Automatic Detection Algorithm of Football Events in Videos.

The purpose is to effectively solve the problems of high time cost, low detection accuracy, and difficult standard training samples in video processing. Based on previous investigations, football game videos are taken as research objects, and their shots are segmented to extract the keyframes. The football game videos are divided into different semantic shots using the semantic annotation method. The key events and data in the football videos are analyzed and processed using a combination of artificial rules and a genetic algorithm. Finally, the performance of the proposed model is evaluated and analyzed by using concrete example videos as data sets. Results demonstrate that adding simple artificial rules based on the classic semantic annotation algorithms can save a lot of time and costs while ensuring accuracy. The target events can be extracted and located initially using a unique lens. The model constructed by the genetic algorithm can provide higher accuracy when the training samples are insufficient. The recall and precision of events using the text detection method can reach 96.62% and 98.81%, respectively. Therefore, the proposed model has high video recognition accuracy, which can provide certain research ideas and practical experience for extracting and processing affective information in subsequent videos.

Characterization of a novel roseophage and the morphological and transcriptional response of the sponge symbiont Ruegeria AU67 to infection.

Sponges have recently been recognized to contain complex communities of bacteriophages; however, little is known about how they interact with their bacterial hosts. Here, we isolated a novel phage, called Ruegeria phage Tedan, and characterized its impact on the bacterial sponge symbiont Ruegeria AU67 on a morphological and molecular level. Phage Tedan was structurally, genomically and phylogenetically characterized to be affiliated with the genus Xiamenvirus of the family Siphoviridae. Through microscopic observations and transcriptomic analysis, we show that phage Tedan upon infection induces a process leading to metabolic and morphological changes in its host. These changes would render Ruegeria AU67 better adapted to inhabit the sponge holobiont due to an improved utilization of ecologically relevant energy and carbon sources as well as a potential impediment of phagocytosis by the sponge through cellular enlargement. An increased survival or better growth of the bacterium in the sponge environment will likely benefit the phage reproduction. Our results point towards the possibility that phages from host-associated environments require, and have thus evolved, different strategies to interact with their host when compared to those phages from free-living or planktonic environments.

The modulation of Smac/DIABLO on mitochondrial apoptosis induced by LPS in Crassostrea gigas.

The mitochondrial pathway of apoptosis is well studied as the major mechanism of physiological cell death in vertebrates. In the present study, a second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis-binding protein (IAP) with low pI protein (DIABLO) (designated as CgSmac) was identified from oyster Crassostrea gigas. The open reading frame of CgSmac was of 966 bp nucleotides encoding a predicted polypeptide of 321 amino acids with a conserved Smac/DIABLO domain containing a potential IAP-binding motif of VMPV. CgSmac proteins were distributed in hemocytes and co-localized with mitochondria. Western blotting analysis revealed that CgSmac proteins mainly existed in the dimer form in hemocytes, and the monomeric precursors and mature monomers were also detected. After lipopolysaccharide (LPS) stimulation, the mRNA expression of CgSmac in hemocytes was significantly up-regulated and peaked at 6 h (12.26-fold, p < 0.05), and the protein level of its dimers was significantly up-regulated at 6 h, 12 h, 24 h, and 48 h, while that of CgSmac monomers was up-regulated at 6 h, 12 h and down-regulated at 24 h, 48 h. The decrease of mitochondrial membrane potential indicated that the occurrence of early stage of apoptosis in primary cultured hemocytes was induced by LPS, and RNA interference (RNAi) of CgSmac could not rescue this decrease. The caspase-3 activity in primary cultured hemocytes was significantly suppressed after RNAi of CgSmac. Correspondingly, the total apoptotic rate of primary cultured hemocytes was also significantly suppressed in dsCgSmac + LPS group (31.57%) compared to dsEGFP + LPS group (40.27%, p < 0.05), which in turn demonstrated the conserved pro-apoptotic function of CgSmac. Furthermore, the early apoptotic rate (10.4% vs. 8.5%, p < 0.05) was significantly higher in dsCgSmac + LPS group than that of dsEGFP + LPS group, while the necrosis (7.7% vs. 10.0%, p < 0.05) and late apoptotic rates (13.4% vs. 21.9%, p < 0.05) were lower in dsCgSmac + LPS group than those of dsEGFP + LPS group. Collectively, CgSmac could activate mitochondrial apoptosis pathway by promoting caspase-3 activity in oyster hemocytes against exogenous LPS invasion. These results provided new insights on oyster apoptosis and the immune defense mechanisms in invertebrates.

Molecular characterization of a cathepsin L1 highly expressed in phagocytes of pacific oyster Crassostrea gigas.

