3D Microfluidic System for Evaluating Inhibitory Effect of Chinese Herbal Medicine Oldenlandia diffusa on Human Malignant Glioma Invasion Combined with Network Pharmacology Analysis.

OBJECTIVE: To investigate the anti-invasion efficacy of the ethanol extract of Oldenlandia diffusa Will. (EEOD) on a three-dimensional (3D) human malignant glioma (MG) cell invasion and perfusion model based on microfluidic chip culture and the possible mechanism of action of Oldenlandia diffusa Will. (OD). METHODS: The comprehensive pharmacodynamic analysis method in this study was based on microfluidic chip 3D cell perfusion culture technology, and the action mechanism of Chinese medicine (CM) on human MG cells was investigated through network pharmacology analysis. First, the components of EEOD were analyzed by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Then, cell viability and apoptosis were assessed to determine the optimum concentration of EEOD for invasion experiments, and two-dimensional (2D) migration and invasion abilities of U87 and U251 MG cells were evaluated using scratch wound and Transwell assays. The possible mechanism underlying the effects of EEOD on glioma was analyzed through a network pharmacology approach. RESULTS: Thirty-five compounds of EEOD were detected by UPLC-Q-TOF/MS. EEOD suppressed the viability of MG cells, promoted their apoptosis, and inhibited their migratory and invasive potentials (all P<0.05). Network pharmacology analysis showed that OD inhibited the invasion of MG cells by directly regulating MAPK and Wnt pathways through MAPK, EGFR, MYC, GSK3B, and other targets. The anti-invasion effect of OD was also found to be related to the indirect regulation of microtubule cytoskeleton organization. CONCLUSIONS: ]EEOD could inhibit the invasion of human MG cells, and the anti-invasion mechanism of OD might be regulating MAPK and Wnt signaling pathways and microtubule cytoskeleton organization.

Genomic and biological characterization of vB_PvuS_Pm34, a novel lytic bacteriophage that infects Proteus vulgaris.

Proteus phage vB_PvuS_Pm34 (Pm34) isolated from the sewage, is a novel virus specific to Proteus vulgaris. Pm34 belonged to the family Siphovirodae with an icosahedron capsid head and a non-contractile tail. Its genome was 39,558 bp in length with a G + C content of 41.4%. Similarity analysis showed that Pm34 shared low identities of 27.6%-38.4% with any other Proteus phages, but had the 96% high identity with Proteus mirabilis AOUC-001. In the genome of Pm34, 70 open reading frames was deduced and 32 had putative functions including integrase and host lysis proteins. No tRNAs, antibiotic resistance and virulence genes were detected. Pm 34 presented a broad pH (4-8) and good temperature tolerance (<40 °C). This is the first report of the bacteriophage specific to P. vulgaris, which can enrich the knowledge of bacteriophages of Prouteus bacteria and provide the possibility for the alternative treatment of P. vulgaris infection.

Mapping theme trends and knowledge structures for human neural stem cells: a quantitative and co-word biclustering analysis for the 2013-2018 period.

Neural stem cells, which are capable of multi-potential differentiation and self-renewal, have recently been shown to have clinical potential for repairing central nervous system tissue damage. However, the theme trends and knowledge structures for human neural stem cells have not yet been studied bibliometrically. In this study, we retrieved 2742 articles from the PubMed database from 2013 to 2018 using "Neural Stem Cells" as the retrieval word. Co-word analysis was conducted to statistically quantify the characteristics and popular themes of human neural stem cell-related studies. Bibliographic data matrices were generated with the Bibliographic Item Co-Occurrence Matrix Builder. We identified 78 high-frequency Medical Subject Heading (MeSH) terms. A visual matrix was built with the repeated bisection method in gCLUTO software. A social network analysis network was generated with Ucinet 6.0 software and GraphPad Prism 5 software. The analyses demonstrated that in the 6-year period, hot topics were clustered into five categories. As suggested by the constructed strategic diagram, studies related to cytology and physiology were well-developed, whereas those related to neural stem cell applications, tissue engineering, metabolism and cell signaling, and neural stem cell pathology and virology remained immature. Neural stem cell therapy for stroke and Parkinson's disease, the genetics of microRNAs and brain neoplasms, as well as neuroprotective agents, Zika virus, Notch receptor, neural crest and embryonic stem cells were identified as emerging hot spots. These undeveloped themes and popular topics are potential points of focus for new studies on human neural stem cells.

Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin.

Cross-species transmission of viruses from wildlife animal reservoirs poses a marked threat to human and animal health 1 . Bats have been recognized as one of the most important reservoirs for emerging viruses and the transmission of a coronavirus that originated in bats to humans via intermediate hosts was responsible for the high-impact emerging zoonosis, severe acute respiratory syndrome (SARS) 2-10 . Here we provide virological, epidemiological, evolutionary and experimental evidence that a novel HKU2-related bat coronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), is the aetiological agent that was responsible for a large-scale outbreak of fatal disease in pigs in China that has caused the death of 24,693 piglets across four farms. Notably, the outbreak began in Guangdong province in the vicinity of the origin of the SARS pandemic. Furthermore, we identified SADS-related CoVs with 96-98% sequence identity in 9.8% (58 out of 591) of anal swabs collected from bats in Guangdong province during 2013-2016, predominantly in horseshoe bats (Rhinolophus spp.) that are known reservoirs of SARS-related CoVs. We found that there were striking similarities between the SADS and SARS outbreaks in geographical, temporal, ecological and aetiological settings. This study highlights the importance of identifying coronavirus diversity and distribution in bats to mitigate future outbreaks that could threaten livestock, public health and economic growth.

