Functional analysis of a viral promoter from a strawberry vein banding virus isolate from China.

BACKGROUND: Promoters are important factors affecting gene expression in cells. The driven activities of viral promoters were generally assessed to screen available promoters for transgenic and research and biotech industries. In this study, we cloned a full-length promoter from a Chinese isolate of strawberry vein banding virus (SVBV) and produced several deletion mutants for evaluation of applications in production of reporter proteins in stable transgenic plants. METHODS: The full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction. The effects of SVBV SP1, SP2, SP3, and SP4 on gene expression were evaluated using ß-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. RESULTS: Transient expression assays showed that the SVBV SP1 promoter and its three deletion mutants all expressed the reporter genes, albeit at very different levels. Interestingly, transcriptional activity driven by the SP1 promoter was much higher than that of the cauliflower mosaic virus (CaMV) 35S promoter. After stable transformation of the GUS gene into Nicotiana tabacum plants, SVBV SP1-driven transgene expression was approximately 2.6-fold higher than CaMV 35S promoter-driven transgene expression. In addition, GUS gene expression levels were enhanced by co-inoculation of the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing SVBV open reading frame (ORF) V or ORF VI. CONCLUSIONS: The SVBV SP1 promoter from the Chinese isolate evaluated in this study could successfully drive transient and stable expression in plants, it was a stronger promoter than the CaMV 35S and FLt-US promoters and may be more useful for the production of stable transgenic plants.

Genome-wide identification and expression analysis of the NAC transcription factor family in tomato (Solanum lycopersicum) during aluminum stress.

BACKGROUND: The family of NAC proteins (NAM, ATAF1/2, and CUC2) represent a class of large plant-specific transcription factors. However, identification and functional surveys of NAC genes of tomato (Solanum lycopersicum) remain unstudied, despite the tomato genome being decoded for several years. This study aims to identify the NAC gene family and investigate their potential roles in responding to Al stress. RESULTS: Ninety-three NAC genes were identified and named in accordance with their chromosome location. Phylogenetic analysis found SlNACs are broadly distributed in 5 groups. Gene expression analysis showed that SlNACs had different expression levels in various tissues and at different fruit development stages. Cycloheximide treatment and qRT-PCR analysis indicated that SlNACs may aid regulation of tomato in response to Al stress, 19 of which were significantly up- or down-regulated in roots of tomato following Al stress. CONCLUSION: This work establishes a knowledge base for further studies on biological functions of SlNACs in tomato and will aid in improving agricultural traits of tomato in the future.

Flapless immediate implant placement into fresh molar extraction socket using platelet-rich fibrin: A case report.

BACKGROUND: There are some challenges concerning immediate implant placement in the molar region. Platelet-rich fibrin (PRF), an autologous biomaterial, has been used widely for periodontal intra-bony defects, sinus augmentation, socket preservation, and gingival recession. However, the literature remains scarce for reports on immediate implants with PRF, particularly in the case of fresh molar extraction socket. CASE SUMMARY: The patient was a 43-year-old woman with maxillary molar vertical crown-root fracture. She underwent flapless immediate implant placement into the fresh molar socket with PRF. At the follow-up visit 15 d post procedure, the vascularization of soft tissue was visible. There was no swelling or pain after the surgery. Six months postoperatively, the regeneration of bone and soft tissues was visible. Subsequently, the definitive restoration was placed. The patient was satisfied with the aesthetic outcomes. CONCLUSION: The flapless immediate implant placement into the fresh molar socket with PRF is a feasible procedure. This case report demonstrates that PRF promotes bone and soft tissue regeneration apart from having an enhanced anti-inflammatory ability. Furthermore, the procedure involves a minimally invasive technique, thus reducing the surgical complexity.

Identification and Analysis of Potential Genes Regulated by an Alphasatellite (TYLCCNA) that Contribute to Host Resistance against Tomato Yellow Leaf Curl China Virus and Its Betasatellite (TYLCCNV/TYLCCNB) Infection in Nicotiana benthamiana.