Cathepsin L1 (CTSL1) is a lysosomal cysteine protease with a papain-like structure. It is known to be implicated in multiple processes of immune response against pathogen infection based on the proteolytic activity. In the present study, a CTSL1 homologue (designated as CgCTSL1) was identified from Crassostrea gigas. It contained a typically single Pept_C1 domain with three conserved catalytically essential residues (Gln25, His135 and Asn178). The mRNA of CgCTSL1 was ubiquitously expressed in oyster tissues with the highest expression level in important immune tissues such as gill and hemocytes. CgCTSL1 proteins were mainly detected in gill and hepatopancreas by immunohistochemistry. Recombinant CgCTSL1 (rCgCTSL1) exhibited proteolytic activity to cleave the substrate Ac-FR-amino-4-trifluoromethyl coumarin (AFC) in a dose-dependent manner, and the inhibitor could reduce its proteolytic activity. After the interference of CgCTSL1 mRNA, the proteolytic activity of oyster hemocytes was significantly down-regulated with the released AFC fluorescence value decreasing from 375.84 to 179.21 (p < 0.05). Flow cytometry analysis revealed that the expression of CgCTSL1 protein was higher in phagocytes with the mean fluorescence intensity (MFI) value of 21,187 (4.13-fold, p < 0.01) compared to the MFI value of 5,130 in non-phagocytic hemocytes. The further confocal analysis demonstrated that the actively phagocytic hemocytes with green bead signals were co-localized with stronger CgCTSL1 positive signals. The mRNA expression levels of CgCTSL1 in phagocyte-like sub-populations of granulocytes and semi-granulocytes were 298.12-fold (p < 0.01) and 2.75-fold (p < 0.01) of that in agranulocytes, respectively. Western blotting analysis of the hemocyte proteins revealed that CgCTSL1 was relatively abundant in granulocytes and semi-granulocytes compared to that in agranulocytes. These results collectively suggested that CgCTSL1, a CTSL1 homologue highly expressed in phagocyte-like hemocytes, was possibly involved in cellular immune response dependent on its conserved proteolytic activity, which might provide clues for the divergence between phagocytes and non-phagocytic hemocytes as well as the identification of promising molecular markers for phagocytes in oyster C. gigas.

A novel GATA-like zinc finger transcription factor involving in hematopoiesis of Eriocheir sinensis.

GATA transcription factor is a family of DNA-binding proteins that can recognize and bind to sequence of (A/T) GATA (A/G). In the present study, a GATA-like protein (named as EsGLP) was characterized from Eriocheir sinensis, including an 834 bp full length open reading frame of EsGLP, encoding a polypeptide of 277 amino acids. The deduced amino acid sequence of EsGLP contained one conserved GATA-type zinc finger of the form Cys-X2-Cys-X17-Cys-X2-Cys, with four cysteine sites. The EsGLP mRNA transcripts were mainly detected in the hematopoietic tissue, hepatopancreas and gonad. The recombinant EsGLP protein was prepared for the antibody production. The EsGLP protein was mainly distributed in the edge of lobules in the HPT and the cytoplasm of hemocytes. The mRNA transcripts of EsGLP in hemocytes were significantly decreased at 24 h (0.39-fold and 0.27-fold, p < .05) and 48 h (0.35-fold and 0.16-fold, p < .05) after LPS and Aeromonas hydrophila stimulation, respectively. However, one peak of EsGLP mRNA transcripts were recorded at 24 h (8.71-fold, p < .05) in HPT after A. hydrophila stimulation. The expression level of EsGLP mRNA in HPT was significantly up-regulated at 2 h, 2.5 h and 9 h (41.74-fold, 45.38-fold and 26.07-fold, p < .05) after exsanguination stimulation. When EsGLP gene expression was inhibited by the injection of double-stranded RNA, both the total hemocytes counts and the rate of EdU-positive hemocytes were significantly decreased (0.32-fold and 0.56-fold compared to that in control group, p < .05). All these results suggested that EsGLP was an important regulatory factor in E. sinensis which involved in the hemocytes generation and the immune response against invading pathogens.

A serotonin receptor (Cg5-HTR-1) mediating immune response in oyster Crassostrea gigas.

