Rapid evaluation of PHD2 inhibitory activity of natural products based on capillary electrophoresis online stacking strategy.

Prolyl hydroxylase domain 2 (PHD2) is an important enzyme in the human body that perceives changes in oxygen concentration and regulates response in hypoxic environments. Evaluation of PHD2 inhibitory activity of natural products is crucial for drug development of hypoxia related diseases. At present, the detection of low concentration of α-ketoglutaric acid (the substrate of PHD2 enzymatic reaction) requires derivatization reactions or sample pretreatment, which undoubtedly increases the workload of PHD2 inhibitory activity evaluation. In this paper, a direct detection approach of α-ketoglutaric acid was established by using the online stacking strategy of capillary electrophoresis to evaluate the PHD2 inhibitory activity of natural products. Under optimized conditions, detection of a single sample can be achieved within 2 min. By calculation, the intraday precision RSD of the apparent electrophoretic mobility and peak areas of α-ketoglutaric acid are 0.92 % and 0.79 %, respectively, and the interday RSD were 1.27 % and 0.96 % respectively. The recoveries of the present approach were 97.9-105.2 %, and the LOQ and LOD were 2.0 µM and 5.0 µM, respectively. Furthermore, this approach was applied for the evaluation of inhibitory activity of PHD2 for 13 natural products, and PHD2 inhibitory activity of salvianolic acid A was firstly reported. The present work not only realizes evaluation of PHD2 inhibitory activity through direct detection of α-ketoglutaric acid, but also provides technical support for the discovery of potential drug molecules in hypoxia related diseases.

Inhibitory Effect of Jinkui Shenqi Pills on Glucocorticoid-Enhanced Axial Length Elongation in Experimentally Myopic Guinea Pigs.

OBJECTIVE: To explore the underlying mechanism of inhibition by Jinkui Shenqi Pills (JKSQP) on glucocorticoid-enhanced axial length elongation in experimental lens-induced myopia (LIM) guinea pigs. METHODS: Sixty 2-week old male guinea pigs were randomly divided into 4 groups with 15 guinea pigs in each group, according to the random numbers generated by SPSS software: control, LIM, saline and JKSQP groups. The control group includes animals with no treatment, while the guinea pigs in the other 3 groups received lens-induced myopization on the right eyes throughout the experiment (for 8 weeks). The saline and JKSQP groups were given daily intraperitoneal injections of 10 mg/kg hydrocortisone for 2 consecutive weeks at the same time, and then orally administered either saline or JKSQP [13.5 g/(kg•d) for 6 consecutive weeks. Body weight, anal temperature and animal appearance were observed and recorded to evaluate the GC-associated symptoms. The ocular parameters, including refraction and axial length, were measured by streak retinoscopy and A-scan ultrasonography, respectively. The levels of plasma hormones associated with the hypothalamic-pituitary-adrenal axis (HPAA), including free triiodothyronine, free thyroxine, estradiol and testosterone, were measured by radioimmunoassay, and cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate were measured by enzyme-linked immunosorbent assay. In addition, the mRNA and protein expressions of retinal amphiregulin (AREG) was measured by quantitative real-time polymerase chain reaction and Western blotting, respectively. RESULTS: JKSQP effectively increased body weight and anal temperature, improved animal appearance and suppressed axial length elongation in glucocorticoid-enhanced myopic guinea pigs with normalization of 4 HPAA-associated plasma hormones (all P<0.05). The plasma level of cAMP was significantly increased, whereas the plasma level of cGMP and the mRNA and protein expressions of retinal AREG were decreased after treatment with JKSQP (all P<0.05). CONCLUSION: JKSQP exhibited a significant inhibitory effect on axial length elongation with decreased expression of AREG in the retina, and normalized 4 HPAA-associated plasma hormones and the expression of cAMP and cGMP in GC-enhanced myopic guinea pigs.

Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model.