Recently, begomovirus/betasatellite disease complexes were found to be associated with alphasatellites, and their presence modulated disease symptoms and/or viral DNA accumulation in infected plants. However, the biological functions of alphasatellites during begomovirus/betasatellite infections remain unclear. Tomato yellow leaf curl China virus (TYLCCNV) associated with a betasatellite (TYLCCNB) is a widespread monopartite begomovirus in China. In the Yunnan province of China, the TYLCCNV/TYLCCNB disease complex is found in association with an alphasatellite (TYLCCNA). In this study, in order to explain the mechanisms underlying TYLCCNV/TYLCCNB infection and reductions in viral DNA accumulation caused by TYLCCNA, we analyzed the transcriptome profiles of Nicotiana benthamiana seedlings challenged by TYLCCNV/TYLCCNB or TYLCCNV/TYLCCNB/TYLCCNA using RNA sequencing. In total, 2272 and 1207 differentially expressed genes (DEGs) were identified to respond to TYLCCNV/TYLCCNB and TYLCCNV/TYLCCNB/TYLCCNA infections, respectively. Compared with the DEGs in the TYLCCNV/TYLCCNB-infected N. benthamiana seedlings, the number of DEGs in plants co-infected with TYLCCNV/TYLCCNB + TYLCCNA was significantly reduced. Additionally, 36 DEGs were identified to be regulated by TYLCCNA, six of which were further analyzed using the virus-induced gene silencing (VIGS) approach. Silencing of these six TYLCCNA responsive DEGs caused more severe disease symptoms and higher viral DNA accumulation levels, suggesting that TYLCCNA responsive DEGs may attenuate TYLCCNV/TYLCCNB infection.

The role of thioredoxin system in cancer: strategy for cancer therapy.

PURPOSE: Cancer, a major public health problem, exhibits significant redox alteration. Thioredoxin (Trx) system, including Trx and Trx reductase (TrxR), as well as Trx-interacting protein (TXNIP) play important roles in controlling the cellular redox balance in cancer cells. In most cancers, Trx and TrxR are usually overexpressed and TXNIP is underexpressed. In recent years, some agents targeting Trx, TrxR, and TXNIP were used to explore a therapy approach for cancer patients. METHODS: A systematic search of PMC and the PubMed Database was conducted to summarize the potential of Trx system inhibitors for cancer treatment. RESULTS: In this article, we first summarize the functions of Trx, TrxR, and TXNIP in cancers. We also review some small molecule inhibitors of Trx/TrxR and D-allose (TXNIP inducer) and discuss their antitumor mechanisms. We highlight the combined inhibition of Trx system and GSH system in cancer therapy. We expect that a highly specific and selective antitumor agent with no cytotoxicity on human normal cells could be developed in the future. CONCLUSION: In conclusion, Trx system may be very promising for clinical therapy of cancer in the future.

Transcription factor WRKY22 promotes aluminum tolerance via activation of OsFRDL4 expression and enhancement of citrate secretion in rice (Oryza sativa).

Whilst WRKY transcription factors are known to be involved in diverse plant responses to biotic stresses, their involvement in abiotic stress tolerance is poorly understood. OsFRDL4, encoding a citrate transporter, has been reported to be regulated by ALUMINUM (Al) RESISTANCE TRANSCRIPTION FACTOR 1 (ART1) in rice, but whether it is also regulated by other transcription factors is unknown. We define the role of OsWRKY22 in response to Al stress in rice by using mutation and transgenic complementation assays, and characterize the regulation of OsFRDL4 by OsWRKY22 via yeas one-hybrid, electrophoretic mobility shift assay and ChIP-quantitative PCR. We demonstrate that loss of OsWRKY22 function conferred by the oswrky22 T-DNA insertion allele causes enhanced sensitivity to Al stress, and a reduction in Al-induced citrate secretion. We next show that OsWRKY22 is localized in the nucleus, functions as a transcriptional activator and is able to bind to the promoter of OsFRDL4 via W-box elements. Finally, we find that both OsFRDL4 expression and Al-induced citrate secretion are significantly lower in art1 oswrky22 double mutants than in the respective single mutants. We conclude that OsWRKY22 promotes Al-induced increases in OsFRDL4 expression, thus enhancing Al-induced citrate secretion and Al tolerance in rice.

Mimic Phosphorylation of a ßC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

Phosphorylation of the ßC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-ßC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-ßC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-ßC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-ßC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-ßC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-ßC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-ßC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-ßC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor.IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, ßC1 (TYLCCNB-ßC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-ßC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N. benthamiana ASYMMETRIC LEAVES 1. To our knowledge, this is the first report establishing an inhibitory effect of phosphorylation of TYLCCNB-ßC1 on its pathogenic functions as both a VSR and a symptom determinant and to provide a mechanistic explanation of how SNF1-related protein kinase 1 acts as a host defense factor. These findings expand the scope of phosphorylation-mediated defense mechanisms and contribute to further understanding of plant defense mechanisms against geminiviruses.

iTRAQ analysis of the tobacco leaf proteome reveals that RNA-directed DNA methylation (RdDM) has important roles in defense against geminivirus-betasatellite infection.

