Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16.

BACKGROUND: Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. METHODS: Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. RESULTS: The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. CONCLUSIONS: The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

Kapok Fiber: A Natural Biomaterial for Highly Specific and Efficient Enrichment of Sialoglycopeptides.

Cancer development and chronic diseases are associated with the overexpression of sialoglycans terminated to the surface proteins and lipids of cancer cells compared with normal cells. The isolation and detection of sialoglycopeptides from complex peptides mixture still remain challenges due to their low abundance, low ionization, and losses of sialic acid residues and water molecule during analytical processes. In this study, kapok fiber, a natural fiber derived from the kapok tree (Bombax ceiba L.), has shown excellent capability to specifically and efficiently enrich sialoglycopeptides, without losses of sialic acid residues and water molecule from sialoglycans. The main components on the surface of kapok fiber are syringyl and guaiacyl lignins which play an important role in isolating sialoglycopeptides from complex peptide mixtures.

Polyethylene microplastics cooperate with Helicobacter pylori to promote gastric injury and inflammation in mice.

Microplastics provide stable habitats for the colonization and survival of pathogenic microorganisms, and cooperate with microorganisms to pose a potential threat to human health. In this study, polyethylene microplastics (PE-MPs) in artificial gastric juice time-dependently decomposed and broke into small-diameter PE-MP fragments that were more stable than those in an aqueous solution. Helicobacter pylori adhered to the surfaces of the PE-MPs to form a biofilm. The gastric tissues of mice treated with PE-MPs first and mixture of PE-MPs and H. pylori were positive for H. pylori infection in the 10th and 14th weeks after treatment, whereas those infected with H. pylori first and H. pylori alone were positive only in the 14th week after treatment. PE-MPs were visible in the gastric, intestinal, and liver tissues of mice treated with PE-MPs. The average diameter of the PE-MP fragments in the liver was greater than those of fragments that entered the gastric or intestinal tissues, and the average diameter of PE-MPs in the PE-MPs only-treated mice was significantly smaller than those of PE-MPs entering the intestinal tissues of the other groups. The infiltration of inflammatory cells was most serious in the mice treated with the mixture of PE-MPs and H. pylori, or with PE-MPs first and then H. pylori. Of all the groups, the gastric organ index and MPO, IL6, and TNF-α levels were highest in the mice treated with the mixture of PE-MPs and H. pylori. These results indicate that the interaction between PE-MPs and H. pylori contributed to the rapid bacterial colonization of gastric mucosal epithelial cells, improved the efficiency of PE-MP entry into tissues, and promoted gastric injury and inflammation in mice. These findings suggest that microplastics may provide a stable habitat for H. pylori, and act synergistically with H. pylori to pose a potential threat to human health.

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.