Circulating exosomes in ophthalmic disease: novel carriers of biological information circulating exosomes in ophthalmic disease.

Exosomes, small membrane vesicles with a diameter of 30-100 nm, transport lipids, proteins, DNA, and RNA. Exosomes originate from endocytic vessels and are processed and released through exocytosis. They can be taken up by target cells and mediate intercellular communication. Initially, exosomes were thought to be waste products excreted by cells. However, with more research, they have been found to play important roles in physiological and pathological processes. Therefore, they are promising biomarkers for the diagnosis and treatment of a variety of disease conditions, including fundus diseases, ocular surface diseases, retinal diseases, tumors, ocular trauma, and light damage. In this review, we discuss the history, biogenesis, release, isolation, characterization, and biological functions of exosomes, as well as their future application prospects in ophthalmic diseases.

LncRNA MIR497HG inhibits proliferation and migration of retinal endothelial cells under high-level glucose treatment via miRNA-128-3p/SIRT1 axis.

OBJECTIVE: The aim of this study was to elucidate the potential influence of MIR497HG on regulating proliferative capacity of human retinal endothelial cells (HRECs). MATERIALS AND METHODS: Relative expression levels of MIR497HG, microRNA-128-3p (miRNA-128-3p) and SIRT1 in HRECs treated with different doses of glucose and mannitol were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Dual-Luciferase reporter gene assay was conducted to assess the interaction among MIR497HG, miRNA-128-3p, and SIRT1. In addition, the potential effects of MIR497HG/miRNA-128-3p/SIRT1 axis on proliferative and migratory capacities in HRECs were evaluated by Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'- deoxyuridine (EdU) and transwell assay, respectively. RESULTS: High-level glucose (HG) treatment significantly downregulated MIR497HG and SIRT1 expression, whereas upregulated miRNA-128-3p expression in HRECs (p<0.05). MiRNA-128-3p was the target gene binding MIR497HG, and SIRT1 was the downstream gene of miRNA-128-3p. Overexpression of MIR497HG significantly attenuated proliferative and migratory abilities of HG-induced HRECs (p<0.05). Furthermore, decreased trends were partially reversed by overexpression of miRNA-128-3p or knockdown of SIRT1. CONCLUSIONS: MIR497HG is downregulated after HG treatment. In addition, it suppresses the proliferation and migration of HRECs by targeting miRNA-128-3p/SIRT1 axis, thus influencing the progression of diabetic retinopathy.

[The genetic factors of non-response to anti-vascular endothelial growth factor therapy].

The response to anti-vascular endothelial growth factor (VEGF) treatment is variable. It is generally measured in terms of changes in correlated functional and/or anatomical outcomes, and patients are then classified as optimal response, poor response and non-response. The precise cause of non-response remains undetermined. A variety of factors could account for poor or non-response to anti-VEGF therapy, such as age, baseline vision, disease course, lesion characteristics and genomic polymorphism. At the present time, many studies on the genetic factors of non-response or poor response to anti-VEGF treatment mainly focus on VEGF genes (VEGF-A, VEGFR-2), complement factor H (CFH), age-related maculopathy susceptibility 2 (LOC387715/ARMS2), high temperature factor A-1 (HTRA1), interleukin-related gene (IL-8 rs4073) and so forth. It is still worthy of further investigations that how to assess genetic reasons for non-response or poor response, so that we can provide individualized treatment sequences and predict the response to anti-VEGF therapy. (Chin J Ophthalmol, 2018, 54:873-878).

A systematic review on delayed absorption of subretinal fluid after scleral buckling for rhegmatogenous retinal detachment.

Rhegmatogenous retinal detachment is a vision-threatening disease and is treated by either scleral buckling or pars planar vitrectomy. Rapid absorption of the subretinal fluid (SRF) helps in the early recovery of the vision. The absorption of SRF after the scleral buckling procedure is rapid, provided that the retinal break or breaks are closed at or after surgery. However, in some patients with rhegmatogenous retinal detachment, complete absorption of the SRF occur several weeks or months after the surgery. In this review, we discuss the factors influencing the rate of SRF absorption and the role of delayed absorption on visual recovery. We also discuss the therapeutic options for delayed SRF absorption and the available additional therapeutic options. Knowledge of the factors that influence the rate of SRF absorption, would enable the surgeon to predict the outcomes more accurately.

