The effect of AAV-mediated downregulation of Claudin-3 on the development of mouse retinal vasculature.

Retinal vascular development is a very tightly regulated and organized process of vessel formation and regression to generate the mature vasculature system. Claudin-3 has been found to be required for the normal development of the neural retina and its vessels in zebrafish in our recent study. In this study, we investigated whether Claudin-3 played a role in the development of mouse retinal vasculature. Immunofluorescent staining was performed to detect the expression and localization of Claudin-3 in the mouse retina. Intravitreal injection of a recombinant adeno-associated virus (AAV) expressing a short hairpin RNA targeting Claudin-3 mRNA was performed to down-regulate Claudin-3 expression in retina in neonatal (Postnatal Day 3, P3) C57BL/6J mice. Retinal vessels were examined by isolectin B4 immunofluorescent staining on the whole-mount retinas and frozen retinal sections at P10. The apoptotic retinal ganglion cells (RGCs) were measured by TdT-mediated dUTP nick-end labelling (TUNEL) staining. Vascular endothelial growth factor A (VEGF-A) expression was detected by immunofluorescent staining. The protein levels of Claudin-3, VEGF-A and B cell lymphoma 2 (Bcl-2) were evaluated by Western blot at P7, P10 and P14. We found that Claudin-3 mainly expressed in the RGCs and progressively increased during the retinal development. The AAV-mediated downregulation of Claudin-3 at P3 impeded the development of retinal deep vascularization of P10 mouse, but without effect on the development of the retinal superficial plexus. Claudin-3 knockdown increased RGC apoptosis and reduced the expression of VEGF-A and Bcl-2 in the retinas. These results suggested that the downregulation of Claudin-3 induced RGC apoptosis and impeded the mouse retinal vascular development by downregulating the levels of VEGF-A and Bcl-2.

High-precision two-dimensional beam steering with a 64-element optical fiber phased array.

Large-scale optical fiber phased arrays (OFPAs) are capable of realizing high-power lasers and high-speed beam steering, which are widely used in long-distance detection and communication. However, dephasing occurring from optical fiber jitter and power amplifier noise can reduce beam quality and steering precision in applications. We demonstrate a two-dimensional 64-element OFPA system that employs a stochastic parallel gradient descent algorithm to synchronize the phases and thus achieve high-quality multi-beam output. Using multi-beam steering, the total scan time for covering a certain field of view can be shorter compared to single-beam steering. Moreover, an avalanche photodiode array is used to enhance the precision of the voltage for beam steering. Experimental results show that the peak sidelobe ratio of the main beam achieves 23.7 dB, and the speed of the beam steering between two discretionary angles is 128 kHz.

The role of cldnh during the early retinal development in zebrafish.

Claudin-3, an integral component of tight junction, has recently been shown to be expressed in retinal ganglion cells, retinal pigment cells, and retinal vascular endothelial cells. However, the role of claudin-3 in the development of the neural retina and its vessels remains undefined. This study aimed to investigate the role of zebrafish claudin-h (cldnh), the closest ortholog of mouse and human claudin-3, in the development of the neural retina and its vessels. Cldnh levels in green fluorescent protein transgenic zebrafish were genetically manipulated by cldnh morpholino oligonucleotide (MO) and cldnh mRNA to investigate gene function. The expression of cldnh was analyzed using polymerase chain reaction and immunofluorescence staining. The altered morphological, cellular and molecular events in the cldnh MO-morphant eyes were detected using hematoxylin-eosin staining, fluorescent dye injection, confocal in vivo imaging, BrdU labeling, TUNEL assay, RNA sequencing, and Western blot. We demonstrated that the cldnh protein was expressed in the neural retina and the hyaloid vessel which is the predecessor of the retinal vessel in zebrafish. Cldnh knockdown delayed lamination of the neural retina and reduced its thickness, which might be associated with the downregulation of the retinal development-related genes of atoh7, pcdh17, crx, neurod1, insm1a, sox9b and cdh11, and the upregulation of the cell cycle and apoptosis-associated genes of tp53, cdkn1a and casp8. Cldnh knockdown also reduced the density and interrupted the lumenization of the hyaloid vessels, which might be owing to the downregulation of the vessel formation-related genes of hlx1 and myl7. In conclusion, cldnh was required for the normal development of the neural retina and its vessels in zebrafish, providing a basis for elucidating its role in the pathogenesis of retinal vascular or inflammatory diseases.

