Characterization of a Hyaluronic Acid Utilization Locus and Identification of Two Hyaluronate Lyases in a Marine Bacterium Vibrio alginolyticus LWW-9

Hyaluronic acid (HA) is a negatively charged and linear polysaccharide existing in the tissues and body fluids of all vertebrates. Some pathogenic bacteria target hyaluronic acid for adhesion and/or infection to host cells. Vibrio alginolyticus is an opportunistic pathogen related to infections of humans and marine animals, and the hyaluronic acid-degrading potential of Vibrio spp. has been well-demonstrated. However, little is known about how Vibrio spp. utilize hyaluronic acid. In this study, a marine bacterium V. alginolyticus LWW-9 capable of degrading hyaluronic acid has been isolated. Genetic and bioinformatic analysis showed that V. alginolyticus LWW-9 harbors a gene cluster involved in the degradation, transport, and metabolism of hyaluronic acid. Two novel PL8 family hyaluronate lyases, VaHly8A and VaHly8B, are the key enzymes for the degradation of hyaluronic acid. VaHly8A and VaHly8B have distinct biochemical properties, reflecting the adaptation of the strain to the changing parameters of the aquatic habitats and hosts. Based on genomic and functional analysis, we propose a model for the complete degradation of hyaluronic acid by V. alginolyticus LWW-9. Overall, our study expands our knowledge of the HA utilization paradigm within the Proteobacteria, and the two novel hyaluronate lyases are excellent candidates for industrial applications

XGNN: Towards Model-Level Explanations of Graph Neural Networks

Graphs neural networks (GNNs) learn node features by aggregating and combining neighbor information, which have achieved promising performance on many graph tasks. However, GNNs are mostly treated as black-boxes and lack human intelligible explanations. Thus, they cannot be fully trusted and used in certain application domains if GNN models cannot be explained. In this work, we propose a novel approach, known as XGNN, to interpret GNNs at the model-level. Our approach can provide high-level insights and generic understanding of how GNNs work. In particular, we propose to explain GNNs by training a graph generator so that the generated graph patterns maximize a certain prediction of the model.We formulate the graph generation as a reinforcement learning task, where for each step, the graph generator predicts how to add an edge into the current graph. The graph generator is trained via a policy gradient method based on information from the trained GNNs. In addition, we incorporate several graph rules to encourage the generated graphs to be valid. Experimental results on both synthetic and real-world datasets show that our proposed methods help understand and verify the trained GNNs. Furthermore, our experimental results indicate that the generated graphs can provide guidance on how to improve the trained GNNs

症例の予後改善のための,電子ビームCT,4列～320列CTを用いた循環器領域の新しい臨床診断学の開発への貢献

I went to the Stanford University Department of Radiology\u27s three-dimensional (3D) imaging laboratory from 1996 to 1999 to study a novel 3D image processing technique using electron beam computed tomography (CT). When I returned to Japan, I found that multi-slice CT had been available in daily practice since 1998. We have published a total of 152 peer-reviewed papers on diagnostic images in the field of cardiovascular disease. In 2003, when 16-slice CT was available for use in general hospitals, we successfully developed a prototype 256-slice cone-beam CT at the National Institute of Radiological Sciences. We produced several papers discussing the utilities of this prototype CT in both animal and phantom experiments, the concepts and ideas that were currently used for cardiac perfusion and myocardium characteristic study. In 2010, our paper was used as a reference in the American College of Cardiology Foundation Expert Consensus Guideline. The our current topics presented include coronary artery stenosis, coronary arterial plaques, the characteristics of the myocardium, the anatomy of structural and congenital heart disease, and the cardiac function, all using 16-320 slice CT with reduced radiation exposure in CT acquisition. Furthermore, we are now performing novel clinical CT studies combined magnetic resonance imaging (MRI), positron emission tomography, and echocardiography. Using previous image data, we analyzed an epidemiology study using CT findings to predict the occurrence of major cardiovascular adverse events over long-term follow-up periods of more than 100 months (median), one of the longest follow-up periods documented in the literature. We also need to obtain accurate diagnoses for subjects with cardiac failure or fatal arrhythmia of unknown origin, allowing them to receive specific effective therapy for their possible cardiac amyloidosis, cardiac sarcoidosis, or Fabry\u27s disease. Of course, in all CT imaging techniques used for evaluation and monitoring of cardiovascular risk

Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria

Recently, many small non-coding RNAs (sRNAs) with important regulatory roles have been identified in bacteria. As their eukaryotic counterparts, a major class of bacterial trans-encoded sRNAs acts by basepairing with target mRNAs, resulting in changes in translation and stability of the mRNA. RNA interference (RNAi) has become a powerful gene silencing tool in eukaryotes. However, such an effective RNA silencing tool remains to be developed for prokaryotes. In this study, we described first the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. Based on the common structural characteristics of natural sRNAs in Gram-negative bacteria, we developed the designing principle of atsRNA. Most of the atsRNAs effectively suppressed the expression of exogenous EGFP gene and endogenous uidA gene in Escherichia coli. Further studies demonstrated that the mRNA base pairing region and AU rich Hfq binding site were crucial for the activity of atsRNA. The atsRNA-mediated gene silencing was Hfq dependent. The atsRNAs led to gene silencing and RNase E dependent degradation of target mRNA. We also designed a series of atsRNAs which targeted the toxic genes in Staphyloccocus aureus, but found no significant interfering effect. We established an effective method for specific gene silencing in Gram-negative bacteria

