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1.
New Phytol ; 225(2): 896-912, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31318448

RESUMO

SCF (Skp1/Cullin1/F-box) complexes are key regulators of many cellular processes. Viruses encode specific factors to interfere with or hijack these complexes and ensure their infection in plants. The molecular mechanisms controlling this interference/hijack are currently largely unknown. Here, we present evidence of a novel strategy used by Rice black-streaked dwarf virus (RBSDV) to regulate ubiquitination in rice (Oryza sativa) by interfering in the activity of OsCSN5A. We also show that RBSDV P5-1 specifically affects CSN-mediated deRUBylation of OsCUL1, compromising the integrity of the SCFCOI1 complex. We demonstrate that the expressions of jasmonate (JA) biosynthesis-associated genes are not inhibited, whereas the expressions of JA-responsive genes are down-regulated in transgenic P5-1 plants. More importantly, application of JA to P5-1 transgenic plants did not reduce their susceptibility to RBSDV infection. Our results suggest that P5-1 inhibits the ubiquitination activity of SCF E3 ligases through an interaction with OsCSN5A, and hinders the RUBylation/deRUBylation of CUL1, leading to an inhibition of the JA response pathway and an enhancement of RBSDV infection in rice.

2.
New Phytol ; 226(1): 205-220, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31815302

RESUMO

Vacuolar (H+ )-PPases (VPs), are key regulators of active proton (H+ ) transport across membranes using the energy generated from PPi hydrolysis. The VPs also play vital roles in plant responses to various abiotic stresses. Their functions in plant responses to pathogen infections are unknown. Here, we show that TaVP, a VP of wheat (Triticum aestivum) is important for wheat resistance to Chinese wheat mosaic virus (CWMV) infection. Furthermore, overexpression of TaVP in plants induces the activity of PPi hydrolysis, leading to plants cell death. A virus-derived small interfering RNA (vsiRNA-20) generated from CWMV RNA1 can regulate the mRNA accumulation of TaVP in wheat. The accumulation of vsiRNA-20 can suppress cell death induced by TaVP in a dosage-dependent manner. Moreover, we show that the accumulation of vsiRNA-20 can affect PPi hydrolysis and the concentration of H+ in CWMV-infected wheat cells to create a more favorable cellular environment for CWMV replication. We propose that vsiRNA-20 regulates TaVP expression to prevent cell death and to maintain a weak alkaline environment in cytoplasm to enhance CWMV infection in wheat. This finding may be used as a novel strategy to minimize virus pathogenicity and to develop new antiviral stratagems.

3.
Sci Rep ; 9(1): 19717, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31873199

RESUMO

Compared with traditional monolayer cell culture, the three-dimensional tumor spheroid has emerged as an essential in vitro model for cancer research due to the recapitulation of the architecture and physiology of solid human tumors. Herein, by implementing the rapid prototyping of a benchtop 3D printer, we developed a new strategy to generate and analyze tumor spheroids on a commonly used multi-well plate. In this method, the printed artifact can be directly mounted on a 96/384-well plate, enables hanging drop-based spheroid formation, avoiding the tedious fabrication process from micromechanical systems. Besides long-term spheroid culture (20 days), this method supports subsequent analysis of tumor spheroid by seamlessly dripping from the printed array, thereby eliminating the need for spheroids retrieval for downstream characterization. We demonstrated several tumor spheroid-based assays, including tumoroid drug testing, metastasis on or inside extracellular matrix gel, and tumor transendothelial (TEM) assay. Based on quantitative phenotypical and molecular analysis without any precarious retrieval and transfer, we found that the malignant breast cancer (MDA-MB-231) cell aggregate presents a more metastatic morphological phenotype than the non-malignant breast cancer (MCF-7) and colonial cancer (HCT-116) cell spheroid, and shows an up-regulation of epithelial-mesenchymal transition (EMT) relevant genes (fold change > 2). Finally, we validated this tumor malignancy by the TEM assay, which could be easily performed using our approach. This methodology could provide a useful workflow for expediting tumoroid modeled in vitro assay, allowing the "Lab-on-a-Cloud" scenario for routine study.