Geminiviruses have caused serious losses in crop production. To investigate the mechanisms underlying host defenses against geminiviruses, an isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic approach was used to explore the expression profiles of proteins in Nicotiana benthamiana (N. benthamiana) leaves in response to tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB) at an early phase. In total, 4155 proteins were identified and 272 proteins were changed differentially in response to TYLCCNV/TYLCCNB infection. Bioinformatics analysis indicated that S-adenosyl-l-methionine cycle II was the most significantly up-regulated biochemical process during TYLCCNV/TYLCCNB infection. The mRNA levels of three proteins in S-adenosyl-l-methionine cycle II were further analyzed by qPCR, each was found significantly up-regulated in TYLCCNV/TYLCCNB-infected N. benthamiana. This result suggested a strong promotion of the biosynthesis of available methyl groups during geminivirus infections. We further tested the potential role of RdDM in N. benthamiana by virus-induced gene silencing (VIGS) and found that a disruption in RdDM resulted in more severe infectious symptoms and higher accumulation of viral DNA after TYLCCNV/TYLCCNB infection. Although the precise functions of these proteins still need to be determined, our proteomic results enhance the understanding of plant antiviral mechanisms. BIOLOGICAL SIGNIFICANCE: One of the major limitations to crop growth in the worldwide is the prevalence of geminiviruses. They are able to infect food and cash crops and cause serious crop failures and economic losses worldwide, especially in Africa and Asia. Tomato yellow leaf curl China virus (TYLCCNV), which causes severe viral diseases in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). However, the mechanisms underlying the TYLCCNV/TYLCCNB defense in plants are still not fully understood at the molecular level. In this study, the combined proteomic, bioinformatic and VIGS analyses revealed that TYLCCNV/TYLCCNB invasion caused complex proteomic alterations in the leaves of N. benthamiana involving the processes of stress and defense, energy production, photosynthesis, protein homeostasis, metabolism, cell structure, signal transduction, transcription, transportation, and cell growth/division. Promotion of available methyl groups via the S-adenosyl-l-methionine cycle II pathway in N. benthamiana appeared crucial for antiviral responses. These findings enhance our understanding in the proteomic aspects of host antiviral defenses against geminiviruses, and also demonstrate that the combination of proteomics with bioinformatics and VIGS analysis is an effective approach to investigate systemic plant responses to geminiviruses and to shed light on plant-virus interactions.

Role of perivascular adipose tissue in nicotine­induced endothelial cell inflammatory responses.

Smoking is considered to be one of the primary causes of atherosclerosis and vascular injury. Previous studies have shown that nicotine in tobacco can lead to vascular inflammation and endothelial dysfunction. Perivascular adipose tissue (PVAT) is known to secrete various types of adipokines to maintain vascular homeostasis. The present study investigated whether nicotine­induced PVAT malfunction can accelerate endothelial inflammation and eventually lead to endothelial dysfunction. The levels of inflammatory adipokines, including nuclear factor (NF)­κB, interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α, the ICAM­1 and VCAM­1 adhesion molecules and secretion of adiponectin were assessed in mature adipocytes and endothelial cells cultured alone or in co­culture under nicotine stimulation. It was found that nicotine reduced the secretion of adiponectin and stimulated secretion of the NF­κB, IL­1ß, IL­6 and TNF­α inflammatory adipokines in mature adipocytes. Although nicotine stimulated endothelial cells to secrete IL­1ß and IL­6, no significant increase in the secretion of TNF­α was observed. The co­culture of mature adipocytes with endothelial cells markedly augmented the expression of the NF­κB, IL­1ß, IL­6 and TNF­α inflammatory adipokines and the ICAM­1 and VCAM­1 adhesion molecules, and significantly lowered the levels of adiponectin. These findings suggested that nicotine induced mature adipocyte dysfunction, which caused the abnormal secretion of adiponectin and inflammatory adipokines, and exacerbated endothelial inflammation. These findings also suggested a mechanism whereby nicotine induced the secretion of adiponectin and inflammatory cytokines by adipocytes. The results of the present study elucidated a novel pathway induced by cigarette smoke, which contributed to atherosclerosis and vascular injury.

OsTCTP, encoding a translationally controlled tumor protein, plays an important role in mercury tolerance in rice.

