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Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish lifelong latent infection in B cells and are associated with a variety of tumors. In addition to protein coding genes, these viruses encode numerous microRNAs (miRNAs) within their genomes. While putative host targets of EBV and KSHV miRNAs have been previously identified, the specific functions of these miRNAs during in vivo infection are largely unknown. Murine gammaherpesvirus 68 (MHV68) is a natural pathogen of rodents that is genetically related to both EBV and KSHV, and thus serves as an excellent model for the study of EBV and KSHV genetic elements such as miRNAs in the context of infection and disease. However, the specific targets of MHV68 miRNAs remain completely unknown. Using a technique known as qCLASH (quick crosslinking, ligation, and sequencing of hybrids), we have now identified thousands of Ago-associated, direct miRNA-mRNA interactions during lytic infection, latent infection and reactivation from latency. Validating this approach, detailed molecular analyses of specific interactions demonstrated repression of numerous host mRNA targets of MHV68 miRNAs, including Arid1a, Ctsl, Ifitm3 and Phc3. Notably, of the 1,505 MHV68 miRNA-host mRNA targets identified in B cells, 86% were shared with either EBV or KSHV, and 64% were shared among all three viruses, demonstrating significant conservation of gammaherpesvirus miRNA targeting. Pathway analysis of MHV68 miRNA targets further revealed enrichment of cellular pathways involved in protein synthesis and protein modification, including eIF2 Signaling, mTOR signaling and protein ubiquitination, pathways also enriched for targets of EBV and KSHV miRNAs. These findings provide substantial new information about specific targets of MHV68 miRNAs and shed important light on likely conserved functions of gammaherpesvirus miRNAs., Competing Interests: The authors have declared that no competing interests exist.

NAC (NAM, ATAF1/2 and CUC2) proteins are key regulators of various plant stress tolerances. However, knowledge of NAC genes remains largely unknown in Miscanthus. Here, we characterized a novel NAC gene MlNAC10 from M. lutarioriparius than plays a role in abiotic stress tolerance. MlNAC10 encodes a nuclear-localized protein with a C-terminal transactivation domain, and has a specific binding affinity to the NAC recognition sequence (NACRS). Ectopic expression of MlNAC10 in Arabidopsis led to increased sensitivity to abscisic acid (ABA) at early seedling growth stages. In addition, the proline content was significantly increased and the reactive oxygen species (ROS) scavenging capability was significantly enhanced in MlNAC10 overexpression lines under ABA treatment. Moreover, the drought and salt stress tolerance was significantly improved in MlNAC10 overexpression lines. Consistently, the activities of three antioxidant enzymes, namely catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), were dramatically stimulated in the overexpression lines compared to the wild type (WT). Correspondingly, the accumulation of ROS was dramatically decreased and malondialdehyde (MDA) was accumulated at a much lower level in the transgenic lines. Meanwhile, the expression of six abiotic stress-related genes was dramatically stimulated in the overexpression lines in comparison to the WT. Together, our results demonstrated that MlNAC10 acts as an important regulator of drought and salinity stress tolerance by stimulating antioxidant enzymes and alleviating ROS damage via the ABA signaling pathway., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

