Objective:To investigate the anti-oxidative stress effects of microRNA 125b (miR-125b) on lens epithelial cells (LECs) and its possible mechanism.Methods:Twenty-four anterior capsule specimens were collected from 24 eyes of 24 age-related cataract patients during phacoemulsification and 20 normal anterior capsule specimens were obtained from 20 eyes of 20 donors in Henan Eye Hospital from July 2018 to March 2019 under the approval of a Medical Ethics Committee of Henan Eye Hospital (No.YKYY20193151).The reverse transcription PCR and Western blot assay were employed to detect and compare the relative expression levels of miR-125b and nuclear factor E2-related factor 2 (Nrf2) in different specimens.The human lens epithelial cell line HLEB-3 was divided into control group and oxidative stress model group.The oxidative stress models were established by coculture with different concentrations (100, 200, 400 μmol/L) of H 2O 2 for 24 hours, and the cells were cultured with normal medium without H 2O 2 in the control group.The reactive oxygen species (ROS) content was detected by DCFH-DA fluorescent probe, and the activities of total-antioxidative capability (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as malondialdehyde (MDA) concentration were detected by ELISA, and compared among the groups.The expression levels of miR-125b and Nrf2 were detected by reverse transcription PCR and Western blot assay, respectively.The cells were transfected with miR-125b mimics, miR-125b control and miR-125b inhibitor for 24 hours, respectively, and ROS content was detected by DCFH-DA fluorescent probe and T-AOC, SOD and GSH-Px activities as well as MDA concentration were detected by ELISA and compared among different transfected groups.A dual luciferase reporter assay was used to assess an association between miR-125b and Nrf2.The expression level of Nrf2 protein was detected by Western blot assay and the expression levels of Nrf2 and Keap1 were assayed and located by immunofluorescence double staining. Results:The relative expression levels of miR-125b and Nrf2 in the normal lens anterior capsule specimens were 0.21±0.03 and 0.27±0.06, which were significantly lower than 0.89±0.05 and 0.84±0.12 in the cataract specimens, respectively ( t=15.355, P<0.05; t=18.647, P<0.05).The relative expression levels of miR-125b and Nrf2 were significantly increased in various H 2O 2 treated groups in comparison with the control group and were gradually elevated with the increase of H 2O 2 concentration (all at P<0.05).Compared with the control group, the T-AOC, SOD and GSH-Px activities were reduced, and ROS content and MDA concentration were significantly ascended (all at P<0.05).Compared with the miR-125b control group, the T-AOC, GSH-Px and SOD activities were increased, and ROS content and MDA concentration were decreased in the miR-125b mimics group (all at P<0.05).In addition, the T-AOC, GSH-Px and SOD activities were significantly weakened, and ROS content and MDA concentration were significantly increased in the miR-125b inhibitor group in comparison with the miR-125b control group (all at P<0.05).Dual luciferase reporter assay showed that miR-125b targeted to the expression of Nrf2 in the H 2O 2 model cells.The fluorescence of Nrf2 in the cytoplasm was the strongest with more nuclear transfer in the miR-125b mimics group, and the expression intensity of Keap1 in the cytoplasm was weaker.The expression of Nrf2 was the weakest with less nuclear transfer in the miR-125b inhibitor group, and the expression level of Keap1 in the cytoplasm was stronger. Conclusions:MiR-125b can enhance the anti-oxidative stress of LECs in age-related cataractous eyes probably by upregulating the expression of Nrf2 and activating the Keap1/Nrf2 signaling pathway.

BACKGROUNDTo construct the Bifidobacterium Infantis/CD tumor targeting gene therapy system.

METHODSPCR expanded CD gene and pGEX-1LambdaT plasmid was transferred into E.Coli. JM109. with TSS solution. Then a dual restriction enzyme (EcoR I and BamH I) digestion was carried out to cut CD gene and pGEX-1LambdaT plasmid. Two segments (4.9 kb and 1.3 kb) were selected and extracted from the gel. T4 DNA Ligase was added to these two segments' mixture. Finally, reconstruction fragment was transferred into Bifidobacterium Infantis by electroporation. The plasmid was extracted from the positive clone selected and testified by an agarose eletrophoresis after enzymed.

RESULTSA positive transferred Bifidobacterium Infantis with 6.2 kb long reconstruction plasmid fragment with CD gene was established which was in accordance with theoretical calculation.

CONCLUSIONSThe Bifidobacterium Infantis/CD tumor targeting gene therapy system could be successfully constructed.