The role of optical coherence tomography angiography in fundus vascular abnormalities.

BACKGROUND: To evaluate the role of optical coherence tomography angiography (OCTA) in observation of fundus vascular abnormalities. METHODS: Patients (n = 50, 10 in each group) with fundus disorders including branch retinal vein occlusion (BRVO), non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), exudative age-related macular degeneration (AMD), and polypoidal choroidal vasculopathy (PCV) were examined. They underwent imaging of OCTA and fluorescein angiography/indocyanine green angiography. The split-spectrum amplitude-decorrelation angiography algorithm was employed to obtain angiography within a 6 × 6 mm scanning area at the posterior retina. Segmentation algorithm was used to obtain 2-dimensional images from arbitrary layers. The OCTA features were analyzed and compared with the findings of conventional angiography. The contralateral eyes of the patients with BRVO and the eyes of 20 healthy volunteers served as controls. RESULTS: OCTA showed precise images of normal and abnormal vasculature in the posterior retina and choroid by the given layers. Vascular abnormalities such as enlarged foveal avascular zone (FAZ), non-perfusion area of retina, microaneurysm, retinal neovascularization, choroidal neovascularization (CNV), branching vascular network and polypoidal lesions in choroid were clearly displayed by OCTA. CONCLUSIONS: OCTA provided a better projection of vascular pathologies of the posterior retina and choroid and could determine the precise location of the vascular lesion. The noninvasive OCTA can benefit the diagnosis of vascular abnormalities in the posterior retina and choroid.

Function and mechanism exploring of icariin in schizophrenia through network pharmacology.

This study aims to explore the therapeutic effect and possible mechanisms of icariin in schizophrenia. SD rats were divided into five groups, a control group, a MK801-induced schizophrenia model group, and three icariin treatment groups, with twelve rats in each group. Morris water maze and open field were used to observe the spatial learning and memory ability of rats. Compared with the control group, rats in the MK801-induced model group showed an increase in stereotypic behavior score, distance of spontaneous activities, escape latency, malondialdehyde (MDA) content, and IL-6, IL-1ß, TNF-α expression, but a decrease in platform crossing times and superoxide dismutase (SOD) activity (P < 0.05). Furthermore, all the above changes of the model group were reversed after icariin treatment in a dose-dependent manner (P < 0.05). Network pharmacology found that icariin can exert anti-schizophrenic effects through some signaling pathways, such as relaxin, estrogen, and TNF signaling pathways. MAPK1, MAPK3, FOS, RELA, TNF, and JUN were the key targets of icariin on schizophrenia, and their expression was detected in animal models, which was consistent with the predicted results of network pharmacology. Icariin treatment may improve the spatial learning and memory ability of schizophrenic rats through TNF signaling pathway.

Comprehensive analysis of the protein phosphatase 2A regulatory subunit B56ε in pan-cancer and its role and mechanism in hepatocellular carcinoma.

BACKGROUND: B56ε is a regulatory subunit of the serine/threonine protein phosphatase 2A, which is abnormally expressed in tumors and regulates various tumor cell functions. At present, the application of B56ε in pan-cancer lacks a comprehensive analysis, and its role and mechanism in hepatocellular carcinoma (HCC) are still unclear. AIM: To analyze B56ε in pan-cancer, and explore its role and mechanism in HCC. METHODS: The Cancer Genome Atlas, Genotype-Tissue Expression, Gene Expression Profiling Interactive Analysis, and Tumor Immune Estimation Resource databases were used to analyze B56ε expression, prognostic mutations, somatic copy number alterations, and tumor immune characteristics in 33 tumors. The relationships between B56ε expression levels and drug sensitivity, immunotherapy, immune checkpoints, and human leukocyte antigen (HLA)-related genes were further analyzed. Gene Set Enrichment Analysis (GSEA) was performed to reveal the role of B56ε in HCC. The Cell Counting Kit-8, plate cloning, wound healing, and transwell assays were conducted to assess the effects of B56ε interference on the malignant behavior of HCC cells. RESULTS: In most tumors, B56ε expression was upregulated, and high B56ε expression was a risk factor for adrenocortical cancer, HCC, pancreatic adenocarcinoma, and pheochromocytoma and paraganglioma (all P < 0.05). B56ε expression levels were correlated with a variety of immune cells, such as T helper 17 cells, B cells, and macrophages. There was a positive correlation between B56ε expression levels with immune checkpoint genes and HLA-related genes (all P < 0.05). The expression of B56ε was negatively correlated with the sensitivity of most chemotherapy drugs, but a small number showed a positive correlation (all P < 0.05). GSEA analysis showed that B56ε expression was related to the cancer pathway, p53 downstream pathway, and interleukin-mediated signaling in HCC. Knockdown of B56ε expression in HCC cells inhibited the proliferation, migration, and invasion capacity of tumor cells. CONCLUSION: B56ε is associated with the microenvironment, immune evasion, and immune cell infiltration of multiple tumors. B56ε plays an important role in HCC progression, supporting it as a prognostic marker and potential therapeutic target for HCC.