Mutations in the extracellular matrix gene Fibrillin-2 (FBN2) are related to genetic macular degenerative disorders including age-related macular degeneration (AMD) and early-onset macular degeneration (EOMD). It was reported that the retinal protein expression of FBN2 was reduced in patients with AMD and EOMD. The effect of exogenously supplied fbn2 recombinant protein on fbn2-deficiency-related retinopathy was not known. Here we investigated the efficacy and molecular mechanism of intravitreally applied fibrin-2 recombinant protein in mice with fbn2-deficient retinopathy. The experimental study included groups (all n = 9) of adult C57BL/6J male mice which underwent no intervention, intravitreal injection of adeno-associated virus (AAV) empty vector or intravitreal injection of AAV-sh-fbn2 (adeno-associated virus for expressing short hairpin RNA for fibrillin-2) followed by three intravitreal injections of fbn2 recombinant protein, given in intervals of 8 days in doses of 0.30 µg, 0.75 µg, 1.50 µg, and 3.00 µg, respectively. Eyes with intravitreally applied AAV-sh-fbn2 as compared to eyes with injection of AAV-empty vector or developed an exudative retinopathy with involvement of the deep retinal layers, reduction in axial length and reduction in ERG amplitudes. After additional and repeated application of fbn2 recombinant protein, the retinopathy improved with an increase in retinal thickness and ERG amplitude, the mRNA and protein expression of transforming growth factor-beta (TGF-ß1) and TGF-ß binding protein (LTBP-1) increased, and axial length elongated, with the difference most marked for the dose of 0.75 µg of fbn2 recombinant protein. The observations suggest that intravitreally applied fbn2 recombinant protein reversed the retinopathy caused by an fbn2 knockdown.

1H-Indazoles derivatives targeting PI3K/AKT/mTOR pathway: Synthesis, anti-tumor effect and molecular mechanism.

The PI3K/AKT/mTOR signaling pathway is one of the most common abnormal activation pathways in tumor cells, and has associated with multiple functions such as tumor cell growth, proliferation, migration, invasion, and tumor angiogenesis. Here, a series of 3-amino-1H-indazole derivatives were synthesized, and their antiproliferative activities against HT-29, MCF-7, A-549, HepG2 and HGC-27 cells were evaluated. Among them, W24 exhibited the broad-spectrum antiproliferative activity against four cancer cells with IC50 values of 0.43-3.88 µM. Mechanism studies revealed that W24 inhibited proliferation by affecting the DNA synthesis, induced G2/M cell cycle arrest and apoptosis by regulating Cyclin B1, BAD and Bcl-xL, meanwhile induced the change of intracellular ROS and mitochondrial membrane potential in HGC-27 cells. Moreover, W24 inhibited the migration and invasion of HGC-27 cells by decreasing EMT pathway related proteins and reducing the mRNA expression levels of Snail, Slug and HIF-1α. Furthermore, W24 displayed low tissue toxicity profile and good pharmacokinetic properties in vivo. Therefore, 3-amino-1H-indazole derivatives might serve as a new scaffold for the development of PI3K/AKT/mTOR inhibitor and anti-gastric cancer reagent.

Discovery of novel VEGFR-2-PROTAC degraders based on the localization of lysine residues via recruiting VHL for the treatment of gastric cancer.

VEGFR-2 is an attractive therapeutic target for antitumor drug research by blocking tumor angiogenesis and PROTAC provides a new technology for targeted protein knockout. Herein, a library of novel VEGFR-2-PROTAC degraders were rationally designed and synthesized based on the Lys residue region on the surface of VEGFR-2 protein using protein structure-based drug design strategy. Among them, P7 exhibited preferable antitumor activity against HGC-27 cells and less toxic to human normal HUVEC, HEK293T and GES-1 cells in vitro, as well as the potent degradation activity of VEGFR-2 protein in HGC-27 cells (DC50: 0.084 ± 0.04 µM, Dmax: 73.7%) and HUVEC cells (DC50: 0.51 ± 0.10 µM, Dmax: 76.6%). Additionally, P7 degraded VEGFR-2 protein by the formation of ternary complex and the ubiquitin proteasome pathway in HGC-27 cells. Furthermore, P7 shortened the half-life of VEGFR-2 protein synthesis and had no inhibitory effect on the expression of VEGFR-2 mRNA in HGC-27 cells. Moreover, P7 inhibited the colony formation, migration and invasion of HGC-27 cells in a time- and dose-dependent manner, and meanwhile induced G2/M phase cycle arrest and apoptosis. All the results demonstrated that P7 could be as a promising VEGFR-2-PROTAC degrader for gastric cancer therapy.

Synaptosomal Actin Dynamics in the Developmental Visual Cortex Regulate Behavioral Visual Acuity in Rats.

Purpose: Synaptosomal actin dynamics are essential for synaptic structural stability. Whether actin dynamics are involved in structural and functional synaptic plasticity within the primary visual cortex (V1) or behavioral visual acuity in rats has still not been thoroughly investigated. Methods: Synaptosome preparation and western blot analysis were used to analyze synaptosomal actin dynamics. Transmission electron microscopy was used to detect synaptic density and mitochondrial area alterations. A visual water maze task was applied to assess behavioral visual acuity. Microinjection of the actin polymerization inhibitor or stabilizer detected the effect of actin dynamics on visual function. Results: Actin dynamics, the mitochondrial area, and synaptic density within the area of V1 are increased during the critical period for the development of binocularity. Microinjection of the actin polymerization inhibitor cytochalasin D into the V1 decreased the mitochondrial area, synaptic density, and behavioral visual acuity. Long-term monocular deprivation reduced actin dynamics, the mitochondrial area, and synaptic density within the V1 contralateral to the deprived eye compared with those ipsilateral to the deprived eye and impaired visual acuity in the amblyopic eye. In addition, the mitochondrial area, synaptic density, and behavioral visual acuity were improved by stabilization of actin polymerization by jasplakinolide microinjection. Conclusions: During the critical period of visual development of binocularity, synaptosomal actin dynamics regulate synaptic structure and function and play roles in behavioral visual acuity in rats.

Short-term effects of intravitreal Conbercept injection combined with laser photocoagulation on macular edema secondary to ischemic retinal vein occlusion.

AIM: To observe changes in the best-corrected visual acuity (BCVA), central macular thickness (CMT), and central choroidal thickness (CCT) of patients with macular edema (ME) secondary to ischemic retinal vein occlusion (iRVO) following intravitreal Conbercept injection. METHODS: This retrospective study included 33 eyes from 33 patients who received intravitreal injections of Conbercept for ME secondary to iRVO. Treatments were performed on a 3+pro re nata (3+PRN) basis. All of the patients were examined by fundus fluorescein angiography and spectral domain optical coherence tomography at the first visit. Laser photocoagulation was performed in the nonperfusion area of the retina of all eyes after the first injection. BCVA, CMT, and CCT were observed before and after 6mo of treatment. The number of injections necessary to achieve improved vision was also noted. RESULTS: Following Conbercept treatment, the mean BCVA significantly improved from 0.81±0.39 at baseline to 0.41±0.25 and 0.43±0.29 logMAR in the third and sixth months, respectively (both P=0.000). The CMT of the patients at baseline was 556.75±98.57 µm; 304.78±68.53 and 306.85±76.77 µm 3 and 6mo after treatment, respectively (both P=0.000 vs baseline). The CCTs of the patients at baseline, 3 and 6mo after treatment were 304.63±57.83, 271.31±45.53, and 272.29±39.93 µm, respectively (P=0.026 and 0.035 vs baseline). No severe adverse event relevant to the therapy was noted, and the average number of injections delivered was 3.35. CONCLUSION: Intravitreal Conbercept injection combined with laser photocoagulation appears to be a safe and effective treatment for ME secondary to iRVO in the short-term.

Synthesis and bioevaluation of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amines as tubulin polymerization inhibitors with anti-angiogenic effects.

A new series of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine derivatives as tubulin polymerization inhibitors were synthesized, and evaluated for the anti-proliferative activities. A structure-activity relationship study revealed that the free amino moiety of 1H-pyrazolo[3,4-b]pyridin-3-amine played an essential role in the anti-proliferative activities. Especially, compound 15c displayed the strongest anti-proliferation against MCF-7 cells with IC50 value of 0.067 ± 0.003 µM, and high selectivity over the normal human embryonic lung WI-38 cells with IC50 value of 23.41 ± 1.53 µM. Further mechanistic studies revealed that 15c showed strong anti-tubulin polymerization activity, changed the morphology of tubulin, and arrested the cell cycle at the G2/M transition in MCF-7 cells. Molecular docking analysis suggested that 15c well occupied the colchicine-binding pocket of tubulin. Additionally, 15c demonstrated anti-angiogenic activities with blocking the migration, invasion and tube formation, disrupting the newly formed tube, and regulating both MMP-9 and TIMP-1 in HUVEC cells. In summary, our results highlight that compound 15c is a potential antitumor compound that are worthy of further development.