Objective:To analyze the clinical phenotypes and pathogenic gene of a Han Chinese family with enhanced S-cone syndrome (ESCS).Methods:The method of pedigree investigation was adopted.A suspected ESCS Han Chinese family including 8 members of 3 generations was recruited in Henan Eye Hospital from June to September 2021.There was one patient in the family.A thorough ophthalmic examination of the proband was carried out to evaluate the phenotypes, including visual acuity, degree of strabismus, anterior segment and fundus, autofluorescence imaging, fluorescein fundus angiography, full-field electroretinogram (ERG), multifocal ERG, optical coherence tomography.DNA was extracted from peripheral blood samples from the proband and family members.The pathogenic gene and variation were screened by whole exome sequencing (WES).The variation and co-segregation were verified by Sanger sequencing.The deleteriousness of the variation was analyzed by SIFT, Polyphen2 and MutationTaster.The pathogenicity of the variation was evaluated in accordance with the American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines.The analysis of amino acid sequence conservation was performed by SIFT.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Henan Eye Hospital (No.HNEECKY-2017[6]).Written informed consent was obtained from each subject.Results:This pedigree was consistent with autosomal recessive inheritance.The proband had clinical features such as night blindness, hyperopia, accommodative esotropia, peripheral retinal pigmentation, retinoschisis, and photopic ERG responses dominated by large-amplitude waves.Variations including a compound heterozygous variation, c.671C>T: p.S224L on exon 5 and c. 955G>A: p.E319K on exon 6 of NR2E3 were identified by WES.The variations were confirmed to be consistent with co-segregation.The both loci were missense variations, the variation frequency of which was 0 in the East Asian population via the gnomAD database.The variations were predicted to be deleterious by SIFT, Polyphen2 and MutationTaster.The c.671C>T variation was recorded with unknown significance in ClinVar database, and the c.955G>A variation was an unreported new locus.According to the ACMG Standards and Guidelines, the both variations were labeled as with uncertain clinical significance, and the corresponding amino acid sequences were highly conservative across multiple species. Conclusions:This family has the clinical characteristics of ESCS and meets the genetic diagnosis criteria.Two novel variations in NR2E3 gene, c.671C>T: p.S224L and 955G>A: p.E319K, are found.

Objective To explore the clinical application value of MRI fat quantification parameter[verte-bral bone marrow fat fraction(FF)]combined with 25-hydroxyvitamin D[25(OH)D]in predicting fracture risk in patients with osteoporosis.Methods A total of 90 patients with osteoporosis who were admitted to the hospital from January 2023 to April 2024 were selected as the subjects.Among them,50 patients with osteoporotic vertebral com-pression fractures were included in the fracture group,and 40 patients without fractures were included in the control group.All patients underwent the iterative decomposition of water and fat with echo asymmetry and least-squares estimation(IDEAL-IQ)method of MRI to measure the FF of each vertebra among L1-5 and the average FF of L1-5.Serum 25(OH)D level was detected by electrochemiluminescence method.FF and serum 25(OH)D levels of the two groups were compared.The correlation of FF,25(OH)D and bone mineral density(BMD)was analyzed.Multi-variate logistic regression analysis was conducted to screen the risk factors for fracture in patients with osteoporosis.Receiver operating characteristic(ROC)curves were used to evaluate the predictive value of FF,25(OH)D,and their combination for fracture in patients with osteoporosis.Results Patients in the fracture group were older than those in the control group.BMD and serum 25(OH)D level were lower than those of the control group(P<0.05).The FF of L2 and average FF of L1-5 in the fracture group were higher than those in the control group(P<0.05).Correlation analysis results showed that the FF of L2 and the average FF of L1-5 were negatively correlated with BMD(P<0.05),while serum 25(OH)D level was positively correlated with BMD(P<0.05).Multivariate logistic regression analysis showed that age and FF of L2 were independent risk factors for fracture in patients with osteoporo-sis,while BMD and 25(OH)D were protective factors(P<0.05).ROC curves indicated that the AUC values of FF of L2 and 25(OH)D for predicting fracture were 0.714(95%CI:0.606～0.822)and 0.774(95%CI:0.672～0.876).The AUC of joint prediction was 0.923(95%CI:0.867～0.978),which was significantly larger than that of separate prediction(P<0.05).Conclusions FF of L2 and serum 25(OH)D are related to fracture in patients with osteopo-rosis.Age and BMD are factors influencing the occurrence of fracture in patients with osteoporosis.FF of L2 and 25(OH)D have certain predictive value for fracture risk in patients with osteoporosis,and combined detection of the two can improve predictive efficiency.