The study on expression of CIAPIN1 interfering hepatocellular carcinoma cell proliferation and its mechanisms.

OBJECTIVE: Liver cancer is one of the common gastrointestinal cancers. This study was designed to investigate the effect of the cytokine-induced apoptosis inhibitor 1 (CIAPIN1) on hepatocellular carcinoma cell proliferation and invasion. MATERIALS AND METHODS: To establish a low and high expression of CIAPIN1 in hepatoma cell lines, pGPU6/GFP/Neo and CIAPIN1 siRNA vectors were constructed. The growth curve of liver cancer cells with a low and high expression of CIAPIN1 was measured by MTT assay and colony formation in soft. The effect of overexpression and inhibition of CIAPIN1 on the expressions of cell cycle proteins Cyclin D1, CDK2, CDK4, and Cyclin E were detected by western blot. RESULTS: As compared with the low expression group, the cells in CIAPIN1 high expression group showed a significant decrease in proliferation (p < 0.05). In addition, the colony-forming ability of cells with high expression of CIAPIN1 was decreased significantly (p < 0.01). Furthermore, the expressions of Cyclin D1 CDK2, CDK4, and Cyclin E in high expression group were significantly increased (p < 0.01). CONCLUSIONS: CIAPIN1 played an important role in the proliferation of liver cancer cells through increasing the expressions of cell cycle related proteins Cyclin D1, CDK2, CDK4, and Cyclin E.

Tumor necrosis factor-like weak inducer of apoptosis association with proliferative diabetic retinopathy and promotes proliferation and collagen synthesis in retinal ARPE-19 cells.

Chronic inflammation develops in the retinal microvasculature under sustained hyperglycemia and is implicated in the pathogenesis of diabetic retinopathy. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 have been reported to promote pro-inflammatory cytokines, which are involved in the pathogenesis of proliferative diabetic retinopathy (PDR). It is therefore possible that the TWEAK/Fn14 pathway can play a regulatory role in PDR. In the present study, we examined the expression of TWEAK and Fn14 in vitreous fluid from PDR patients. To confirm the correlation between the TWEAK expression and clinical pathological characteristics of PDR, we investigated the regulatory role of the TWEAK/Fn14 pathway in cell proliferation and collagen synthesis in retinal ARPE-19 cells. The results demonstrated that vitreous fluid from patients with PDR had higher levels of TWEAK and Fn14 than that from T2DM patients without PDR, thus suggesting an important regulatory role of TWEAK/Fn14 signaling in the pathogenesis of PDR. Furthermore, overexpression of TWEAK in ARPE-19 cells also promoted proliferation of and collagen synthesis in these retinal cells. It is possible that TWEAK/Fn14 upregulation in PDR may contribute to PDR progression by promoting the proliferation or fibrosis of retinal cells.

Iterative ensemble Kalman filter for atmospheric dispersion in nuclear accidents: An application to Kincaid tracer experiment.

Information about atmospheric dispersion of radionuclides is vitally important for planning effective countermeasures during nuclear accidents. Results of dispersion models have high spatial and temporal resolutions, but they are not accurate enough due to the uncertain source term and the errors in meteorological data. Environmental measurements are more reliable, but they are scarce and unable to give forecasts. In this study, our newly proposed iterative ensemble Kalman filter (EnKF) data assimilation scheme is used to combine model results and environmental measurements. The system is thoroughly validated against the observations in the Kincaid tracer experiment. The initial first-guess emissions are assumed to be six magnitudes underestimated. The iterative EnKF system rapidly corrects the errors in the emission rate and wind data, thereby significantly improving the model results (>80% reduction of the normalized mean square error, r=0.71). Sensitivity tests are conducted to investigate the influence of meteorological parameters. The results indicate that the system is sensitive to boundary layer height. When the heights from the numerical weather prediction model are used, only 62.5% of reconstructed emission rates are within a factor two of the actual emissions. This increases to 87.5% when the heights derived from the on-site observations are used.