Enhancement of the carboxymethylation of corn starch via induced electric field.

This study aimed to enhance the synthesis of carboxymethyl starch (CMS) by induced electric field (IEF). Corn starch was alkalized, pumped into IEF system, and then reacted with monochloroacetic acid at excitation voltages of 0-400 V. IEF enhanced the carboxymethylation by accelerating the rate of OH- and ClCH2COO- attacking starch particles and slightly intensifying the thermal effect by ~7.1 °C (30 min). Compared with the control (0 V), IEF increased the degree of substitution and reaction efficiency by 0.056-0.148 and 9.37-24.56 %, caused more destruction in starch granular and crystal structure, and thus increased its water solubility, swelling power, and paste transparency. Furthermore, some new crystals were formed during IEF treatment, which enhanced the thermostability of CMS, showing an increase of the maximum decomposition temperature by 16-26 °C. Overall, the results classified that IEF could improve the carboxymethylation and enhance the thermostability of products, which provided guides for the applications of electro-techniques in starch modification involving charged species.

MicroRNA-92a-3p Regulates Retinal Angiogenesis by Targeting SGK3 in Vascular Endothelial Cells.

Purpose: The purpose of this study was to investigate the effects and mechanism of microRNA (miR)-92a-3p in retinal angiogenesis in vitro and in vivo. Methods: The expression of miR-92a-3p was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Agomir-92a-3p was intravitreally injected into the right eye on postnatal day 3 (P3), P5, and P8 in the mice, with the agomir-NC injected left eye as the control. At P7, P9, and P12, immunofluorescence was performed to examine the retinal superficial vascular plexus, deep vascular plexus, proliferation, and apoptosis in retinal vascular endothelial cells (ECs). Human retinal microvascular endothelial cells (HRMECs) were treated with mimic-NC and mimic-92a-3p, then the tube formation, cell migration, and wound healing assays were used to detect the effect of miR-92a-3p on retinal angiogenesis in vitro. Agomir-92a-3p was also intravitreally injected into the right eye of oxygen-induced retinopathy (OIR) mice at P12, with the agomir-NC injected left eye as the control, the neovascularization was observed by retinal flatmount staining with isolectin B4 at P17. Bioinformatics and high-throughput sequencing were performed to identify potential target genes of miR-92a-3p. RT-qPCR and Western blot were carried out to detect the expression of SGK3, p-GSK3ß, GSK3ß, Bcl-xL, and cleaved caspase-3 in the HRMECs and mouse retinas. Results: The overexpression of miR-92a-3p inhibited the development of retinal superficial vascular plexus and deep vascular plexus, decreased the expression of Ki67, and increased the expression of cleaved caspase-3 in isolectin B4-labeled retinal vascular ECs. In vitro, the overexpression of miR-92a-3p markedly suppressed the tube formation, cell migration, and wound healing of cultured ECs. Overexpression of miR-92a-3p inhibited both in vivo and in vitro physiological angiogenesis by downregulating the expression of SGK3, p-GSK3ß/GSK3ß, and Bcl-xL. In addition, agomir-92a-3p inhibited the pathological retinal neovascularization of OIR mice, by targeting SGK3, p-GSK3ß/GSK3ß, and Bcl-xL. Conclusions: The miR-92a-3p could affect retinal angiogenesis by targeting SGK3 pathway, suggesting that miR-92a-3p may be a potential anti-angiogenic factor for retinal vascular disease.

Identification of key miRNAs and genes for mouse retinal development using a linear model.