Design, synthesis and biological evaluation of novel 2-(4-(1H-indazol-6-yl)-1H-pyrazol-1-yl)acetamide derivatives as potent VEGFR-2 inhibitors.

Vascular endothelial growth factor-2 (VEGFR-2) plays a pivotal role in tumor angiogenesis. Herein, a library of novel 2-(4-(1H-indazol-6-yl)-1H-pyrazol -1-yl)acetamide derivatives were designed and synthesized as VEGFR-2 inhibitors based on scaffold hopping strategy. These compounds exhibited the excellent inhibitory in both VEGFR-2 and tumor cells proliferation. Especially, compound W13 possessed potent VEGFR-2 inhibition with IC50 = 1.6 nM and anti-proliferation against HGC-27 tumor cells with IC50 = 0.36 ± 0.11 µM, as well as less toxicity against normal GES-1 cells with IC50 = 187.46 ± 10.13 µM. Moreover, W13 obviously inhibited colony formation, migration and invasion of HGC-27 cells by adjusting the expression of MMP-9 and E-cadherin, and induced HGC-27 cells apoptosis by increasing ROS production and regulating the expression of apoptotic proteins. Furthermore, W13 blocked the PI3K-Akt-mTOR signaling pathway in HGC-27 cells. In addition, anti-angiogenesis of W13 was proved by inhibiting tube formation and the expression of p-VEGFR-2 in HUVEC cells. All the results demonstrated that W13 could be developing as a promising anticancer agent for gastric cancer therapy.

Design, synthesis and biological evaluation of indole-2-one derivatives as potent BRD4 inhibitors.

Bromodomain protein 4 (BRD4) plays a crucial role in transcriptional regulation and is considered to be a viable drug target for cancer treatment. Herein, we designed and synthesized a series of indole-2-one derivatives through scaffold hopping drug design. Most of the compounds showed potent BRD4 inhibitory activities and anti-proliferation activities in cancer cell lines. Especially, compound 12j exhibited excellent BRD4 inhibitory activities (BD1 IC50 = 19 nM, BD2 IC50 = 28 nM) and anti-proliferation potency with IC50 values of 4.75 µM and 1.35 µM in HT-29 and HL-60 cells, respectively. Additionally, docking studies showed that the hydrophobic pocket next to KAc region and WPF shelf were critical to the activity of the compound. Compound 12j could arrest the cell-cycle progression of HT-29 cells into the G1 phase and reduce the expression of c-Myc. Moreover, compound 12j exhibited favorable oral pharmacokinetic properties. All the results demonstrated that compound 12j was a potent BRD4 inhibitor and had merely potential for colon cancer treatment.

Synthesis, biological evaluation and molecular modeling study of 2-amino-3,5-disubstituted-pyrazines as Aurora kinases inhibitors.

Serine/threonine protein kinases Aurora A, B, and C play essential roles in cell mitosis and cytokinesis, and a number of Aurora kinase inhibitors have been evaluated in the clinic. Herein we report the synthesis and their antiproliferation of 3,5-disubstituted-2-aminopyrazines as kinases inhibitors. Amongst, 4-((3-amino-6- (3,5-dimethylisoxazol-4-yl)pyrazin-2-yl)oxy)-N-(3-chlorophenyl) benzamide (12Aj) exhibited the strongest antiproliferative activities against U38, HeLa, HepG2 and LoVo cells with IC50 values were 11.5 ± 3.2, 1.34 ± 0.23, 7.30 ± 1.56 and 1.64 ± 0.48 µM, as well as inhibited Aurora A and B with the IC50 values were 90 and 152 nM, respectively. Molecular docking studies indicated that 12Aj appeared to form stable hydrogen bonds with either Aurora A or Aurora B. Furthermore, 12Aj arrested HeLa cell cycle in G2/M phase by regulating protein levels of cyclinB1 and cdc2. In addition, the bioinformatics prediction further revealed that 12Aj possessed good drug likeness using SwissADME. These results suggested that 12Aj was worthy of future development of potent anticancer agents as pan-Aurora kinases.