Ensemble-based simultaneous emission estimates and improved forecast of radioactive pollution from nuclear power plant accidents: application to ETEX tracer experiment.

The accidental release of radioactive materials from nuclear power plant leads to radioactive pollution. We apply an augmented ensemble Kalman filter (EnKF) with a chemical transport model to jointly estimate the emissions of Perfluoromethylcyclohexane (PMCH), a tracer substitute for radionuclides, from a point source during the European Tracer Experiment, and to improve the forecast of its dispersion downwind. We perturb wind fields to account for meteorological uncertainties. We expand the state vector of PMCH concentrations through continuously adding an a priori emission rate for each succeeding assimilation cycle. We adopt a time-correlated red noise to simulate the temporal emission fluctuation. The improved EnKF system rapidly updates (and reduces) the excessively large initial first-guess emissions, thereby significantly improves subsequent forecasts (r = 0.83, p < 0.001). It retrieves 94% of the total PMCH released and substantially reduces transport error (>80% average reduction of the normalized mean square error).

Modified ensemble Kalman filter for nuclear accident atmospheric dispersion: prediction improved and source estimated.

Atmospheric dispersion models play an important role in nuclear power plant accident management. A reliable estimation of radioactive material distribution in short range (about 50 km) is in urgent need for population sheltering and evacuation planning. However, the meteorological data and the source term which greatly influence the accuracy of the atmospheric dispersion models are usually poorly known at the early phase of the emergency. In this study, a modified ensemble Kalman filter data assimilation method in conjunction with a Lagrangian puff-model is proposed to simultaneously improve the model prediction and reconstruct the source terms for short range atmospheric dispersion using the off-site environmental monitoring data. Four main uncertainty parameters are considered: source release rate, plume rise height, wind speed and wind direction. Twin experiments show that the method effectively improves the predicted concentration distribution, and the temporal profiles of source release rate and plume rise height are also successfully reconstructed. Moreover, the time lag in the response of ensemble Kalman filter is shortened. The method proposed here can be a useful tool not only in the nuclear power plant accident emergency management but also in other similar situation where hazardous material is released into the atmosphere.

[The expression of tPA directed by the bovine BLG regulatory elements in the mammary gland of transgenic mice].

In order to get the regulatory elements which are essential for generating mammary gland bioreactors, the whole 8.4 kb bovine BLG gene was obtained by PCR amplification. The 1.6 kb chicken lysozyme matrix attachment region (MAR) was used to overcome position effects. The bovine BLG-tPA expression vector was constructed and the BLG-tPA fusion gene was introduced into fertilized eggs of mice by microinjection to generate transgenic mouse. 170 offsprings were obtained, of which 9 were proved to be transgenic mice based on PCR and Southern-blot analysis. The tPA expression level amounted to 12 micrograms/mL in the milk of mice. The bovine BLG-tPA fusion gene integrated in the founders was inheritable.

[Immunologic properties of coli surface antigen 6(CS6) of enterotoxigenic Escherichia coli and cholera toxin subunit B expressed in Shigella flexneri 2a strain T32].

A host-plasmid balancing system composed with a delta asd mutant (FaD) of an avirulent strain (T32) of Shigella flexneri 2a and plasmid harboring asd gene was used to express enterotoxigenic E. coli surface antigen 6(CS6) and V. cholerae toxin subunit B (CTB). The results of Western blotting and ELISA showed that all of recombinant plasmids (pYX201, pYX202 and pYX203) could be maintained stably and expressed CS6 and CTB respectively in T32 without any antibiotic selection. All the recombinant bacterial strains could elicit the corresponding antibodies in rabbits. The antibodies against CTB elicited by both FaD/pYX201 and FaD/pYX203 showed to be high level, and had long prolongation time, in otherwise, the antibodies against CS6 showed to be low level, indicating that higher expression level of foreign antigen may be benefit for construction of genetic multivalent vaccine.

