Distinct tomato yellow leaf curl Chuxiong virus isolated from whiteflies and plants in China and its symptom determinant and suppressor of post-transcriptional gene silencing.

A begomovirus isolated from whiteflies (Bemisia tabaci) and tomato, sweet potato in China was found to be representative of a distinct begomovirus species, for which the name tomato yellow leaf curl Chuxiong virus (TYLCCxV) is proposed. The results of genomic identification and sequence comparison showed that TYLCCxV shares the highest complete nucleotide sequence identity (88.3%) with croton yellow vein mosaic virus (CroYVMV), and may have originated from the recombination between synedrella leaf curl virus (SyLCV) and squash leaf curl Yunnan virus (SLCuYV). Agrobacterium-mediated inoculation showed that TYLCCxV is highly infectious for a range of plant species, producing upward leaf curling, leaf crumpling, chlorosis, distortion, and stunt symptoms in Solanum lycopersicum plants. The results of Southern blot indicated that TYLCCxV is capable of efficiently replicating two heterologous betasatellites. The inoculation of PVX::C4 on Nicotiana benthamiana induced upward leaf curling and stem elongation symptoms, suggesting that TYLCCxV C4 functions as a symptom determinant. TYLCCxV V2 is an important virulence factor that induces downward leaf curling symptoms, elicits systemic necrosis, and suppresses local and systemic GFP silencing in co-agroinfiltrated N. benthamiana and transgenic 16c plants. Considering the multifunctional virulence proteins V2 and C4, the possibility of TYLCCxV causing devastating epidemics on tomato in China is discussed.

Effects and mechanisms of Zhizi Chuanxiong herb pair against atherosclerosis: an integration of network pharmacology, molecular docking, and experimental validation.

BACKGROUND: The Zhizi Chuanxiong herb pair (ZCHP) can delay pathological progression of atherosclerosis (AS); however, its pharmacological mechanism remains unclear because of its complex components. The purpose of current study is to systematically investigate the anti-AS mechanism of ZCHP. METHODS: The databases of TCMSP, STITCH, SwissTargetPrediction, BATMAN-TCM, and ETCM were searched to predict the potential targets of ZCHP components. Disease targets associated with AS was retrieved from the GEO database. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analyses were executed using DAVID 6.8. Molecular docking method was employed to evaluate the core target binding to blood components, and animal experiments were performed to test action mechanism. RESULTS: A ZCHP-components-targets-AS network was constructed by using Cytoscape, included 11 main components and 52 candidate targets. Crucial genes were shown in the protein-protein interaction network, including TNF, IL-1ß, IGF1, MMP9, COL1A1, CCR5, HMOX1, PTGS1, SELE, and SYK. KEGG enrichment illustrated that the NF-κB, Fc epsilon RI, and TNF signaling pathways were important for AS treatment. These results were validated by molecular docking. In ApoE-/- mice, ZCHP significantly reduced intima-media thickness, pulse wave velocity, plaque area, and serum lipid levels while increasing the difference between the end-diastolic and end-systolic diameters. Furthermore, ZCHP significantly decreased the mRNA and protein levels of TNF-α and IL-1ß, suppressed NF-κB activation, and inhibited the M1 macrophage polarization marker CD86 in ApoE-/- mice. CONCLUSION: This study combining network pharmacology, molecular biology, and animal experiments showed that ZCHP can alleviate AS by suppressing the TNF/NF-κB axis and M1 macrophage polarization.

A Randomized Controlled Trial on the Efficacy of Xinnaoning Capsule in the Treatment of CSAP Complicated With Qi Stagnation and Blood Stasis Syndrome.

