NADPH oxidase 4 mediates insulin-stimulated HIF-1α and VEGF expression, and angiogenesis in vitro

Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-β and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis in vitro induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment

TransVCL: Attention-enhanced Video Copy Localization Network with Flexible Supervision

Video copy localization aims to precisely localize all the copied segments within a pair of untrimmed videos in video retrieval applications. Previous methods typically start from frame-to-frame similarity matrix generated by cosine similarity between frame-level features of the input video pair, and then detect and refine the boundaries of copied segments on similarity matrix under temporal constraints. In this paper, we propose TransVCL: an attention-enhanced video copy localization network, which is optimized directly from initial frame-level features and trained end-to-end with three main components: a customized Transformer for feature enhancement, a correlation and softmax layer for similarity matrix generation, and a temporal alignment module for copied segments localization. In contrast to previous methods demanding the handcrafted similarity matrix, TransVCL incorporates long-range temporal information between feature sequence pair using self- and cross- attention layers. With the joint design and optimization of three components, the similarity matrix can be learned to present more discriminative copied patterns, leading to significant improvements over previous methods on segment-level labeled datasets (VCSL and VCDB). Besides the state-of-the-art performance in fully supervised setting, the attention architecture facilitates TransVCL to further exploit unlabeled or simply video-level labeled data. Additional experiments of supplementing video-level labeled datasets including SVD and FIVR reveal the high flexibility of TransVCL from full supervision to semi-supervision (with or without video-level annotation). Code is publicly available at https://github.com/transvcl/TransVCL.Comment: Accepted by the Thirty-Seventh AAAI Conference on Artificial Intelligence(AAAI2023

Learning Segment Similarity and Alignment in Large-Scale Content Based Video Retrieval

With the explosive growth of web videos in recent years, large-scale Content-Based Video Retrieval (CBVR) becomes increasingly essential in video filtering, recommendation, and copyright protection. Segment-level CBVR (S-CBVR) locates the start and end time of similar segments in finer granularity, which is beneficial for user browsing efficiency and infringement detection especially in long video scenarios. The challenge of S-CBVR task is how to achieve high temporal alignment accuracy with efficient computation and low storage consumption. In this paper, we propose a Segment Similarity and Alignment Network (SSAN) in dealing with the challenge which is firstly trained end-to-end in S-CBVR. SSAN is based on two newly proposed modules in video retrieval: (1) An efficient Self-supervised Keyframe Extraction (SKE) module to reduce redundant frame features, (2) A robust Similarity Pattern Detection (SPD) module for temporal alignment. In comparison with uniform frame extraction, SKE not only saves feature storage and search time, but also introduces comparable accuracy and limited extra computation time. In terms of temporal alignment, SPD localizes similar segments with higher accuracy and efficiency than existing deep learning methods. Furthermore, we jointly train SSAN with SKE and SPD and achieve an end-to-end improvement. Meanwhile, the two key modules SKE and SPD can also be effectively inserted into other video retrieval pipelines and gain considerable performance improvements. Experimental results on public datasets show that SSAN can obtain higher alignment accuracy while saving storage and online query computational cost compared to existing methods.Comment: Accepted by ACM MM 202

Engineering human ventricular heart muscles based on a highly efficient system for purification of human pluripotent stem cell-derived ventricular cardiomyocytes

Background Most infarctions occur in the left anterior descending coronary artery and cause myocardium damage of the left ventricle. Although current pluripotent stem cells (PSCs) and directed cardiac differentiation techniques are able to generate fetal-like human cardiomyocytes, isolation of pure ventricular cardiomyocytes has been challenging. For repairing ventricular damage, we aimed to establish a highly efficient purification system to obtain homogeneous ventricular cardiomyocytes and prepare engineered human ventricular heart muscles in a dish. Methods The purification system used TALEN-mediated genomic editing techniques to insert the neomycin or EGFP selection marker directly after the myosin light chain 2 (MYL2) locus in human pluripotent stem cells. Purified early ventricular cardiomyocytes were estimated by immunofluorescence, fluorescence-activated cell sorting, quantitative PCR, microelectrode array, and patch clamp. In subsequent experiments, the mixture of mature MYL2-positive ventricular cardiomyocytes and mesenchymal cells were cocultured with decellularized natural heart matrix. Histological and electrophysiology analyses of the formed tissues were performed 2 weeks later. Results Human ventricular cardiomyocytes were efficiently isolated based on the purification system using G418 or flow cytometry selection. When combined with the decellularized natural heart matrix as the scaffold, functional human ventricular heart muscles were prepared in a dish. Conclusions These engineered human ventricular muscles can be great tools for regenerative therapy of human ventricular damage as well as drug screening and ventricular-specific disease modeling in the future. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0651-x) contains supplementary material, which is available to authorized users

Research on Index System for Disabled Elders Evaluation and Grey Clustering Model Based on End-point Mixed Possibility Functions

The file attached to this record is the Publisher's final version.An operational ability assessment system for older adults is of great help to address health and social challenges for ageing. In this paper, the main problems in currently available ADL and ability evaluation systems have been analyzed. The basic principles to build an index system for disability elders evaluation have been put forwarded. Then，an improved Barthel index system for ADL evaluation and a new older adults ability evaluation system consisted of 4 first-level indexes and 14 secondary indexes based on experts’ opinion and the ability assessment system for older adults by Ministry of Civil Affairs of China have been built. The grey clustering model based on end-point mixed triangular possibility function has been introduced. And three living examples of adults’ disability evaluation have been conducted. It is confirmed clearly that the three older adults belong to different categories of "severe disability", "mild disability", and "ability passable" respectively. The research results can be used as reference for government to formulate the elderly-care policies, to run and allocate the elderly-care resources, as well as reference for various nursing or elderly-care institutions

Molecular Cloning and Expression Analysis of P-Selectin from Zebrafish (Danio rerio)

The glycoprotein P-selectin belongs to the selectin family of cell adhesion molecules. In this study, we cloned the full-length cDNA of P-selectin from zebrafish (Danio rerio) by the method of rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). Zebrafish P-selectin cDNA is 2,800 bp and encodes a putative 868 amino acid protein with a theoretical molecular weight of 122.36 kDa and isoelectric point of 6.27. A signal peptide of 25 amino acids is predicted at the N-terminus of the putative protein. All structural domains involved in P-selectin function are conserved in the putative protein. The amino acid sequence of zebrafish P-selectin is 37% to 39% identical to that of mammalian P-selectins. Real-time quantitative PCR and whole-mount in situ hybridization analysis revealed that P-selectin was expressed in early embryonic development, the expression increased from 0.2 hpf (1-cell stage) to 72 hpf, and the expression significantly upregulated within 30 minutes of ADP induction. The results indicate that the structure of P-selectin protein is highly conserved among species and zebrafish P-selectin plays an important role in early embryonic development and probably has similar biological function to mammalian P-selectins

Efficient Transduction of Vascular Endothelial Cells with Recombinant Adeno-Associated Virus Serotype 1 and 5 Vectors

Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human α1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding β-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies