Identification and Characterization of a Novel Lectin from the Clam Glycymeris yessoensis and Its Functional Characterization under Microbial Stimulation and Environmental Stress

Lectin from the bivalve Glycymeris yessoensis (GYL) was purified by affinity chromatography on porcine stomach mucin–Sepharose. GYL is a dimeric protein with a molecular mass of 36 kDa, as established by SDS-PAGE and MALDI-TOF analysis, consisting of 18 kDa subunits linked by a disulfide bridge. According to circular dichroism data, GYL is a β/α-protein with the predominance of β-structure. GYL preferentially agglutinates enzyme-treated rabbit erythrocytes and recognizes glycoproteins containing O-glycosidically linked glycans, such as porcine stomach mucin (PSM), fetuin, thyroglobulin, and ovalbumin. The amino acid sequences of five segments of GYL were acquired via mass spectrometry. The sequences have no homology with other known lectins. GYL is Ca2+-dependent and stable over a range above a pH of 8 and temperatures up to 20 °C for 30 min. GYL is a pattern recognition receptor, as it binds common pathogen-associated molecular patterns, such as peptidoglycan, LPS, β-1,3-glucan and mannan. GYL possesses a broad microbial-binding spectrum, including Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Vibrio proteolyticus), but not the fungus Candida albicans. Expression levels of GYL in the hemolymph were significantly upregulated after bacterial challenge by V. proteolyticus plus environmental stress (diesel fuel). Results indicate that GYL is probably a new member of the C-type lectin family, and may be involved in the immune response of G. yessoensis to bacterial attack

Spatial Structure of Lectin from the Mussel <i>Mytilus trossulus</i>: In-Sights from Molecular Modelling and Practical Proof

Most proteins have the ability to self-associate into homooligomeric protein complexes, which consist of two or more identical subunits. Today, modern methods of molecular modeling are an integral part of the study of many biologically active molecules. In silico methods are widely used in structure establishing and function and activity prediction of lectins – carbohydrate-binding proteins. Here, we described by computer simulation the spatial organization of lectin isolated from the mantle of the mussel Mytilus trossulus (MTL). It was shown that the dimerization of MTL gives a total of six ligand binding sites that may be important for the manifestation its biological properties. The ability of MTL to form a dimeric and oligomeric structure was confirmed by dynamic light scattering and SDS-PAGE methods

Reconstruction of Equivalent Electrical Sources on Heart Surface

We consider the problem of increasing the informative value of electrocardiographic (ECG) surveys using data from multichannel electrocardiographic leads, that include both recorded electrocardiosignals and the coordinates of the electrodes placed on the surface of the human torso. In this area, we were interested in reconstruction of the surface distribution of the equivalent sources during the cardiac cycle at relatively low hardware cost. In our work, we propose to reconstruct the equivalent electrical sources by numerical methods, based on integral connection between the density of electrical sources and potential in a conductive medium. We consider maps of distributions of equivalent electric sources on the heart surface (HSSM), presenting source distributions in the form of a simple or double electrical layer. We indicate the dynamics of the heart electrical activity by the space-time mapping of equivalent electrical sources in HSSM

Immunomodulatory Properties of Polysaccharides from the Coral Pseudopterogorgia americana in Macrophages

Polysaccharides from marine organisms produce an important regulatory effect on the mammalian immune system. In this study, the immunomodulatory properties of a polysaccharide that was isolated from the coral Pseudopterogorgia americana (PPA) were investigated. PPA increased the expression levels of tumour necrosis factor-&alpha; (TNF-&alpha;), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), but not inducible nitric oxide synthase and nitric oxide, in macrophages. A mechanistic study revealed that PPA activated macrophages through the toll-like receptor-4 and induced the generation of reactive oxygen species (ROS), increased the phosphorylation levels of protein kinase C (PKC)-&alpha;, PKC-&delta; and mitogen-activated protein kinases (MAPK), and activated NF-&kappa;B. The inhibition of ROS and knockdown of PKC-&alpha; reduced PPA-mediated TNF-&alpha; and IL-6 expression; however, the knockdown of PKC-&delta; significantly increased PPA-mediated TNF-&alpha; expression. In addition, the inhibition of c-Jun N-terminal kinase-1/2 and NF-&kappa;B reduced PPA-mediated TNF-&alpha;, IL-6 and COX-2 expression. Furthermore, the inhibition of ROS, MAPK and PKC-&alpha;/&delta; reduced PPA-mediated NF-&kappa;B activation, indicating that ROS, MAPK and PKC-&alpha;/&delta; function as upstream signals of NF-&kappa;B. Finally, PPA treatment decreased the phagocytosis activity of macrophages and reduced cytokine expression in bacteria-infected macrophages. Taken together, our current findings suggest that PPA can potentially play a role in the development of immune modulators in the future

Molecular Cloning and Characteristics of a Lectin from the Bivalve Glycymeris yessoensis

C-type lectins (CTLs) are a family of carbohydrate-binding proteins that mediate multiple biological events, including adhesion between cells, the turnover of serum glycoproteins, and innate immune system reactions to prospective invaders. Here, we describe the cDNA cloning of lectin from the bivalve Glycymeris yessoensis (GYL), which encodes 161 amino acids and the C-type carbohydrate recognition domain (CRD) with EPN and WND motifs. The deduced amino acid sequence showed similarity to other CTLs. GYL is a glycoprotein containing two N-glycosylation sites per subunit. N-glycans are made up of xylose, mannose, D-glucosamine, 3-O-methylated galactose, D-quinovoses, and 3-O-methylated 6-deoxy-D-glucose. The potential CRD tertiary structure of the GYL adopted CTL-typical long-form double-loop structure and included three disulfide bridges at the bases of the loops. Additionally, when confirming the GYL sequence, eight isoforms of this lectin were identified. This fact indicates the presence of a multigene family of GYL-like C-type lectins in the bivalve G. yessoensis. Using the glycan microarray approach, natural carbohydrate ligands were established, and the glycotope for GYL was reconstructed as &ldquo;Gal&beta;1&ndash;4GlcNAc&beta; obligatory containing an additional fragment&rdquo;, like a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues

Molecular Cloning and Characteristics of a Lectin from the Bivalve <i>Glycymeris yessoensis</i>

C-type lectins (CTLs) are a family of carbohydrate-binding proteins that mediate multiple biological events, including adhesion between cells, the turnover of serum glycoproteins, and innate immune system reactions to prospective invaders. Here, we describe the cDNA cloning of lectin from the bivalve Glycymeris yessoensis (GYL), which encodes 161 amino acids and the C-type carbohydrate recognition domain (CRD) with EPN and WND motifs. The deduced amino acid sequence showed similarity to other CTLs. GYL is a glycoprotein containing two N-glycosylation sites per subunit. N-glycans are made up of xylose, mannose, D-glucosamine, 3-O-methylated galactose, D-quinovoses, and 3-O-methylated 6-deoxy-D-glucose. The potential CRD tertiary structure of the GYL adopted CTL-typical long-form double-loop structure and included three disulfide bridges at the bases of the loops. Additionally, when confirming the GYL sequence, eight isoforms of this lectin were identified. This fact indicates the presence of a multigene family of GYL-like C-type lectins in the bivalve G. yessoensis. Using the glycan microarray approach, natural carbohydrate ligands were established, and the glycotope for GYL was reconstructed as “Galβ1–4GlcNAcβ obligatory containing an additional fragment”, like a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues

Ginsenoside M1 Induces Apoptosis and Inhibits the Migration of Human Oral Cancer Cells

