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.

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.

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.

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.

Supplementation of xanthophylls increased antioxidant capacity and decreased lipid peroxidation in hens and chicks.

The present study investigated the effects of xanthophyll supplementation on production performance, antioxidant capacity (measured by glutathione peroxidase, superoxide dismutase (SOD), catalase, total antioxidant capacity (T-AOC), and reduced glutathione:oxidised glutathione ratio (GSH:GSSG)) and lipid peroxidation (measured by malondialdehyde (MDA)) in breeding hens and chicks. In Expt 1, 432 hens were fed diets supplemented with 0 (control group), 20 or 40 mg xanthophyll/kg diet. Blood samples were taken at 7, 14, 21, 28 and 35 d of the trial. Liver and jejunal mucosa were sampled at 35 d. Both xanthophyll groups improved serum SOD at 21 and 28 d, serum T-AOC at 21 d and liver T-AOC, and serum GSH:GSSG at 21, 28 and 35 d and liver GSH:GSSG. Xanthophylls also decreased serum MDA at 21 d in hens. Expt 2 was a 2 × 2 factorial design. Male chicks hatched from 0 or 40 mg in ovo xanthophyll/kg diet of hens were fed a diet containing either 0 or 40 mg xanthophyll/kg diet. Liver samples were collected at 0, 7, 14 and 21 d after hatching. Blood samples were also collected at 21 d. In ovo-deposited xanthophylls increased antioxidant capacity and decreased MDA in the liver mainly within 1 week after hatching. Maternal effects gradually vanished during 1-2 weeks after hatching. Dietary xanthophylls increased antioxidant capacity and decreased MDA in the liver and serum mainly from 2 weeks onwards. Data suggested that xanthophyll supplementation enhanced antioxidant capacity and reduced lipid peroxidation in different tissues of hens and chicks.

Molecular characterization and phylogenetic analysis of infectious bursal disease viruses isolated from chicken in South China in 2011.

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus that causes immunosuppressive disease in young chickens. Thousands of cases of IBDV infection are reported each year in South China, and these infections can result in considerable economic losses to the poultry industry. To monitor variations of the virus during the outbreaks, 30 IBDVs were identified from vaccinated chicken flocks from nine provinces in South China in 2011. VP2 fragments from different virus strains were sequenced and analyzed by comparison with the published sequences of IBDV strains from China and around the world. Phylogenetic analysis of hypervariable regions of the VP2 (vVP2) gene showed that 29 of the isolates were very virulent (vv) IBDVs, and were closely related to vvIBDV strains from Europe and Asia. Alignment analysis of the deduced amino acid (aa) sequences of vVP2 showed the 29 vv isolates had high uniformity, indicated low variability and slow evolution of the virus. The non-vvIBDV isolate JX2-11 was associated with higher than expected mortality, and had high deduced aa sequence similarity (99.2 %) with the attenuated vaccine strain B87 (BJ). The present study has demonstrated the continued circulation of IBDV strains in South China, and emphasizes the importance of reinforcing IBDV surveillance.

Characterization of cytotoxicity-related gene expression in response to virulent Marek's disease virus infection in the bursa of Fabricius.

Cell-mediated cytotoxic responses are critical for control of Marek's disease virus (MDV) infection and tumour development. However, the mechanisms of virus clearance mediated by cytotoxic responses in the bursa of Fabricius of chickens during MDV infection are not fully understood. In this study, the host cytotoxic responses during MDV infection in the bursa were investigated by examining the expression of genes in the cell lysis pathways. Partial up-regulation existed in the expression of the important cytolytic molecule granzyme A (GzmA), Fas, NK lysin and DNA repair enzyme Ape1, whereas little or no expression appeared in other cytolytic molecules, including perforin (PFN) and Fas ligand (FasL), and molecules involved in DNA repair and apoptosis in the bursa during MDV infection. These results suggest that less sustained cytotoxic activities are generated in the bursa of MDV-infected chickens. The findings of this study provide a more detailed insight into the host cytotoxic responses to MDV infection.

Complete genome sequence of novel porcine epidemic diarrhea virus strain GD-1 in China.

Porcine epidemic diarrhea virus (PEDV) infection, which causes acute diarrhea and dehydration in suckling piglets, has become a serious problem for the swine industry of China in recent years. In this study, a virulent PEDV strain, GD-1, was obtained from fecal samples from suckling piglets that suffered from severe diarrhea in 2011 in Guangdong, China. Here we describe the complete genome sequence of strain GD-1, which may be helpful in further understanding the molecular epidemiology and genetic diversity of PEDV field isolates in China.