4.
Biology (Basel) ; 8(4)2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31652738

RESUMO

Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly reliant on host factors to fulfill their infection. However, few host factors have been identified to participate in wheat yellow mosaic virus (WYMV) infection. Here, we demonstrate that wheat (Triticum aestivum) light-induced protein (TaLIP) interacts with the WYMV nuclear inclusion b protein (NIb). A bimolecular fluorescence complementation (BIFC) assay displayed that the subcellular distribution patterns of TaLIP were altered by NIb in Nicotiana benthamiana. Transcription of TaLIP was significantly decreased by WYMV infection and TaLIP-silencing wheat plants displayed more susceptibility to WYMV in comparison with the control plants, suggesting that knockdown of TaLIP impaired host resistance. Moreover, the transcription level of TaLIP was induced by exogenous abscisic acid (ABA) stimuli in wheat, while knockdown of TaLIP significantly repressed the expression of ABA-related genes such as wheat abscisic acid insensitive 5 (TaABI5), abscisic acid insensitive 8 (TaABI8), pyrabatin resistance 1-Llike (TaPYL1), and pyrabatin resistance 3-Llike (TaPYL3). Collectively, our results suggest that the interaction of NIb with TaLIP facilitated the virus infection possibly by disturbing the ABA signaling pathway in wheat.

5.
Biochem Biophys Res Commun ; 520(3): 544-550, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31615655

RESUMO

Anaplastic thyroid carcinoma (ATC) is the most aggressive type of thyroid cancer, with no effective treatment available. Identification of new anti-ATC drugs represents an urgent need. In this study, we find that ATC cells are highly sensitive to THZ531, a potent inhibitor of the transcriptional cyclin-dependent kinase (CDK), CDK12. Cell-based assays demonstrate that CDK12 inhibition significantly impedes cell cycle progression, induces apoptotic cell death, and impairs colony formation in ATC cells. THZ531 causes a loss of elongating RNA polymerase II and suppresses gene expression in ATC cells. An integrative analysis of gene expression profiles and super-enhancer landscape, combining with functional assays, leads to the discovery of two new ATC cancer genes, ZC3H4 and NEMP1. Furthermore, CDK12 inhibition enhances the sensitivity of ATC cells to doxorubicin-mediated chemotherapy. Thus, these findings indicate that CDK12 is a potential therapeutic target for ATC treatment and its inhibition may help to overcome the chemoresistance in patients with ATC.

6.
Thyroid ; 29(6): 809-823, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30924726

RESUMO

Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive malignancies, with no effective treatment currently available. The molecular mechanisms of ATC carcinogenesis remain poorly understood. The objective of this study was to investigate the mechanisms and functions of super-enhancer (SE)-driven oncogenic transcriptional addiction in the progression of ATC and identify new drug targets for ATC treatments. Methods: High-throughput chemical screening was performed to identify new drugs inhibiting ATC cell growth. Cell viability assay, colony formation analysis, cell-cycle analysis, and animal study were used to examine the effects of drug treatments on ATC progression. Chromatin immunoprecipitation sequencing was conducted to establish a SE landscape of ATC. Integrative analysis of RNA sequencing, chromatin immunoprecipitation sequencing, and CRISPR/Cas9-mediated gene editing was used to identify THZ1 target genes. Drug combination analysis was performed to assess drug synergy. Patient samples were analyzed to evaluate candidate biomarkers of prognosis in ATC. Results: THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), was identified as a potent anti-ATC compound by high-throughput chemical screening. ATC cells, but not papillary thyroid carcinoma cells, are exceptionally sensitive to CDK7 inhibition. An integrative analysis of both gene expression profiles and SE features revealed that the SE-mediated oncogenic transcriptional amplification mediates the vulnerability of ATC cells to THZ1 treatment. Combining this integrative analysis with functional assays led to the discovery of a number of novel cancer genes of ATC, including PPP1R15A, SMG9, and KLF2. Inhibition of PPP1R15A with Guanabenz or Sephin1 greatly suppresses ATC growth. Significantly, the expression level of PPP1R15A is correlated with CDK7 expression in ATC tissue samples. Elevated expression of PPP1R15A and CDK7 are both associated with poor clinical prognosis in ATC patients. Importantly, CDK7 or PPP1R15A inhibition sensitizes ATC cells to conventional chemotherapy. Conclusions: Taken together, these findings demonstrate transcriptional addiction in ATC pathobiology and identify CDK7 and PPP1R15A as potential biomarkers and therapeutic targets for ATC.