Antibiofilm Activity of an Exopolysaccharide from Marine Bacterium Vibrio sp. QY101

Bacterial exopolysaccharides have always been suggested to play crucial roles in the bacterial initial adhesion and the development of complex architecture in the later stages of bacterial biofilm formation. However, Escherichia coli group II capsular polysaccharide was characterized to exert broad-spectrum biofilm inhibition activity. In this study, we firstly reported that a bacterial exopolysaccharide (A101) not only inhibits biofilm formation of many bacteria but also disrupts established biofilm of some strains. A101 with an average molecular weight of up to 546 KDa, was isolated and purified from the culture supernatant of the marine bacterium Vibrio sp. QY101 by ethanol precipitation, iron-exchange chromatography and gel filtration chromatography. High performance liquid chromatography traces of the hydrolyzed polysaccharides showed that A101 is primarily consisted of galacturonic acid, glucuronic acid, rhamnose and glucosamine. A101 was demonstrated to inhibit biofilm formation by a wide range of Gram-negative and Gram-positive bacteria without antibacterial activity. Furthermore, A101 displayed a significant disruption on the established biofilm produced by Pseudomonas aeruginosa, but not by Staphylococcus aureus. Importantly, A101 increased the aminoglycosides antibiotics' capability of killing P. aeruginosa biofilm. Cell primary attachment to surfaces and intercellular aggregates assays suggested that A101 inhibited cell aggregates of both P. aeruginosa and S. aureus, while the cell-surface interactions inhibition only occurred in S. aureus, and the pre-formed cell aggregates dispersion induced by A101 only occurred in P. aeruginosa. Taken together, these data identify the antibiofilm activity of A101, which may make it potential in the design of new therapeutic strategies for bacterial biofilm-associated infections and limiting biofilm formation on medical indwelling devices. The found of A101 antibiofilm activity may also promote a new recognition about the functions of bacterial exopolysaccharides

YsHyl8A, an Alkalophilic Cold-Adapted Glycosaminoglycan Lyase Cloned from Pathogenic Yersinia sp. 298

A high enzyme-yield strain Yersinia sp. 298 was screened from marine bacteria harvested from the coastal water. The screening conditions were extensive, utilizing hyaluronic acid (HA)/chondroitin sulfate (CS) as the carbon source. A coding gene yshyl8A of the family 8 polysaccharide lyase (PL8) was cloned from the genome of Yersinia sp. 298 and subjected to recombinant expression. The specific activity of the recombinase YsHyl8A was 11.19 U/mg, with an optimal reaction temperature of 40 °C and 50% of its specific activity remaining after thermal incubation at 30 °C for 1 h. In addition, its optimal reaction pH was 7.5, and while it was most stable at pH 6.0 in Na2HPO4-citric acid buffer, it remained highly stable at pH 6.0–11.0. Further, its enzymatic activity was increased five-fold with 0.1 M NaCl. YsHyl8A, as an endo-lyase, can degrade both HA and CS, producing disaccharide end-products. These properties suggested that YsHyl8A possessed both significant alkalophilic and cold-adapted features while being dependent on NaCl, likely resulting from its marine source. Yersinia is a typical fish pathogen, with glycosaminoglycan lyase (GAG lyase) as a potential pathogenic factor, exhibiting strong hyaluronidase and chondroitinase activity. Further research on the pathogenic mechanism of GAG lyase may benefit the prevention and treatment of related diseases

Cloning, Expression, and Characterization of a New Glycosaminoglycan Lyase from Microbacterium sp. H14

Glycosaminoglycan (GAG) lyase is an effective tool for the structural and functional studies of glycosaminoglycans and preparation of functional oligosaccharides. A new GAG lyase from Microbacterium sp. H14 was cloned, expressed, purified, and characterized, with a molecular weight of approximately 85.9 kDa. The deduced lyase HCLaseM belonged to the polysaccharide lyase (PL) family 8. Based on the phylogenetic tree, HCLaseM could not be classified into the existing three subfamilies of this family. HCLaseM showed almost the same enzyme activity towards hyaluronan (HA), chondroitin sulfate A (CS-A), CS-B, CS-C, and CS-D, which was different from reported GAG lyases. HCLaseM exhibited the highest activities to both HA and CS-A at its optimal temperature (35 °C) and pH (pH 7.0). HCLaseM was stable in the range of pH 5.0–8.0 and temperature below 30 °C. The enzyme activity was independent of divalent metal ions and was not obviously affected by most metal ions. HCLaseM is an endo-type enzyme yielding unsaturated disaccharides as the end products. The facilitated diffusion effect of HCLaseM is dose-dependent in animal experiments. These properties make it a candidate for further basic research and application