[Microbial ecology analysis of the biofilm from two biological contact oxidation processes with different performance].

This study investigated the performance of one-step aerobic biological oxidation process and anoxic/aerobic two-step biological oxidation process treating modeled river water containing low carbon and rich ammonia. Biofilm microbial ecology was analyzed with multiple molecular technologies including PCR-DGGE, FISH/CLSM and FISH/FCM to investigate the succession of bacteria community and space distribution along with abundance of the main functional bacteria, and to research the micro-influential factors and the mechanism of different biological contact oxidation processes for their performance. Results showed that two-step contact oxidation process achieved higher removal percentage than that of the one-step process, with COD and NH4(+) -N removal enhanced about 10% and 32%-59%, respectively. A much thicker biofilm was obtained by the one-step process compared to the two-step process, and nitrobacterium was mainly distributed in the depth of 180-200 microm and 105-125 microm, respectively. PCR-DGGE results found that the two-step process demonstrated less microbial diversity than that of the one-step process, FISH/FCM results showed that ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) abundance increased in the two-step process with the increase of operation time, while that of the one-step process declined. Experiment results demonstrate that functional partitioning of the anoxic-aerobic two-step biological contact oxidation process could be in favor for harvesting nitrobacteria and other special bacteria in different reactor spaces, which can improve removal efficiency for organics and ammonia finally.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China.

A total of 127 porcine samples were collected from 48 farms in six provinces in south China. The positive rate of porcine epidemic diarrhea virus (PEDV) was 43.0 % (55/127), and the co-infection rate of PEDV and transmissible gastroenteritis virus (TGEV) was 12.0 % (15/127). The partial S gene and complete M gene were amplified from PEDV-positive strains by RT-PCR, cloned, sequenced and compared with each other, as well as with the reference strains in GenBank. Sequence homology results of the partial S gene and complete M gene showed that all south China field PEDV strains had nucleotide (deduced amino acid) sequence identities of 86.7-98.7 % (83.2-99.3 %) and 96.1-100 % (95.0-100%), respectively, with the foreign reference strains reported in GenBank. Phylogenetic analysis of the partial S gene showed that all the south China PEDV strains and two Thailand strains (08UB01 and 08RB07) belong to the same group and differ genetically from European strains and early domestic strains. Phylogenetic analysis of the complete M gene showed that all south China PEDV strains have a close relationship with most of the strains in Korea and Thailand, but differ genetically from the vaccine strain (CV777).

Phylogenetic analysis of hemagglutinin genes of 40 H9N2 subtype avian influenza viruses isolated from poultry in China from 2010 to 2011.

Avian influenza virus (H9N2) infection is a major problem of product performance in poultry worldwide. Vaccination is used to limit spread, but more knowledge is needed on the epidemiology of virus subtypes to improve vaccine design. In this study, 40 H9N2 subtype avian influenza viruses (AIVs) were isolated from vaccinated poultry flocks in China from 2010 to 2011. Hemagglutinin (HA) from different virus strains was sequenced and analyzed. We found that the HA genes of these strains shared nucleotide and deduced amino acid homologies that ranged from 90.1 to 92.9 and 91.4 to 95.0 %, respectively, when compared with vaccine strains. Phylogenetic analysis showed that the strains tested could be divided into two major groups. Group I consisted of 24 strains isolated mainly from Eastern and Central China. Group II consisted of 20 strains isolated from Southern China. The cleavage site within the HA protein contained two basic motifs, PSRSSR↓GLF for group I, and PARSSR↓GLF for group II. Additional potential glycosylation sites were found at amino acid position 295 in the HA1 of the isolates in group I, compared with isolates in group II and the vaccine strains. Furthermore, 38 out of the 40 isolates had a leucine residue at position 216 (aa 226 in H3), which was characteristic of human influenza virus-like receptor specificity. In the present study we found that geographical factors play a significant role in virus evolution, and emphasize the importance of continuing surveillance of H9N2 AIVs in chickens in China.

Supplementation of xanthophylls decreased proinflammatory and increased anti-inflammatory cytokines in hens and chicks.