NAC (NAM, ATAF1/2 and CUC2) transcription factors play critical roles in plant abiotic stress responses. However, knowledge regarding the functional roles of NACs in abiotic stress tolerance and its underlying mechanisms is relatively limited in Miscanthus. In this study, we functionally characterized a novel Miscanthus NAC gene MlNAC12 by ectopic expression in Arabidopsis. MlNAC12 was localized in the nucleus. It could specifically binds to the NAC recognition sequence (NACRS) and has a transactivation activity in the C-terminus. Overexpression of MlNAC12 in Arabidopsis conferred hypersensitivity to exogenous Abscisic acid (ABA) at seed germination and root elongation stages. In addition, MlNAC12 overexpression enhanced germination and root growth under salt (NaCl) stress. Furthermore, MlNAC12 overexpression lines exhibited significantly enhanced drought stress tolerance, which was evidenced by a higher survival rate and a lower water loss rate compared to the wild type (WT). Accordingly, the stomata aperture was remarkably reduced in MlNAC12 overexpression lines in comparison to the WT under drought stress. Furthermore, the accumulation of the reactive oxygen species (ROS) and malondialdehyde (MDA) under abiotic stresses was significantly decreased, accompanied by dramatically enhanced activities of several antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in the transgenic plants. Correspondingly, the expression of six stress-responsive genes was significantly up-regulated in MlNAC12 overexpression lines. Together, our results indicate that MlNAC12 is a positive regulator of drought and salt stress tolerance through activating ROS scavenging enzymes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cellular hypoxia has been proposed as a major factor in the pathogenesis of chronic renal injury, yet to date there has been no direct evidence to support its importance. Therefore, we examined cortical hypoxia in an animal model of chronic renal injury (murine adriamycin nephrosis; AN) by assessing nuclear localization of the oxygen-dependent alpha-subunit of hypoxia-inducible factor-1 (HIF-1alpha) in animals 7, 14, and 28 days after adriamycin. Results were assessed in conjunction with quantitation of the cortical microvasculature (by CD34 immunostaining) and cortical expression of VEGF. Cortical apoptosis was also examined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. A dramatic and significant increase in nuclear localization of HIF-1alpha was seen 28 days after adriamycin in the context of severe glomerular and tubulointerstitial damage. Areas of nuclear HIF-1alpha staining did not colocalize with areas of cellular apoptosis. AN was also associated with a significant attenuation of the peritubular capillaries that was significant at 14 and 28 days after adriamycin. Cortical VEGF expression fell in a stepwise manner from day 7 until day 28 after adriamycin. In conclusion, these data are consistent with a significant increase in cellular hypoxia occurring in the advanced stages of murine AN. Increased cortical hypoxia was preceded by significant reductions in both the number of peritubular capillaries (i.e., oxygen supply) and the angiogenic cytokine VEGF. Apart from providing the first direct evidence for cellular hypoxia in a model of chronic renal disease, these results suggest that a primary dysregulation of angiogenesis may be the cause of increased hypoxia in this model.

Anthocyanins widely accumulate in the vegetative and reproductive tissues of strawberries and play an important role in stress resistance and fruit quality. Compared with other fruits, little is known about the molecular mechanisms regulating anthocyanin accumulation in strawberry vegetative tissues. In this study, we revealed an R2R3–MYB transcription factor, FaMYB10-like (FaMYB10L), which positively regulated anthocyanin accumulation and was induced by light in the petiole and runner of cultivated strawberry. FaMYB10L is a homologue of FveMYB10-like and a nuclear localization protein. Transient overexpression of FaMYB10L in a white fruit strawberry variety (myb10 mutant) rescued fruit pigmentation, and further qR–PCR analysis revealed that FaMYB10L upregulated the expression levels of anthocyanin biosynthesis-related genes and transport gene. A dual luciferase assay showed that FaMYB10L could activate the anthocyanin transport gene FaRAP. Anthocyanin accumulation was observed in FaMYB10L-overexpressing strawberry calli, and light treatment enhanced anthocyanin accumulation. Furthermore, transcriptomic profiling indicated that the DEGs involved in the flavonoid biosynthesis pathway and induced by light were enriched in FaMYB10L-overexpressing strawberry calli. In addition, yeast two-hybrid assays and luciferase complementation assays indicated that FaMYB10L could interact with bHLH3. These findings enriched the light-involved regulatory network of anthocyanin metabolism in cultivated strawberries. [ABSTRACT FROM AUTHOR]

MYB and BBX transcription factors play important roles in flavonoid biosynthesis. Here, we obtained transgenic woodland strawberry with stable overexpression of FaMYB5, demonstrating that FaMYB5 can increase anthocyanin and proanthocyanidin content in roots, stems and leaves of woodland strawberry. In addition, bimolecular fluorescence complementation assays and yeast two-hybridization demonstrated that the N-terminal (1-99aa) of FaBBX24 interacts with FaMYB5. Transient co-expression of FaBBX24 and FaMYB5 in cultivated strawberry 'Xiaobai' showed that co-expression strongly promoted the expression of F3′H, 4CL-2, TT12, AHA10 and ANR and then increased the content of anthocyanin and proanthocyanidin in strawberry fruits. We also determined that FaBBX24 is also a positive regulator of anthocyanin and proanthocyanidin biosynthesis in strawberry. The results reveal a novel mechanism by which the FaMYB5–FaBBX24 module collaboratively regulates anthocyanin and proanthocyanidin in strawberry fruit. [ABSTRACT FROM AUTHOR]