MicroRNAs (miRNAs) are upstream regulators of gene expression and are involved in several biological processes. The purpose of the present study was to obtain a detailed spatiotemporal miRNA expression profile in mouse retina, to identify one or more miRNAs that are key to mouse retinal development and to investigate the roles and mechanisms of these miRNAs. The miRNA expression pattern of the developing mouse retina was acquired from Locked Nucleic Acid microarrays. Data were processed to identify differentially expressed miRNAs (DE­miRNAs) using the linear model in Python 3.6. Following bioinformatics analysis and reverse transcription­quantitative polymerase chain reaction validation, 8 miRNAs (miR­9­5p, miR­130a­3p, miR­92a­3p, miR­20a­5p, miR­93­5p, miR­9­3p, miR­709 and miR­124) were identified as key DE­miRNAs with low variability during mouse retinal development. Gene Ontology analysis revealed that the target genes of the DE­miRNAs were enriched in cellular metabolic processes. Kyoto Encyclopedia of Genes and Genomes analysis demonstrated that the target genes of the DE­miRNAs were significantly enriched in PI3K/AKT/mTOR, class O of forkhead box transcription factors, mitogen­activated protein kinase (MAPK), neurotrophin and transforming growth factor (TGF)­ß signaling, as well as focal adhesion and the axon guidance pathway. PI3K, AKT, PTEN, MAPK1, Son of Sevenless, sphingosine­1­phosphate receptor 1, BCL­2L11, TGF­ß receptor type 1/2 and integrin α (ITGA)/ITGAB, which are key components of the aforementioned pathways and were revealed to be target genes of several of the DE­miRNAs. The present study used a linear model to identify several DE­miRNAs, as well as their target genes and associated pathways, which may serve crucial roles in mouse retinal development. Therefore, the results obtained in the present study may provide the groundwork for further experiments.

Effect of Choroidal Vessel Density on the Ellipsoid Zone and Visual Function in Retinitis Pigmentosa Using Optical Coherence Tomography Angiography.

Purpose: We evaluate the effect of choroidal vessel density on the residual length of the ellipsoid zone (EZ) and visual function in patients with retinitis pigmentosa (RP) using optical coherence tomography angiography (OCTA). Methods: Fifty-three patients with RP (n = 101 eyes) and 53 normal participants (n = 76 eyes) were enrolled in this study. Patients with RP were assigned to three groups according to their best-corrected visual acuity (BCVA). All patients underwent ophthalmologic examinations, including BCVA, fundus examination performed with a slit-lamp using an indirect 90 diopter (D) lens, OCTA, full-field electroretinogram (ERG), and visual field. The choroidal vessel density in the choriocapillaris-Sattler's layer (DC-S), Haller's layer (DH), horizontal length of the ellipsoid (HEL), and vertical length of the ellipsoid (VEL) were assessed using OCTA and Adobe Photoshop CS3 extended software. Results: A significantly increasing impairment of choroidal vessel density (DC-S and DH) was characterized in the RP groups compared to those of the controls (P < 0.05 for all). The magnitude of the reduction in the DC-S and DH was much easier to identify for more severely impaired BCVA in the RP groups (P < 0.05 for all). The DC-S had the strongest correlation with the HEL, VEL, BCVA, visual field, and b-wave amplitude (r = 0.735, r = 0.753, r = -0.843, r = 0.579, and r = 0.671, respectively). Conclusions: Using noninvasive OCTA, choroidal microcirculation, especially in the small/middle choroidal vessel layers, was a prominent factor affecting the EZ, visual acuity, visual field, and recordable ERG b-wave amplitude of patients with RP. This may provide new insights into the progress mechanism and treatment of RP.

Acquired Nonpigmented Vitreous Cyst Associated With Lattice Degeneration.

A 63-year-old male presented with a round-shaped floater and visual obscuration in the right eye. Clinical evaluation showed a nonpigmented vitreous cyst connected to a lattice degeneration by a stalk. Immunostaining of the vitreous cyst obtained from vitrectomy showed its origin of retinal neuroepithelium. The cyst was formed by continuous vitreous traction, which might tear up the disrupted retina at the area of lattice degeneration. This report added the lattice degeneration to the list of causes for the acquired vitreous cyst. [Ophthalmic Surg Lasers Imaging Retina. 2017;48:856-858.].