all microbial genomic DNAs in certain environmental habitat, constructing and screening metagenomic libraries to seek novel functional genes. It serves as an effective tool for studying these uncultured microorganisms. Therefore, mining novel biocatalysts from metagenome has drawn the attention of researchers in the world. In this paper, environment sample category, genomic DNA extraction, library construction and screening strategies were reviewed. Recent examples of isolated biocatalysts from metagenomic libraries were presented. Future research directions of metagenomics were also discussed.
No preview · Article · Apr 2012 · Sheng wu gong cheng xue bao = Chinese journal of biotechnology
[Show abstract][Hide abstract] ABSTRACT: Prebiotic galacto-oligosaccharides (GOS) were effectively synthesized from lactose in organic-aqueous biphasic media by a novel metagenome-derived β-galactosidase BgaP412. A maximum GOS yield of 46.6% (w/w) was achieved with 75.4% lactose conversion rate in the cyclohexane/buffer system [95:5 (v/v) cyclohexane/buffer] under the optimum reaction conditions (initial lactose concentration = 30% (w/v), T = 50 °C, pH 7.0, and t = 8 h). The corresponding productivity of GOS was approximately 17.5 g L(-1) h(-1). The GOS mixture consisted of tri-, tetra-, and pentasaccharides. Trisaccharides were the chief component of reaction products. These experimental results showed that a low water content, a high initial lactose concentration, and an elevated reaction temperature could significantly promote the transgalactosylation activity of β-galactosidase BgaP412; at the same time, the enhanced GOS yield in an organic-aqueous biphasic system is because of the fact that thermodynamic equilibrium can be shifted to the synthetic direction by reversing the normal hydrolysis.
No preview · Article · Mar 2012 · Journal of Agricultural and Food Chemistry
[Show abstract][Hide abstract] ABSTRACT: Pyrethroid pesticides are broad-spectrum pest control agents in agricultural production. Both agricultural and residential usage is continuing to grow, leading to the development of insecticide resistance in the pest and toxic effects on a number of nontarget organisms. Thus, it is necessary to hunt suitable enzymes including hydrolases for degrading pesticide residues, which is an efficient "green" solution to biodegrade polluting chemicals. Although many pyrethroid esterases have consistently been purified and characterized from various resources including metagenomes and organisms, the thermostable pyrethroid esterases have not been reported up to the present.
In this study, we identified a novel pyrethroid-hydrolyzing enzyme Sys410 belonging to familyV esterases/lipases with activity-based functional screening from Turban Basin metagenomic library. Sys410 contained 280 amino acids with a predicted molecular mass (Mr) of 30.8 kDa and was overexpressed in Escherichia coli BL21 (DE3) in soluble form. The optimum pH and temperature of the recombinant Sys410 were 6.5 and 55°C, respectively. The enzyme was stable in the pH range of 4.5-8.5 and at temperatures below 50°C. The activity of Sys410 decreased a little when stored at 4°C for 10 weeks, and the residual activity reached 94.1%. Even after incubation at 25°C for 10 weeks, it kept 68.3% of its activity. The recombinant Sys410 could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl acetate with the highest activity (772.9 U/mg). The enzyme efficiently degraded cyhalothrin, cypermethrin, sumicidin, and deltamethrin under assay conditions of 37°C for 15 min, with exceeding 95% hydrolysis rate.
This is the first report to construct metagenomic libraries from Turban Basin to obtain the thermostable pyrethroid-hydrolyzing enzyme. The recombinant Sys410 with broad substrate specificities and high activity was the most thermostable one of the pyrethroid-hydrolyzing esterases studied before, which made it an ideal candidate for the detoxification of pyrethroids.
Preview · Article · Mar 2012 · Microbial Cell Factories
[Show abstract][Hide abstract] ABSTRACT: A novel organic solvent-stable and thermotolerant lipase gene (designated ostl28) was cloned from a metagenomic library and overexpressed in Escherichia coli BL21 (DE3) in soluble form. OSTL28 contained 262 amino acids with relative molecular mass 30.1kDa and isoelectric point 9.7. The optimum pH and temperature of the OSTL28 were 7.5 and 60°C, respectively. OSTL28 was stable in the pH range of 4.5–9.5 and at temperatures below 65°C. The enzyme could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl laurate with the highest activity of 236U/mg (54,000U/L). The recombinant OSTL28 was highly resisted to organic solvents, especially glycerol and methanol. The metal ions, with the exception of Hg2+ and Ag+, did not have any influence on enzyme activity, whereas non-ionic surfactants and Al3+ slightly activated the enzyme. These features indicate that it is a potential biocatalyst for biodiesel production.
No preview · Article · Nov 2011 · Journal of Molecular Catalysis B Enzymatic