Differences in ocular biometry between urban and rural children matched by refractive error: the Shandong Children Eye Study.

PURPOSE: To determine the differences in mean ocular dimensions between urban and rural children and identify possible influencing factors. METHODS: This work uses previously published data from the Shandong Children Eye Study, which was based on a random cluster sampling applied to a cross-sectional school-based study design in the rural Guanxian County and Weihai city. All children underwent auto-refractometry and biometry under cycloplegia. RESULTS: The study included 3290 children (aged 9.35 ± 2.93 years), consisting of 888 pairs of boys and 757 pairs of girls matched by sex, age and refractive error (each pair matching one child from urban cohort with one from the rural cohort). Overall urban children were significantly taller and heavier than rural children (t-test; p < 0.001), which was confirmed for all age groups for weight. Urban ocular axial lengths were significantly longer by 0.23 mm compared to the rural population (t-test; p < 0.001), mostly in younger children and boys. Meanwhile, corneal curvatures were flatter in the urban cohort by 0.08 mm (p < 0.001). This association of axial length with urban vs rural region was reduced in magnitude by 69.7% after accounting for height. CONCLUSIONS: For the same, matched refractive error, children from urban regions had significantly longer eyes and flatter corneal curvature than rural children. Since corneal curvature is defined during the first 2 years of life, early environmental factors may be the source of these differences in ocular dimensions.

Novel conjugates of podophyllotoxin and coumarin: Synthesis, cytotoxicities, cell cycle arrest, binding CT DNA and inhibition of Topo IIß.

A series of conjugates of podophyllotoxin and coumarin were prepared using the click reaction, and their cytotoxicities against A549, HepG2, HeLa, and LoVo cells were evaluated. Among them, compound 14e exhibited the strongest cytotoxicities against these cancer cells with IC50 values of 4.9-17.5 µM. Furthermore, 14e disrupted microtubules and induced cell cycle arrest at G1 phase by regulating P21 and Cyclin D1 in LoVo cells. In addition, 14e bond CT DNA and selectively inhibited Topo IIß over Topo IIα. Molecular docking model showed that 14e appeared to form stable hydrogen bonds with several DNA bases and residue Gln778. Taken together, these conjugates have the potential to be developed as anti-tumor drugs.

Metabolic profile analysis of free amino acids in experimental autoimmune uveoretinitis rat plasma.

AIM: To determine the differences of amino acid (AA) levels in experimental autoimmune uveoretinitis (EAU). METHODS: AA analysis of the plasma samples in EAU rats induced by interphotoreceptor retinoid-binding protein emulsion were performed with high performance liquid chromatography (HPLC) and phenylisothiocyanate (PITC) pre-column derivation methods were performed. Using partial least squares discriminant analysis (PLS-DA), the potential biomarkers were identified in EAU rat plasma, and the metabolic pathways related to EAU were further analyzed. RESULTS: The method results showed that linear (r≥0.9957), intra-day reproducible [relative standard deviation (RSD)=0.04%-1.33%], inter-day reproducible (RSD=0.06%-2.07%), repeatability (RSD=0.03%-0.89%), stability (RSD=0.05%-2.48%) and recovery (RSD=1.98%-4.39%), with detection limits of 0.853-11.4 ng/mL. The metabolic profile in EAU rats was different from that in the control groups five AAs concentrations were increased and nine AAs were reduced. Moreover, five metabolic pathways were related to the development of EAU. CONCLUSION: The developed method is a simple, rapid and convenient for determination of AAs in EAU rat plasma, and these findings will provide a comprehensive insight on the metabolic profiling of the pathological changes in EAU.

Synthesis and identification of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl as potential Aurora A inhibitors.

The Aurora kinases are a family of serine/threonine kinases that interact with components of the mitotic apparatus and serve as potential therapeutic targets in oncology. Herein, we reported a series of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl with selective inhibition of Aurora A in either enzymatic assays or cellular phenotypic assays, and displaying more potent anti-proliferation compared with that of VX-680. The most potent compound 10a forms better interaction with Aurora A than Aurora B in molecular docking. Mechanistic studies revealed that 10a disrupt the spindle formation, block the cell cycle progression in the G2/M phase and induce apoptosis in HeLa cell. These results suggested that the produced series of compounds are potential anticancer agents for further development as selective Aurora A inhibitors.