Objective: This study was designed to explore the efficacy and safety of Xinnaoning Capsule (XNNC) in the treatment of patients with chronic stable angina pectoris (CSAP) complicated with Qi stagnation and blood stasis syndrome. Methods: A total of 240 patients with CSAP complicated with Qi stagnation and blood stasis syndrome who met the inclusion criteria were enrolled from 8 medical centers across China. The trial treatment lasted 14 weeks, including a 2-week lead-in period and a 12-week double-blind treatment period. Patients in the experimental group were treated with XNNC, while patients in the control group were treated with placebos. Thereafter, examinations were conducted on the efficacy of angina pectoris before and after treatment and the relief of symptoms, followed by the recording of grading changes in angina severity, changes in the number of angina pectoris, and the amount of taken nitroglycerin. Finally, adverse events were assessed. Results: Compared with the control group, the total score and the effective rate of angina pectoris were significantly increased in the experimental group, accompanied by the statistically significant improvement in the severity of angina pectoris (all p < 0.05). Furthermore, there was no statistically significant difference in the adverse events and serious adverse events between the experimental group and the control group (p = 1.0000) before and after treatment. Conclusion: XNNC is a safe and effective medicine for patients with CSAP complicated with Qi stagnation and blood stasis syndrome.

Enrichment of Wee1/CDC2 and NF-κB Signaling Pathway Constituents Mutually Contributes to CDDP Resistance in Human Osteosarcoma.

PURPOSE: Osteosarcoma (OS) universally exhibits heterogeneity and cisplatin (CDDP) resistance. Although the Wee1/CDC2 and nuclear factor кB (NF-κB) pathways were reported to show abnormal activation in some tumor cells with CDDP resistance, whether there is any concrete connection is currently unclear. We explored it in human OS cells. MATERIALS AND METHODS: Multiple OS cell lines were exposed to a Wee1 inhibitor (AZD1775) and CDDP to assess the half-maximal inhibitory concentration values. Western blot, coimmunoprecipitation, confocal immunofluorescence, cell cycle, and Cell Counting Kit-8assays were performed to explore the connection between the Wee1/CDC2 and NF-κB pathways and their subsequent physiological contribution to CDDP resistance. Finally, CDDP-resistant PDX-OS xenograft models were established to confirm that AZD1775 restores the antitumor effects of CDDP. RESULTS: A sensitivity hierarchy of OS cells to CDDP and AZD1775 exists. In the highly CDDP-tolerant cell lines, Wee1 and RelA were physically crosslinked, which resulted in increased abundance of phosphorylated CDC2 (Y15) and RelA (S536) and consequent modulation of cell cycle progression, survival, and proliferation. Wee1 inhibition restored the effects of CDDP on these processes in CDDP-resistant OS cells. In addition, animal experiments with CDDP-resistant PDX-OS cells showed that AZD1775 combined with CDDP not only restored CDDP efficacy but also amplified AZD1775 in inhibiting tumor growth and prolonged the median survival of the mice. CONCLUSION: Simultaneous enrichment of molecules in the Wee1/CDC2 and NF-κB pathways and their consequent coactivation is a new molecular mechanism of CDDP resistance in OS cells. OS with this molecular signature may respond well to Wee1 inhibition as an alternative treatment strategy.

PLK2 modulation of enriched TAp73 affects osteogenic differentiation and prognosis in human osteosarcoma.

There are three subtypes of undifferentiated human conventional osteosarcoma (HCOS): osteoblastic osteosarcoma (OOS), chondroblastic osteosarcoma (COS), and fibroblastic osteosarcoma (FOS). HCOS also exhibits heterogeneous pathological maldifferentiation in individual patients. Currently, the mechanism regulating HCOS differentiation remains unclear, and therapies are ineffective. Osteopontin (OPN) and osteocalcin (OCN) are markers of osteoblast maturation, and their expression is inhibited in HCOS. A previous study found that PLK2 inhibited TAp73 phosphorylation and consequent anti-OS function of TAp73 in OS cells with enriched TAp73. TAp73 was also reported to regulate bone cell calcification. Here, OOS was found to have higher TAp73 levels and PLK2 expression than those in COS, which is correlated with HCOS maldifferentiation according to Spearman analysis and affects patient prognosis according to Kaplan-Meier survival analysis. In the conventional OS cell-line Saos2 and in patient-derived xenograft OS (PDX-OS) cells, increased PLK2 expression owing to abundant TAp73 levels affected OPN and OCN content as measured by RT-PCR and Western blotting, and alizarin red staining showed that PLK2 affected calcium deposition in OS cells. In addition, PLK2 inhibition in PDX-OS cells prohibited clone formation, as indicated by a clonogenic assay, and sensitized OS cells to cisplatin (CDDP) (which consequently limited proliferation), as shown by the CCK-8 assay. In an established PDX animal model with abundant TAp73 levels, PLK2 inhibition or CDDP treatment prevented tumor growth and prolonged median survival. The combined therapeutic effect of PLK2 inhibition with CDDP treatment was better than that of either monotherapy. These results indicate that increased PLK2 levels due to enriched TAp73 affect osteogenic differentiation and maturation and OS prognosis. In conclusion, PLK2 is a potential target for differentiation therapy of OS with enriched TAp73.