Expression of Shiga toxin B subunit at cell surface in E. coli K-12.

The three parts (Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the LamB fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.

Extracellular export of Shiga toxin B-subunit/haemolysin A (C-terminus) fusion protein expressed in Salmonella typhimurium aroA-mutant and stimulation of B-subunit specific antibody responses in mice.

The Shiga toxin B-subunit has been fused to the 23-kD C-terminus of Escherichia coli haemolysin A (HlyA) and exported from attenuated antigen carrier strain of Salmonella typhimurium aroA (SL3261). The expression of the gene fusion under the control of a synthetic modified beta-lactamase promoter (constitutive expression) and under the iron-regulated aerobactin promoter showed that the fusion protein could be stably expressed and exported out of the bacterial cell in significant amounts so long as high copy number plasmids were not used. Oral and i.p. immunization of mice with the hybrid salmonellae resulted in significant B-subunit specific mucosal and serum antibody responses. A comparative analysis of the location of hybrid proteins in the antigen carrier bacterial cell (i.e. cytoplasmic expression and extracellular export) has shown that both modes of expression result in antigen-specific immune responses. This is the first report demonstrating that foreign polypeptides fused to the 23-kD C-terminus of E. coli haemolysin A can be exported from attenuated Salmonella vaccine strains and that such exported polypeptides can result in antigen-specific immune responses.

Construction of stable LamB-Shiga toxin B subunit hybrids: analysis of expression in Salmonella typhimurium aroA strains and stimulation of B subunit-specific mucosal and serum antibody responses.

The complete Shiga toxin B subunit and two N-terminal segments of the B subunit have been inserted into a cell surface exposed loop of the LamB protein, and expression of the hybrid proteins from three different promoter systems, i.e., (i) an in vitro-inducible tac promoter that provides high-level expression, (ii) the iron-regulated aerobactin promoter presumably induced in vivo under the iron-limiting conditions of the intestinal mucosal environment, and (iii) a synthetic, modified beta-lactamase promoter providing moderate level constitutive expression, has been analyzed in Escherichia coli, Salmonella typhimurium, and attenuated antigen carrier strains of S. typhimurium (aroA mutants). The hybrid vaccine strains were used to immunize mice by the oral and intraperitoneal routes. S. typhimurium aroA mutants apparently have a membrane export defect which prevents the transport of LamB and its derivatives across the cytoplasmic membrane. High-level expression of hybrid proteins through use of the tac promoter proved deleterious to the vaccine strains and prevented the production of viable cells at reasonable cell densities. The lower levels of gene expression observed with the beta-lactamase and aerobactin promoters did not have this effect. Immunization of mice with S. typhimurium aroA strains carrying the hybrid genes expressed from these two promoters resulted in significant B subunit-specific mucosal and serum antibody responses. This suggests that such expression systems may be useful when incorporated into candidate antidysentery live oral vaccines for inducing protection against the effect of Shiga toxin in infections caused by Shigella dysenteriae 1 and other Shiga toxin-or Shiga-like toxin-producing pathogens.

[Cloning of the gene relative to invasive ability of Shigella flexneri 5].

A genomic library of S. flexneri 5 large plasmid (140 Md) was constructed using the cosmid pJB8 as vector. There were more than 4,000 transformants in the library. 66 clones were picked out from the library by hybridization with 17kb gene probe relative to invasive ability. A few clones were analyzed. The results indicated that all these clones tested contained recombinant plasmids, they could hybridize with 17 kb gene probe. When these recombinant plasmids were digested with EcoR1, 17 kb fragments which hybridized with 17 kb gene probe always existed in these recombinant plasmids. It showed that all these recombinant plasmids contained the DNA fragments relative to invasive ability. It provides the possibility to construct an oral living vaccine against S. flexneri.