Development of a TaqMan-based real-time RT-PCR assay for the detection of SADS-CoV associated with severe diarrhea disease in pigs.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus which was first reported in southern China in 2017. It can cause severe diarrhea disease in pigs. In order to detect this new emerging virus rapidly and reliably, a TaqMan-based real-time RT-PCR assay was established in this study. Specific primers and probe were designed and synthesized based on the conserved region within the N gene of the viral genome. Results showed that the lowest limit of detection was 3.0 × 101 copies/µL. This approach was specific for SADS-CoV, and there were no cross-reaction observed against other 15 swine viruses. It was 10 times more sensitive than the conventional PCR and gave higher SADS-CoV positive detection rate (70.69%, 123/174) than the conventional PCR (51.15%, 89/174) from clinical samples. These data indicated that the TaqMan-based real-time RT-PCR assay established here was an effective method with high sensitivity, specificity and reproducibility for faster and more accurate detection and quantification of SADS-CoV.

Phylogenetic and Genome Analysis of 17 Novel Senecavirus A Isolates in Guangdong Province, 2017.

Senecavirus A (SVA), an emerging RNA virus, is considered to be associated with porcine idiopathic vesicular disease (PIVD). From February to September 2017, 17 novel SVA strains were isolated from samples with the vesicular disease from Guangdong Province, China. Full-length genomes and individual genes of the 17 new SVA isolates were genetically and phylogentically analyzed. Results showed that complete genomes, VP1, 3C, and 3D genes of these 17 novel SVA isolates revealed 96.5-99.8%, 95.1-99.9%, 95.6-100%, and 96.9-99.7% nucleotides identities, respectively. Phylogenetic analyses based on sequences of full-length genomes, VP1, 3C, and 3D genes indicated that 17 novel SVA isolates separated to three well-defined groups. Meanwhile, phylogenetic analysis for all available Chinese SVA strains demonstrated that 45 Chinese SVA strains clustered into five distinct groups with no significant relationship between strains from different provinces and/or years, including a newly emerging branch in China. This is the first comprehensive study of phylogenetic analysis for all available Chinese SVA strains, indicating the appearance of a new type of SVA strains and the complicated circulations with at least five different types of SVA strains in pigs in China.

Development of a SYBR green-based real-time quantitative PCR assay to detect PCV3 in pigs.

Porcine circovirus 3 (PCV3) has been reported in cases of porcine dermatitis and nephropathy syndrome, reproductive failure, cardiac and multi-systemic inflammation. A SYBR green-based real-time quantitative PCR (qPCR) assay was established in this study to detect PCV3 in 203 clinical samples from suckling piglets affected by congenital tremors in China. The limit of detection (LOD) of PCV3 was 1.73×104 copies/µL for gel electrophoresis and 1.73×102 copies/µL for SYBR green-based real-time qPCR. The melt curve analysis showed a single melt peak at 82.5°C.The intra-assay coefficient of variation was up to 1.83% and the inter-assay coefficient of variation was up to 2.27%. The PCV3 positive detection rate of 203 clinical samples for the SYBR green-based real-time qPCR and the conventional PCR was 86.70% (176/203) and 26.60% (54/203), respectively. Each tissue detected in the SYBR green-based real-time qPCR showed a higher positive rate than that detected in the conventional PCR. These results indicated that the SYBR green-based real-time qPCR assay is a powerful method with high sensitivity, specificity and reproducibility for epidemiological investigations of PCV3.

Dried Rehmannia root protects against glutamate-induced cytotoxity to PC12 cells through energy metabolism-related pathways.

Rehmannia has been shown to be clinically effective in treating neurodegenerative diseases; however, the neuroprotective mechanisms remain unclear. In this study, we established a model of neurodegenerative disease using PC12 cytotoxic injury induced by glutamate. The cells were treated with 20 mM glutamate in the absence or presence of water extracts of dried Rehmannia root of varying concentrations (70%, 50% and 30%). The different concentrations of Rehmannia water extract significantly increased the activity of glutamate-injured cells, reduced the release of lactate dehydrogenase, inhibited apoptosis, increased the concentrations of NADH, NAD and ATP in cells, ameliorated mitochondrial membrane potential, and reduced the levels of light chain 3. Taken together, our findings demonstrate that Rehmannia water extracts exert a cytoprotective effect against glutamate-induced PC12 cell injury via energy metabolism-related pathways.

Re-emerging of porcine respiratory and reproductive syndrome virus (lineage 3) and increased pathogenicity after genomic recombination with vaccine variant.

Porcine reproductive and respiratory syndrome virus (PRRSV) was first reported in China since late 1995 and several variants were further reported in subsequence years, causing huge economic losses to the Chinese swine industry. To date, three major lineages (lineage 3, 5.1 and 8.7) of Type 2 PRRSV were reported in China based on our global genotyping. The present study provides the epidemiology of the PRRSV in South China based on the isolates collected during 2009-2012, indicating three lineages (lineage 3, 5.1 and 8.7) of Type 2 PRRSV were still circulating in this area. Our phylogenetic reconstruction indicated that lineage 3 re-emerged in 2010 formed a huge cluster with closely related to the 2004 isolates from Hong Kong. Furthermore, the inter-lineage genomic recombination between MLV vaccine strain (lineage 5) and a recently re-emerged lineage 3 virus (QYYZ) has also been found in a farm practicing MLV vaccination. Our in vivo experiment comparing the pathogenicity and clinical presentations among currently isolated viruses indicated that pigs infected with recombinant lineage 3 virus (GM2) showed persistent higher fever compared to pigs infected by its wild counterpart (QYYZ). This study enhanced our understanding on potential importance of the recombination of PRRSV along with their evolution.