The present study investigated the effects of xanthophylls (containing 40 % of lutein and 60 % of zeaxanthin) on proinflammatory cytokine (IL-1ß, IL-6, interferon (IFN)-γ and lipopolysaccharide-induced TNF-α factor (LITAF)) and anti-inflammatory cytokine (IL-4 and IL-10) expression of breeding hens and chicks. In Expt 1, a total of 432 hens were fed diets supplemented with 0 (as the control group), 20 or 40 mg/kg xanthophylls (six replicates per treatment). The liver, duodenum, jejunum and ileum were sampled at 35 d of the trial. The results showed that both levels of xanthophyll addition decreased IL-1ß mRNA in the liver and jejunum, IL-6 mRNA in the liver, IFN-γ mRNA in the jejunum and LITAF mRNA in the liver compared to the control group. Expt 2 was a 2 × 2 factorial design. Male chicks hatched from 0 or 40 mg/kg xanthophyll diet of hens were fed a diet containing either 0 or 40 mg/kg xanthophylls. The liver, duodenum, jejunum and ileum were collected at 0, 7, 14 and 21 d after hatching. The results showed that in ovo xanthophylls decreased proinflammatory cytokine expression (IL-1ß, IL-6, IFN-γ and LITAF) in the liver, duodenum, jejunum and ileum and increased anti-inflammatory cytokine expression (IL-4 and IL-10) in the liver, jejunum and ileum mainly at 0-7 d after hatching. In ovo effects gradually vanished and dietary effects began to work during 1-2 weeks after hatching. Dietary xanthophylls modulated proinflammatory cytokines (IL-1ß, IL-6 and IFN-γ) in the liver, duodenum, jejunum and ileum and anti-inflammatory cytokine (IL-10) in the liver and jejunum mainly from 2 weeks onwards. In conclusion, xanthophylls could regulate proinflammatory and anti-inflammatory cytokine expression in different tissues of hens and chicks.

Effects of gene-augmentation on the formation, characteristics and microbial community of 2,4-dichlorophenoxyacetic acid degrading aerobic microbial granules.

Development of 2,4-dichlorophenoxyacetic acid (2,4-D) degrading aerobic granular sludge was conducted in two sequencing batch reactors (SBR) with one bioaugmented with a plasmid pJP4 donor strain Pseudomonas putida SM1443 and the other as a control. Half-matured aerobic granules pre-grown on glucose were used as the starting seeds and a two-stage operation strategy was applied. Granules capable of utilizing 2,4-D (about 500 mg/L) as the sole carbon source was successfully cultivated in both reactors. Gene-augmentation resulted in the enhancement of 2,4-D degradation rates by the percentage of 65-135% for the granules on Day 18, and 6-24% for the granules on Day 105. Transconjugants receiving plasmid pJP4 were established in the granule microbial community after bioaugmentation and persisted till the end of operation. Compared with the control granules, the granules in the bioaugmented reactor demonstrated a better settling ability, larger size, more abundant microbial diversity and stronger tolerance to 2,4-D. The finally obtained granules in the bioaugmented and control reactor had a granule size of around 600 µm and 500 µm, a Shannon-Weaver diversity index (H) of 0.96 and 0.55, respectively. A shift in microbial community was found during the granulation process.

[Bioaugmentation of bioreactors with a pJP4 receiving transconjugant to enhance the removal of 2,4-D].

The paper first investigated horizontal transfer of a conjugative plasmid pJP4 to two pure strains of E. coli DH5alpha and Alcaligenes sp., and a mixed culture of aerobic granular sludge, respectively. With a pJP4 receiving transconjugant Alcaligenes sp. :: pJP4 as the bioaugmented bacteria, bioaugmentation experiments were conducted in an aerobic granular sludge reactor and a biofilm reactor, respectively, to enhance the removal of a recalcitrant compound 2,4-dichlorophenoxyacetic acid(2,4-D). Results showed that pJP4 successfully transferred to E. coli DH5alpha, Alcaligenes sp. and the mixed culture of aerobic granules. For the aerobic granular sludge reactor operated in semi-continuous mode and fed with 2,4-D sole carbon source wastewater, bioaugmentation with Alcaligenes sp. : : pJP4 increased 2,4-D average removal rate significantly with an enhancement of 12% -1 498%. For the biofilm reactor operated in sequence batch mode and fed with mixed carbon sources wastewater, supplementation of the transconjugant reduced system start-up time greatly from 16 d to 5 d. It is a feasible strategy to obtain special degradative transconjugants through gene augmentation and put them into bioreactor as bioaugmentation agent to enhance the removal of some specific pollutants.