Efficacy and safety of Xinnaoning capsule in treating chronic stable angina (qi stagnation and blood stasis syndrome): Study protocol for a multicenter, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Chronic stable angina (CSA) is a cardiovascular disease with high prevalence. At present, drug treatment is still the main measure of stable angina pectoris. Traditional Chinese medicine has a long history in the treatment of CSA. Qi stagnation and Blood stasis syndrome is a common syndrome of CSA. Xinnaoning (XNN) capsule is considered as an effective adjuvant treatment for CSA with the efficacy of promoting qi and blood circulation but lack of high-quality clinical evidence. The purpose of this study is to evaluate the efficacy and safety of XNN capsule compared with placebo by clinical trial. METHODS: This multicenter, randomized, double-blind, placebo-controlled trial will be conducted with a total of 240 participants diagnosed with chronic stable angina (qi stagnation and blood stasis syndrome). The participants will be randomized (1:1) into groups receiving either XNN or placebo for 12 weeks. After a 2-week run-in period, they will receive either XNN or placebo (3 pills, 3 times daily) for 12 weeks on the basis of conventional therapy. The primary outcomes include changes in the integral scores of angina symptoms. The secondary outcome measures include changes in the total score of traditional Chinese medicine syndrome, severity grading of angina pectoris, the number of angina pectoris per week, nitroglycerin dosage, score of seattle angina scale, serum homocysteine, incidence of cardiovascular events. Safety outcomes will also be assessed. Adverse events will be monitored throughout the trial. RESULTS: This study will investigate whether XNN capsule can alleviate clinical symptoms, and improve quality of life of patients with chronic stable angina (qi stagnation and blood stasis syndrome). The results of this study will provide clinical evidence for the application of XNN capsule in the treatment of chronic stable angina. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03914131.

Effects of Zhizi Chuanxiong Capsule () on the Abnormal Methylation in Rabbits with Atherosclerosis.

OBJECTIVE: To investigate the effects of Zhizi Chuanxiong Capsule (ZCC, ) on abnormal DNA methylation in a rabbit model of atherosclerosis (AS). METHODS: After 1 week of adaptive feeding, 48 New Zealand white rabbits were randomly divided into 4 groups: a control group (n=12) fed with normal diet for 22 weeks; a model group (n=12) fed with high fat diet for 14 weeks followed by 8 weeks of normal diet feeding; a low-dose ZCC group (n=12) fed with high fat diet and low-dose ZCC for 14 weeks, followed by 8 weeks of normal diet and low-dose drug; a high-dose ZCC group (n=12) fed with high fat diet and high-dose drug for 14 weeks, followed by 8 weeks of normal diet and high-dose drug. After 22 weeks of feeding, blood samples were taken from the rabbit ear vein, and the genomic DNA was extracted for methylation immunoprecipitation sequencing (Medip-seq). The aorta tissues were collected for hematoxylin-eosin (HE) staining. RESULTS: Eight rabbits died during the feeding process. HE staining showed that the size of the lipid deposition on vessel wall and atherosclerotic plaque formation were reduced in both low- and high-dose group. The Medip-seq results showed that there were 146 abnormally methylated genes (including both hypermethylated gene and hypomethylated genes) in the model group, compared with the control group. Gene Ontology (GO) and Pathway analysis showed that these abnormally methylated genes were found to be involved in multiple AS-related functions and pathways, such as protein kinase C activity, cholesterol transport, mitogen-activated protein kinase (MAPK) signaling pathway, peroxisome proliferater-activated receptor signaling pathway, vascular smooth muscle contraction, inflammation and so on. The abnormal methylated genes in AS model group were altered in both low- and high-dose groups: low-dose ZCC could change 72 of the 146 abnormally methylated genes, high-dose ZCC could change 71. Through GO and Pathway analysis, these altered methylated genes were involved in protein kinase C activity, inflammatory pathway, MAPK signaling pathway, vascular endothelial growth factor signaling pathway, etc. CONCLUSION: ZCC could treat AS through regulating the abnormal hypermethylated and hypomethylated genes in AS rabbit model.