Corneal diameter and associated parameters in Chinese children: the Shandong Children Eye Study.

BACKGROUND: To assess the corneal diameter and its associations in children. DESIGN: Cross-sectional school-based study. PARTICIPANTS: Six thousand twenty-six children aged 4-18 years were included in the Shandong Children Eye Study. METHODS: Horizontal corneal diameter was measured by laser interferometry-based ocular biometry. MAIN OUTCOME MEASURES: Horizontal corneal diameter. RESULTS: Corneal diameter measurements were available for 5970 (99.1%) children. In multivariate analysis, larger horizontal corneal diameter (mean: 12.02 ± 0.38 mm; range: 10.1-15.0 mm) was associated with longer corneal curvature radius longer axial length, male gender, younger maternal age, rural region of habitation and lower intraocular pressure measurements. Higher prevalence of abnormally large corneas (macrocorneas; horizontal diameter ≥ 12.76 mm; mean value +2 × standard deviations; mean: 2.6%; 95% CI: 2.2, 3.0) was associated with longer corneal curvature radius, longer axial length, younger maternal age and male gender. Higher prevalence of abnormally small corneas (horizontal diameter ≤ 11.24mm; mean value -2 × standard deviations; mean: 2.4%; 95% CI: 2.0, 2.8) was correlated with shorter corneal curvature radius, shorter axial length and urban region of habitation. Neither abnormally large nor small corneas were correlated with time spent indoors/outdoors. CONCLUSIONS: In 4 to 18-year-old children, larger corneal diameter was associated most strongly with flatter corneal curvature, followed by longer axial length and male gender. Corneal diameter was independent of age beyond an age of 4 years. Abnormally large and abnormally small corneas may be defined as being ≥12.76 and ≤11.24 mm in diameter, respectively. Corneal diameter was not correlated with time spent indoors/outdoors.

Stereoacuity and Related Factors: The Shandong Children Eye Study.

OBJECTIVE: To assess stereoacuity in a population-based sample of children and to examine ocular and systemic parameters related to stereoacuity. METHODS: Using a random cluster sampling method, four- to 18-year-old children from kindergartens, elementary schools, junior high schools and senior high schools from a rural area and an urban area in the East Chinese province of Shandong were included in the school-based cross-sectional study. All participants underwent a comprehensive eye examination including assessment of cycloplegic refraction and measurement of stereoacuity using the Titmus Stereo test. RESULTS: Out of 6364 eligible children, 5780 (90.8%) children with a mean age of 10.1 ± 3.2 years (range: 4 to 18 years) participated. Mean (± standard deviation) stereoacuity was 50.2 ± 50.6 arc seconds. Stereoacuity improved significantly (P<0.01) from the age group of 4 years to the age group of 6 to 7 years, then showed a plateau, deteriorated (P = 0.001) for both sexes from the age group of 9 years to the age group of 12 years (P<0.001), after which it improved (P = 0.001) again in the age group of 16 years or older to the pre-puberty values. In multivariate analysis, larger angle of binocular disparity (i.e., lower stereoacuity) was significantly associated with lower best corrected visual acuity (logMAR; P<0.001), higher intereye difference in refractive error (spherical equivalent) (P<0.001), higher cylindrical refractive error (P<0.001), higher refractive error (spherical value; P<0.001), higher intereye difference in best corrected visual acuity (logMAR) (P = 0.001), higher intereye difference in axial length (P = 0.001), and rural region of habitation (P = 0.006). CONCLUSIONS: Stereoacuity as tested with the Titmus Stereo test improved significantly from an age of 4 years to an age of 6 and 7 years, then remained constant, temporarily deteriorated for both sexes in pre-puberty and puberty, after which it improved again to pre-puberty or better values at the age of 16 years or older. Lower stereoacuity was associated with lower best corrected visual acuity and higher intereye difference in best corrected visual acuity, higher cylindrical and spherical refractive errors, higher inter-eye difference in refractive error, higher intereye difference in axial length, and rural region of habitation.