B-box transcription factors (TFs) play a vital role in light-induced anthocyanin accumulation. Here, the FaBBX22 gene encoding 287 amino acids B-box TF was isolated from the cultivated strawberry variety 'Benihoppe' and characterized functionally. The expression analysis showed that FaBBX22 was expressed in the roots, stems, leaves, flowers and fruits, and its transcription level was upregulated under the red- or blue-light irradiation. FaBBX22 was localized in the nucleus and showed trans-acting activity in yeast cells. Ectopic overexpression of FaBBX22 in Arabidopsis enhanced the accumulation of anthocyanin. Additionally, we obtained transgenic strawberry calli that overexpressed the FaBBX22 gene, and strawberry calli coloration assays showed that FaBBX22 increased anthocyanin accumulation by upregulating the expression of anthocyanin biosynthetic genes (FaPAL, FaANS, FaF3′H, FaUFGT1) and transport gene FaRAP in a light-dependent manner. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays indicated that FaBBX22 interacted with FaHY5. Furthermore, mutation of the 70th Asp residue in FaBBX22 protein to an Ala residue disrupted the interaction between FaBBX22 and FaHY5. Further, a transient expression assay demonstrated that the co-expression of FaBBX22 and FaHY5 could strongly promote anthocyanin accumulation in strawberry fruits. Collectively, these results revealed the positive regulatory role of FaBBX22 in light-induced anthocyanin accumulation. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to investigate the B cell-associated transcription factors, Ets-1 and microRNA, miR-326 in systemic lupus erythematosus (SLE) patients, and their correlation with the pathogenesis of SLE.Method: A total of 44 SLE patients and 20 healthy controls were enrolled in this research, all patients fulfilled the American College of Rheumatology classification criteria for SLE. The mRNA expression of Ets-1 and miR-326 in CD19+B cells from SLE patients were examined by qRT-PCR. The percentages of CD19+CD138+plasma cells were analyzed by Flow cytometry.Results: We found decreased expression of Ets-1 mRNA in SLE patients compared with the healthy controls ([0.228 (0.145, 0.507)] vs [0.583 (0.452, 0.763)], p = 0.001),while increased expression of miR-326 mRNA in CD19+B cells SLE patients compared with the healthy controls([1.092 (0.457, 2.855)] vs [0.685 (0.274, 0.819)], p = 0.008). The percentage of CD19+CD138+plasma cells in SLE patients was higher than that of healthy controls (0.55 ± 0.21% vs 0.36 ± 0.21%, p = 0.002). Moreover, a negative correlation between expression of Ets-1 mRNA and miR-326 mRNA in CD19+B cells was detected (r = − 0.334, p = 0.027). A significant association between the occurrences of CD19+CD138+plasma cells and the levels of Ets-1 mRNA and miR-326 mRNA was observed (r = − 0.417, p = 0.005 and r = 0.482, p = 0.001, respectively).Conclusions: Our results suggest that miR-326 might promote B cells differentiation by targeting Ets-1, a negative regulator of B cells differentiation and therefore participate in the pathogenesis of SLE. [ABSTRACT FROM AUTHOR]

When cells are grown to confluence, cell–cell contact inhibition occurs and drives the cells to enter reversible quiescence rather than senescence. Confluent retinal pigment epithelial (RPE) cells exhibiting contact inhibition was used as a model in this study to examine the role of overexpression of transcription factor AP4, a highly expressed transcription factor in many types of cancer, in these cells during long-term culture. We generated stable inducible RPE cell clones expressing AP4 or AP4 without the DNA binding domain (DN-AP4) and observed that, when cultured for 24 days, RPE cells with a high level of AP4 exhibit a large, flattened morphology and even cease proliferating; these changes were not observed in DN-AP4-expressing cells or non-induced cells. In addition, AP4-expressing cells exhibited senescence-associated β-galactosidase activity and the senescence-associated secretory phenotype. We demonstrated that the induced cellular senescence was mediated by enhanced p53 expression and that AP4 regulates the p53 gene by binding directly to two of the three E-boxes present on the promoter of the p53 gene. Moreover, we showed that serum is essential for AP4 in inducing p53-associated cellular senescence. Collectively, we showed that overexpression of AP4 mediates cellular senescence involving in activation of p53 in long-term post-confluent RPE cells. [ABSTRACT FROM AUTHOR]