Identification and characterization of a maize-associated mastrevirus in China by deep sequencing small RNA populations.

BACKGROUND: Maize streak Reunion virus (MSRV) is a member of the Mastrevirus genus in the family Geminiviridae. Of the diverse and increasing number of mastrevirus species found so far, only Wheat dwarf virus and Sweetpotato symptomless virus 1 have been discovered in China. Recently, a novel, unbiased approach based on deep sequencing of small interfering RNAs followed by de novo assembly of siRNA, has greatly offered opportunities for plant virus identification. METHODS: Samples collected from maize leaves was deep sequencing for virus identification. Subsequently, the assay of PCR, rolling circle amplification and Southern blot were used to confirm the presence of a mastrevirus. RESULTS: Maize streak Reunion virus Yunnan isolate (MSRV-[China:Yunnan 06:2014], abbreviated to MSRV-YN) was identified from maize collected from Yunnan Province, China, by small RNA deep sequencing. The complete genome of this virus was ascertained as 2,880 nucleotides long by conventional sequencing. A phylogenetic analysis showed it shared 96.3 % nucleotide sequence identity with the isolate of Maize streak Reunion virus from La Reunion Island. To our knowledge, this is the first identification of MSRV in China. Analyses of the viral derived small interfering RNAs (vsiRNAs) profile showed that the most abundant MSRV-YN vsiRNAs were 21, 22 and 24 nt long and biased for A and G at their 5' terminal residue. There was a slightly higher representation of MSRV-YN siRNAs derived from the virion-sense strand genome than the complementary-sense strand genome. Moreover, MSRV-YN vsiRNAs were not uniformly distributed along the genome, and hotspots were detected in the movement protein and coat protein-coding region. CONCLUSIONS: A mastrevirus MSRV-YN collected in Yunnan Province, China, was identified by small RNA deep sequencing. This vsiRNAs profile derived from MSRV-YN was characterized, which might contribute to get an insight into the host RNA silencing defense induced by MSRV-YN, and provide guidelines on designing antiviral strategies using RNAi against MSRV-YN.

Chloroquine enhances replication of influenza A virus A/WSN/33 (H1N1) in dose-, time-, and MOI-dependent manners in human lung epithelial cells A549.

Anti-malaria drug, chloroquine, has been reported to be effective against influenza A virus (IAV) in vitro and used in in-vivo experiments and clinical trial for prevention or treatment of influenza. In this study, it has been shown by immunofluorescence, hemagglutination, and plaque assays that chloroquine enhanced A/WSN/33 (H1N1) replication with pronounced cytopathic effect in dose-, time-, and MOI-dependent manners in human lung epithelial cells A549. Time-of-addition assay showed that inhibitory effect on virus replication by chloroquine pre-treatment was indistinctive, and virus productions were enhanced when the drug was applied after viral adsorption. The effectiveness of chloroquine as an anti-influenza drug is questioned, and caution in its use is recommended.

PI3K/Akt signaling pathway modulates influenza virus induced mouse alveolar macrophage polarization to M1/M2b.