Oral squamous cell carcinoma (OSCC) accounts for 5.8% of all malignancies in Taiwan, and the incidence of OSCC is on the rise. OSCC is also a common malignancy worldwide, and the five-year survival rate remains poor. Therefore, new and effective treatments are needed to control OSCC. In the present study, we prepared ginsenoside M1 (20-O-beta-d-glucopyranosyl-20(S)-protopanaxadiol), a major deglycosylated metabolite of ginsenoside, through the biotransformation of Panax notoginseng leaves by the fungus SP-LSL-002. We investigated the anti-OSCC activity and associated mechanisms of ginsenoside M1 in vitro and in vivo. We demonstrated that ginsenoside M1 dose-dependently inhibited the viability of human OSCC SAS and OEC-M1 cells. To gain further insight into the mode of action of ginsenoside M1, we demonstrated that ginsenoside M1 increased the expression levels of Bak, Bad, and p53 and induced apoptotic DNA breaks, G1 phase arrest, PI/Annexin V double-positive staining, and caspase-3/9 activation. In addition, we demonstrated that ginsenoside M1 dose-dependently inhibited the colony formation and migration ability of SAS and OEC-M1 cells and reduced the expression of metastasis-related protein vimentin. Furthermore, oral administration or subcutaneous injection of ginsenoside M1 significantly reduced tumor growth in SAS xenograft mice. These results indicate that ginsenoside M1 can be translated into a potential therapeutic against OSCC

Improvement of the Model of Object Recognition in Aero Photographs Using Deep Convolutional Neural Networks

Detection and recognition of objects in images is the main problem to be solved by computer vision systems. As part of solving this problem, the model of object recognition in aerial photographs taken from unmanned aerial vehicles has been improved. A study of object recognition in aerial photographs using deep convolutional neural networks has been carried out. Analysis of possible implementations showed that the AlexNet 2012 model (Canada) trained on the ImageNet image set (China) is most suitable for this problem solution. This model was used as a basic one. The object recognition error for this model with the use of the ImageNet test set of images amounted to 15 %. To solve the problem of improving the effectiveness of object recognition in aerial photographs for 10 classes of images, the final fully connected layer was modified by rejection from 1,000 to 10 neurons and additional two-stage training of the resulting model. Additional training was carried out with a set of images prepared from aerial photographs at stage 1 and with a set of VisDrone 2021 (China) images at stage 2. Optimal training parameters were selected: speed (step) (0.0001), number of epochs (100). As a result, a new model under the proposed name of AlexVisDrone was obtained. The effectiveness of the proposed model was checked with a test set of 100 images for each class (the total number of classes was 10). Accuracy and sensitivity were chosen as the main indicators of the model effectiveness. As a result, an increase in recognition accuracy from 7 % (for images from aerial photographs) to 9 % (for the VisDrone 2021 set) was obtained which has indicated that the choice of neural network architecture and training parameters was correct. The use of the proposed model makes it possible to automate the process of object recognition in aerial photographs. In the future, it is advisable to use this model at ground stations of unmanned aerial vehicle complex control when processing aerial photographs taken from unmanned aerial vehicles, in robotic systems, in video surveillance complexes and when designing unmanned vehicle system

The Unusual Lipid A Structure and Immunoinhibitory Activity of LPS from Marine Bacteria Echinicola pacifica KMM 6172T and Echinicola vietnamensis KMM 6221T

Gram-negative bacteria experiencing marine habitats are constantly exposed to stressful conditions dictating their survival and proliferation. In response to these selective pressures, marine microorganisms adapt their membrane system to ensure protection and dynamicity in order to face the highly mutable sea environments. As an integral part of the Gram-negative outer membrane, structural modifications are commonly observed in the lipopolysaccharide (LPS) molecule; these mainly involve its glycolipid portion, i.e., the lipid A, mostly with regard to fatty acid content, to counterbalance the alterations caused by chemical and physical agents. As a consequence, unusual structural chemical features are frequently encountered in the lipid A of marine bacteria. By a combination of data attained from chemical, MALDI-TOF mass spectrometry (MS), and MS/MS analyses, here, we describe the structural characterization of the lipid A isolated from two marine bacteria of the Echinicola genus, i.e., E. pacifica KMM 6172T and E. vietnamensis KMM 6221T. This study showed for both strains a complex blend of mono-phosphorylated tri- and tetra-acylated lipid A species carrying an additional sugar moiety, a d-galacturonic acid, on the glucosamine backbone. The unusual chemical structures are reflected in a molecule that only scantly activates the immune response upon its binding to the LPS innate immunity receptor, the TLR4-MD-2 complex. Strikingly, both LPS potently inhibited the toxic effects of proinflammatory Salmonella LPS on human TLR4/MD-2