BACKGROUND: Mercury (Hg) is not only a threat to public health but also a growth risk factor to plants, as it is readily accumulated by higher plants. Accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development; however, the detoxification and tolerance mechanisms of plants to Hg stress are still not fully understood. Exposure to toxic Hg also occurs in some crops cultivated under anoxic conditions, such as rice (Oryza sativa L.), a model organism and one of the most important cultivated plants worldwide. In this study, we functionally characterized a rice translationally controlled tumor protein gene (Os11g43900, OsTCTP) involved in Hg stress tolerance. RESULTS: OsTCTP was ubiquitously expressed in all examined plant tissues, especially in actively dividing and differentiating tissues, such as roots and nodes. OsTCTP was found to localize both the cytosol and the nucleus. OsTCTP was induced by mercuric chloride, cupric sulfate, abscisic acid, and hydrogen peroxide at the protein level in a time-dependent manner. Overexpression of OsTCTP potentiated the activities of several antioxidant enzymes, reduced the Hg-induced H2O2 levels, and promoted Hg tolerance in rice, whereas knockdown of OsTCTP produced opposite effects. And overexpression of OsTCTP did not prevent Hg absorption and accumulation in rice. We also demonstrated that Asn 48 and Asn 97 of OsTCTP amino acids were not the potential N-glycosylation sites. CONCLUSIONS: Our results suggest that OsTCTP is capable of decreasing the Hg-induced reactive oxygen species (ROS), therefore, reducing the damage of ROS and enhancing the tolerance of rice plants to Hg stress. Thus, OsTCTP is a valuable gene for genetic engineering to improve rice performance under Hg contaminated paddy soils.

Protective effect of melatonin on cigarette smoke-induced restenosis in rat carotid arteries after balloon injury.

Vascular restenosis after the interventional angioplasty remains the main obstacle to a favorable long-term patency. Many researches suggest cigarette smoking is one of the most important causes of restenosis. This study was designed to investigate whether melatonin could protect against the cigarette smoke-induced restenosis in rat carotid arteries after balloon injury. Three groups of male rats (normal condition, cigarette smoke exposed, cigarette smoke exposed, and melatonin injected) were used in this study. An established balloon-induced carotid artery injury was performed, and the carotid arteries were harvested from these three groups 14 days later. The ratio of intima to media, the infiltration of inflammatory cells, the expression of inflammatory cytokines (NF-κB, IL-1ß, IL-6, TNF-α, MCP-1), adhesion molecules (ICAM-1, VCAM-1), and eNOS were measured. The results showed that cigarette smoke exposure aggravated the stenosis of the lumen, promoted the infiltration of inflammatory cells and induced the expression of the inflammatory cytokines and adhesion molecules after the balloon-induced carotid artery injury. Moreover, cigarette smoke exposure can inhibit the expression of eNOS. Particularly, we surprised that melatonin could minimize this effect caused by cigarette smoke. These results suggested that melatonin could prevent the cigarette smoke-induced restenosis in rat carotid arteries after balloon injury and the mechanism of its protective effect may be the inhibition of the inflammatory reaction. This also implies melatonin has the potential therapeutic applicability in prevention of restenosis after the vascular angioplasty in smokers.

Root proteome of rice studied by iTRAQ provides integrated insight into aluminum stress tolerance mechanisms in plants.

One of the major limitations to crop growth on acid soils is the prevalence of soluble aluminum ions (Al(3+)). Rice (Oryza sativa L.) has been reported to be highly Al tolerant; however, large-scale proteomic data of rice in response to Al(3+) are still very scanty. Here, we used an iTRAQ-based quantitative proteomics approach for comparative analysis of the expression profiles of proteins in rice roots in response to Al(3+) at an early phase. A total of 700 distinct proteins (homologous proteins grouped together) with >95% confidence were identified. Among them, 106 proteins were differentially expressed upon Al(3+) toxicity in sensitive and tolerant cultivars. Bioinformatics analysis indicated that glycolysis/gluconeogenesis was the most significantly up-regulated biochemical process in response to excess Al(3+). The mRNA levels of eight proteins mapped in the glycolysis/gluconeogenesis were further analyzed by qPCR and the expression levels of all the eight genes were higher in tolerant cultivar than in sensitive cultivar, suggesting that these compounds may promote Al tolerance by modulating the production of available energy. Although the exact roles of these putative tolerance proteins remain to be examined, our data lead to a better understanding of the Al tolerance mechanisms in rice plants through the proteomics approach. BIOLOGICAL SIGNIFICANCE: Aluminum (mainly Al(3+)) is one of the major limitations to the agricultural productivity on acid soils and causes heavy yield loss every year. Rice has been reported to be highly Al tolerant; however, the mechanisms of rice Al tolerance are still not fully understood. Here, a combined proteomics, bioinformatics and qPCR analysis revealed that Al(3+) invasion caused complex proteomic changes in rice roots involving energy, stress and defense, protein turnover, metabolism, signal transduction, transport and intracellular traffic, cell structure, cell growth/division, and transcription. Promotion of the glycolytic/gluconeogenetic pathway in roots appeared crucially important for Al tolerance. These results lead to a better understanding of the Al tolerance mechanisms in rice and help to improve plant performance on acid soils, eventually to increase the crop production.