7.
Front Plant Sci ; 9: 1627, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30487803

RESUMO

Virus-induced gene silencing (VIGS) is an important tool for functional genomics studies in plants. With this method, it is possible to target most endogenous genes and downregulate the messenger RNA (mRNA) in a sequence-specific manner. Chinese wheat mosaic virus (CWMV) has a bipartite, single-strand positive RNA genome, and can infect both wheat and Nicotiana benthamiana, and the optimal temperature for systemic infection in plants is 17°C. To assess the potential of the virus as a vector for gene silencing at low temperature, a fragment of the N. benthamiana or wheat phytoene desaturase (PDS) gene was expressed from a modified CWMV RNA2 clone and the resulting photo bleaching in infected plants was used as a reporter for silencing. Downregulation of PDS mRNA was also measured by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). In experiments using fragments of PDS ranging from 500 to 1500 nucleotides, insert length influenced the stability and the efficiency of VIGS. The CWMV induced silencing system was also used to suppress miR165/166 and miR3134a through expression of miRNA target mimics. The relative expression levels of mature miR165/166 and miR3134a decreased whereas the transcript levels of their target genes increased. Interestingly, we also found the CWMV-induced silencing system was more efficient compare with the vector based on Barley stripe mosaic virus (BSMV) or Foxtail mosaic virus (FoMV) in wheat or the vector based on TRV in N. benthamiana at 17°C. In summary, the CWMV vector is effective in silencing endogenous genes and miRNAs at 17°C, thereby providing a powerful tool for gene function analysis in both N. benthamiana and wheat at low temperature.

8.
Huan Jing Ke Xue ; 34(10): 4024-30, 2013 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-24364326

RESUMO

Fifty-eight urban road dust samples were collected in June of 2009 from Suzhou, Wuxi and Nantong, South Jiangsu Province. Eight polybrominated diphenyl ether (PBDE) congeners and thirty-two polychlorinated biphenyl (PCB) congeners were measured using the gas chromatography-mass spectrometry. Concentrations of Sigma8PBDEs and Sigma32PCBs ranged from 4. 21 to 1 471 microg.kg-1 and ND was 14. 1 microg.kg-1 in the road dust samples, respectively. On the average, levels of PBDEs were much higher than PCBs in samples. Compared with the levels of PBDEs and PCBs in urban soil samples, results indicated that PBDEs levels observed in road dust samples were much higher than urban soil samples collected from other cities. Road dust PBDEs from combustion sources and vehicle emission should be attracted concerns. No significant difference was found between the levels of PBDEs or PCBs in the industrial areas and the urban center area, but was higher than landscape area. The results showed that there were exited unintentional emissions of PCBs in industrial areas. Investigation of compositional pattern of PBDEs indicated that BDE209 was the predominant congener in all samples, contributed 96.7% (64. 1% -99.8% ) of Sigma8 PBDEs . The dominant congeners in the Sigma32PCBs were tetra-PCBs and hexa-PCBs. The compositional pattern of PCBs congeners found in the road dust was not consistent with that of the commercial PCBs product and other environmental matrix.


Assuntos
Poeira/análise , Monitoramento Ambiental , Poluição Ambiental/análise , Éteres Difenil Halogenados/análise , Bifenilos Policlorados/análise , China , Cidades , Cromatografia Gasosa-Espectrometria de Massas
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