Novel outer membrane protein involved in cellulose and cellooligosaccharide degradation by Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and -glucosidase activity was distributed on the cell surface of C. hutchinsonii.Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the -glucosidase activity,it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the -glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface -glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii.

A novel locus essential for spreading of Cytophaga hutchinsonii colonies on agar.

Cytophaga hutchinsonii is an aerobic cellulolytic gliding bacterium. The mechanism of its cell motility over surfaces without flagella and type IV pili is not known. In this study, mariner-based transposon mutagenesis was used to identify a new locus CHU_1797 essential for colony spreading on both hard and soft agar surfaces through gliding. CHU_1797 encodes a putative outer membrane protein of 348 amino acids with unknown function, and proteins which have high sequence similarity to CHU_1797 were widespread in the members of the phylum Bacteroidetes. The disruption of CHU_1797 suppressed spreading toward glucose on an agar surface, but had no significant effect on cellulose degradation for cells already in contact with cellulose. SEM observation showed that the mutant cells also regularly arranged on the surface of cellulose fiber similar with that of the wild type strain. These results indicated that the colony spreading ability on agar surfaces was not required for cellulose degradation by C. hutchinsonii. This was the first study focused on the relationship between cell motility and cellulose degradation of C. hutchinsonii.

A new locus affects cell motility, cellulose binding, and degradation by Cytophaga hutchinsonii.

Cytophaga hutchinsonii is a Gram-negative gliding bacterium, which can rapidly degrade crystalline cellulose via a novel strategy without any recognizable processive cellulases. Its mechanism of cellulose binding and degradation is still a mystery. In this study, the mutagenesis of C. hutchinsonii with the mariner-based transposon HimarEm3 and gene complementation with the oriC-based plasmid carrying the antibiotic resistance gene cfxA or tetQ were reported for the first time to provide valuable tools for mutagenesis and genetic manipulation of the bacterium. Mutant A-4 with a transposon mutation in gene CHU_0134, which encodes a putative thiol-disulfide isomerase exhibits defects in cell motility and cellulose degradation. The cellulose binding ability of A-4 was only half of that of the wild-type strain, while the endo-cellulase activity of the cell-free supernatants and on the intact cell surface of A-4 decreased by 40%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins binding to cellulose in the outer membrane showed that most of them were significantly decreased or disappeared in A-4 including some Gld proteins and hypothetical proteins, indicating that these proteins might play an important role in cell motility and cellulose binding and degradation by the bacterium.

Influence of polyethylene terephthalate/ polyamide (PET/PA) bilayer structure on physical and strength-related properties of superpressure angioplasty balloons.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA), including balloon angioplasty, is a standard clinical invasive treatment for coronary artery disease. The coronary lesion with calcification is difficult to dilate and the prevention of balloon failure is especially important. OBJECTIVE: A novel superpressure balloon was fabricated with bilayered structure of polyethylene terephthalate (PET) and PA12 (polyamide). METHODS: The structures of bilayer balloon were adjusted to achieve overall excellent performance. Physicochemical, thermal and mechanical properties of bilayer balloons were characterized by X-ray diffraction, differential scanning calorimeter, hydraulic tester and theoretical simulation. RESULTS: Compared with pure PA12 and PET balloons, PA12 outer layer: PET inner layer balloon with layer ratio of 3:7 shows the highest burst stress and relatively low compliance due to an increase in crystallinity and orientation. CONCLUSIONS: The produced bilayer balloon proved to possess a small folding dimension thanks to its ultrathin bilayer structure, which is good for crossing cramped vessels. We believe these optimally fabricated bilayer balloons are proved to provide attractive opportunities for preparation, performance enhancement, and practical applications of super-pressure balloon catheters and cryoablation balloons, that will significantly promote the development of percutaneous transluminal coronary angioplasty for clinical applications.

Tissue distribution of polystyrene nanoplastics in mice and their entry, transport, and cytotoxicity to GES-1 cells.

With the widespread use of plastics and nanotechnology products, nanoplastics (NPs) have become a potential threat to human health. It is of great practical significance to study and evaluate the distribution of NPs in mice as mammal models and their entry, transport, and cytotoxicity in human cell lines. In this study, we detected the tissue distribution of fluorescent polystyrene nanoplastics (PS-NPs) in mice and assessed their endocytosis, transport pathways, and cytotoxic effects in GES-1 cells. We found that PS-NPs were clearly visible in gastric, intestine, and liver tissues of mice and in GES-1 cells treated with PS-NPs. Entry of PS-NPs into GES-1 cells decreased with the inhibition of caveolae-mediated endocytosis (nystatin), clathrin-mediated endocytosis (chlorpromazine HCl), micropinocytosis (ethyl-isopropyl amiloride), RhoA (CCG-1423), and F-actin polymerization (lantrunculin A). Rac1 inhibitors (NSC 23766) had no significant effect on PS-NPs entering GES-1 cells. F-actin levels significantly decreased in CCG-1423-pretreated GES-1 cells exposed to PS-NPs. GES-1 cell ultrastructural features indicated that internalized PS-NPs can be encapsulated in vesicles, autophagosomes, lysosomes, and lysosomal residues. RhoA, F-actin, RAB7, and LAMP1 levels in PS-NPs-treated GES-1 cells were remarkably up-regulated and the Rab5 level was significantly down-regulated compared to levels in untreated cells. PS-NPs treatment decreased cell proliferation rates and increased cell apoptosis. The formation of autophagosomes and autolysosomes and levels of LC3II increased with the length of PS-NPs treatment. The results indicated that cells regulated endocytosis in response to PS-NPs through the RhoA/F-actin signaling pathway and internalized PS-NPs in the cytoplasm, autophagosomes, or lysosomes produced cytotoxicity. These results illustrate the potential threat of NPs pollution to human health.

Polyethylene microplastics affect the distribution of gut microbiota and inflammation development in mice.

Environmental pollution caused by plastics has become a public health problem. However, the effect of microplastics on gut microbiota, inflammation development and their underlying mechanisms are not well characterized. In the present study, we assessed the effect of exposure to different amounts of polyethylene microplastics (6, 60, and 600 µg/day for 5 consecutive weeks) in a C57BL/6 mice model. Treatment with a high concentration of microplastics increased the numbers of gut microbial species, bacterial abundance, and flora diversity. Feeding groups showed a significant increase in Staphylococcus abundance alongside a significant decrease in Parabacteroides abundance, as compared to the blank (untreated) group. In addition, serum levels of interleukin-1α in all feeding groups were significantly greater than that in the blank group. Of note, treatment with microplastics decreased the percentage of Th17 and Treg cells among CD4+ cells, while no significant difference was observed between the blank and treatment groups with respect to the Th17/Treg cell ratio. The intestine (colon and duodenum) of mice fed high-concentration microplastics showed obvious inflammation and higher TLR4, AP-1, and IRF5 expression. Thus, polyethylene microplastics can induce intestinal dysbacteriosis and inflammation, which provides a theoretical basis for the prevention and treatment of microplastics-related diseases.