Serotonin receptors, including ligand-gated ion channel (LGICs) and G protein-coupled receptors (GPCR), play vital roles in modulating physiological processes and immunoreaction. In the present study, a homologue of serotonin (5-HT) receptor was identified from oyster Crassostrea gigas (designated Cg5-HTR-1). Its open reading frame (ORF) was of 1239 bp, encoding a polypeptide of 412 amino acids with a seven transmembrane region. Cg5-HTR-1 shared high similarity with the 5-HTRs from other animals. The cAMP contents in HEK293T cells decreased significantly after Cg5-HTR-1 transfection and 5-HT incubation (p < .05), while blocking Cg5-HTR-1 with specific receptor antagonist reversed this downtrend. The intracellular Ca2+ concentrations increased significantly (p < .05) after cell transfection and 5-HT incubation, and the antagonist treatment also arrested this process. Cg5-HTR-1 transcripts were widely distributed in various tissues, with the highest level in hepatopancreas and lowest level in mantle and gill. The mRNA expression of Cg5-HTR-1 in hemocyte increased significantly after lipopolysaccharide (LPS) stimulation and reached the peak level (6.47-fold, p < .05) at 6 h post treatment. The inhibition of Cg5-HTR-1 significantly reduced the expression of tumor necrosis factor (TNF) mRNA in hemocyte, down-regulated the superoxide dismutase (SOD) activity in serum, and induced the apoptosis of hemocyte (p < .05). These results suggested that Cg5-HTR-1 was a novel member of 5-HT1 receptor family and it mediated serotonergic immunomodulation on both cellular and humoral immune responses.

The cyclin-dependent kinase 2 (CDK2) mediates hematopoiesis through G1-to-S transition in Chinese mitten crab Eriocheir sinensis.

Cyclin-dependent kinases (CDKs), a family of cell cycle-related serine/threonine kinases, participate in various biological processes, and play crucial roles in the innate immunity. In the present study, a CDK2 (designed as EsCDK2) with a serine/threonine protein kinase catalytic domain was identified from Chinese mitten crab (Eriocheir sinensis). The full-length cDNA sequence of EsCDK2 was of 2405 bp with an open reading frame (ORF) of 909 bp. EsCDK2 shared 66%-81% sequence similarities with previously identified CDK2s. It was clustered with the CDK2 from Penaeus monodon in the invertebrate branch of the phylogenetic tree. The mRNA transcripts of EsCDK2 were highly expressed in hematopoietic tissue (HPT) and gonad, while lower in hemocytes, heart, gills, and muscle. EsCDK2 protein distributed in both cytoplasm and nucleus of HPT cells. The expression of EsCDK2 mRNA in HPT was significantly up-regulated and peaked at 3 h post stimulations with Aeromonas hydrophila (2.31-fold, p < 0.05) and Lipopolysaccharide (LPS) (2.02-fold, p < 0.05). After exsanguination, the total hemocyte counts (THC) decreased significantly to 0.42 × 107/ml (0.39-fold, p < 0.05) at 0.5 h, then returned to a normal level at 6 h, while the mRNA expression of EsCDK2 in HPT cells was up-regulated at the early phase from 0.5 h to 6 h. After injection of EsCDK2-dsRNA, the mRNA expression level of EsCDK2 in HPT and THC both decreased to 0.53-fold (p < 0.01) and 0.78-fold (p < 0.05) at 24 h, respectively, and the percentage of new-born hemocytes in HPT also decreased significantly from 37.7% to 16.3% (0.43-fold, p < 0.01). After knocking down of EsCDK2, THC decreased dramatically at 6 h (0.65-fold, p < 0.01) post exsanguination, while returned normal at 6 h in PBS group. After interference of EsCDK2 mRNA expression, the percentage of G0-G1 phase cells significantly increased to 85.01% (1.26-fold, p < 0.01), while S phase and G2-M phase cells significantly decreased to 7.92% (0.46-fold, p < 0.01) and 7.07% (0.43-fold, p < 0.01) respectively, indicating that the cell cycle of HPT cells arrested at G1 phase. These results collectively demonstrated that EsCDK2 participated in the regeneration of hemocytes or hematopoiesis by regulating the transition from G1 to S phase in the cell cycle, and involves in the innate immune responses of E. sinensis.

Glycogen synthase kinase-3 (GSK3) regulates TNF production and haemocyte phagocytosis in the immune response of Chinese mitten crab Eriocheir sinensis.

Glycogen synthase kinase-3 (GSK3) is a serine/threonine protein kinase firstly identified as a regulator of glycogen synthesis. Recently, it has been proved to be a key regulator of the immune reaction. In the present study, a GSK3 homolog gene (designated as EsGSK3) was cloned from Chinese mitten crab, Eriocheir sinensis. The open reading frame (ORF) was 1824 bp, which encoded a predicted polypeptide of 607 amino acids. There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3. Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3-ß from oriental river prawn Macrobrachium nipponense in the invertebrate branch, while GSK3s from vertebrates formed the other distinct branch. EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas, eyestalk, muscle, gonad, haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas. And EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis. The expression levels of EsGSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge (p < 0.05) in comparison with control group, and then gradually decreased to the initial level at 48 h (p > 0.05). The mRNA expression of lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (EsLITAF) was also induced by A. hydrophila challenge. However, the mRNA expression of EsLITAF and TNF-α production was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium, while the phagocytosis of crab haemocytes was significantly promoted. These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E. sinensis by promoting TNF-α production and inhibiting haemocyte phagocytosis.