Macrophages polarized to M1 (pro-inflammation) or M2 (anti-inflammation) phenotypes in response to environmental signals. In this study, we examined the polarization of alveolar macrophage (AM), following induction by different influenza virus strains (ST169 (H1N1), ST602 (H3N2) and HKG9 (H9N2)). Macrophages from other tissues or cell line exert alternative responding pattern, and AM is necessary for investigating the respiratory system. AM polarized toward the M1 phenotype after 4 hours of infection by all three virus strains, and AM to presented M2b phenotype after 8 hours induction, and immunosuppressive phenotype after 24 hours of induction. Protein expression assay showed similar results as the gene expression analysis for phenotype verification. The ELISA assay showed that TNF-α secretion was up-regulated after 4 and 8 hours of infection by influenza viruses, and it returned to basal levels after 24 hours of infection. IL-10 expression was elevated after 8 and 24 hours of infection. Immunofluorescence showed that iNOS expression was up-regulated but not Arg1 expression. Influenza virus notably increased phospho-Akt but not phospho-Erk1/2 or phospho-p38, and the AM polarization pattern have been changed by LY294002 (PI3K inhibitor). In conclusion, our results demonstrate the dynamic polarization of AM induced by influenza viruses, and suggested that PI3K/Akt signaling pathway modulates AM polarization to M1/M2b.

Identification of 23-(s)-2-amino-3-phenylpropanoyl-silybin as an antiviral agent for influenza A virus infection in vitro and in vivo.

It has been reported that autophagy is involved in the replication of many viruses. In this study, we screened 89 medicinal plants, using an assay based on the inhibition of the formation of the Atg12-Atg5/Atg16 heterotrimer, an important regulator of autophagy, and selected Silybum marianum L. for further study. An antiviral assay indicated that silybin (S0), the major active compound of S. marianum L., can inhibit influenza A virus (IAV) infection. We later synthesized 5 silybin derivatives (S1 through S5) and found that 23-(S)-2-amino-3-phenylpropanoyl-silybin (S3) had the best activity. When we compared the polarities of the substituent groups, we found that the hydrophobicity of the substituent groups was positively correlated with their activities. We further studied the mechanisms of action of these compounds and determined that S0 and S3 also inhibited both the formation of the Atg12-Atg5/Atg16 heterotrimer and the elevated autophagy induced by IAV infection. In addition, we found that S0 and S3 could inhibit several components induced by IAV infection, including oxidative stress, the activation of extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and IκB kinase (IKK) pathways, and the expression of autophagic genes, especially Atg7 and Atg3. All of these components have been reported to be related to the formation of the Atg12-Atg5/Atg16 heterotrimer, which might validate our screening strategy. Finally, we demonstrated that S3 can significantly reduce influenza virus replication and the associated mortality in infected mice. In conclusion, we identified 23-(S)-2-amino-3-phenylpropanoyl-silybin as a promising inhibitor of IAV infection.

Inability of NS1 protein from an H5N1 influenza virus to activate PI3K/Akt signaling pathway correlates to the enhanced virus replication upon PI3K inhibition.

BACKGROUND: Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, activated during influenza A virus infection, can promote viral replication via multiple mechanisms. Direct binding of NS1 protein to p85ß subunit of PI3K is required for activation of PI3K/Akt signaling. Binding and subsequent activation of PI3K is believed to be a conserved character of influenza A virus NS1 protein. Sequence variation of NS1 proteins in different influenza A viruses led us to investigate possible deviation from the conservativeness. RESULTS: In the present study, NS1 proteins from four different influenza A virus subtypes/strains were tested for their ability to bind p85ß subunit of PI3K and to activate PI3K/Akt. All NS1 proteins efficiently bound to p85ß and activated PI3K/Akt, with the exception of NS1 protein from an H5N1 virus (A/Chicken/Guangdong/1/05, abbreviated as GD05), which bound to p85ß but failed to activate PI3K/Akt, implying that as-yet-unidentified domain(s) in NS1 may alternatively mediate the activation of PI3K. Moreover, PI3K inhibitor, LY294002, did not suppress but significantly increased the replication of GD05 virus. CONCLUSIONS: Our study indicates that activation of PI3K/Akt by NS1 protein is not highly conserved among influenza A viruses and inhibition of the PI3K/Akt pathway as an anti-influenza strategy may not work for all influenza A viruses.