NACs are one of the largest transcription factor families in plants and are involved in the response to abiotic stress. A new stress-responsive NAC transcription factor gene, LpNAC13 , was isolated from Lilium pumilum bulbs. The expression of LpNAC13 was induced by drought, salt, cold and ABA treatments. LpNAC13 overexpressing plants were generated to explore the function of LpNAC13 in response to drought and salt stress. Overexpression of LpNAC13 in tobacco displayed a reduced drought tolerance but exhibited an enhanced salt tolerance. The LpNAC13 overexpression plants had decreased antioxidant enzyme activities, content of proline and chlorophyll, increased MDA content under drought condition, the results in the LpNAC13 plants under salt condition were opposite to those under drought condition. The seed germination and root length assays of overexpression of LpNAC13 showed decreased sensitivity to ABA. Functional analyses demonstrate that LpNAC13 plays opposite roles in drought and salt stress tolerance, acting as a negative regulator of drought response but as a positive regulator of salt response in tobacco. • LpNAC13 is a stress-responsive NAC transcription factor gene, which was isolated from Lilium pumilum bulbs. • The expression of LpNAC13 was induced by drought, salt, cold and ABA treatments. • Overexpression of LpNAC13 in tobacco displayed a reduced drought tolerance but exhibited an enhanced salt tolerance. • The study of LpNAC13 provides some new view of the molecular mechanisms of drought and salt resistance in the lily family. [ABSTRACT FROM AUTHOR]

NAC transcription factors have multiple biological functions in plants. In this study, a new NAC transcription factor, LpNAC6 , was cloned from Lilium pumilum , and its salt and drought resistance functions were identified. We treated LpNAC6 transgenic tobacco plants with different intensities of alkali and drought stress. Results showed that LpNAC6 transgenic tobacco had enhanced alkali tolerance, but decreased drought tolerance. Antioxidant enzyme (SOD, POD, CAT) activity, chlorophyll content, proline content, and photosynthetic capacity of transgenic tobacco were significantly higher than those of wild-type tobacco, while the contents of MDA, H 2 O 2 , and O 2 − were significantly lower than those of wild-type tobacco. The expression level of stress-related genes in transgenic tobacco increased significantly, and the alkali tolerance was enhanced, but the opposite was true under drought stress. Our findings suggest that LpNAC6 has a reverse regulatory effect on alkaline and drought tolerance in plants, which is of great significance for plant screening and stress tolerance regulation of transgenic plants in arid saline–alkali land. • The function of a novel NAC transcription factor LpNAC6 cloned from Lilium pumilum was verified and analyzed. • LpNAC6 positively regulated plant responses to alkali and negatively regulated their responses to drought. • This study provides some new insights into the molecular mechanisms of alkali and drought resistance in the lily family. [ABSTRACT FROM AUTHOR]

Abstract: Oxidized low-density lipoprotein (ox-LDL) is an instrumental factor in atherogenesis, however, the effects of ox-LDL on the balance of Th17/Treg in acute coronary syndrome [ACS, including unstable angina (UA) and acute myocardial infarction (AMI)] is still unclear. CD4+CD25+ regulatory T (Treg) cells and Th17 cells, subsets of T-helper cells, play important roles in peripheral immunity and their imbalance leads to the development of tissue inflammation and autoimmune diseases. However, few studies have explored the effect of Th17/Treg balance in plaque destabilization and the onset of ACS. To explore the shift of Th17/Treg balance in ACS patients and the effect of ox-LDL on the balance, we examined the frequencies of Th17 and Treg cells, key transcription factors and relevant cytokines in patients with AMI, UA, stable angina (SA) and controls. We analysed the correlations of serum ox-LDL to Th17/Treg frequency, and the effects of ox-LDL on Th17/Treg cells in vitro. Our study demonstrated that ACS patients have shown a significant increase of Th17 frequency, RORγt expression and serum Interleukin 17 (IL-17), and a obvious decline of Treg frequency, Foxp3 expression, suppressive function, and serum IL-10. Serum ox-LDL positively correlated with the frequency of Th17 cells and negatively correlated with the frequency of Treg cells. In vitro incubation of peripheral blood mononuclear cells from controls with ox-LDL resulted in a significant reduction of Treg cells and a significant elevation of Th17 cells in a dose- and time-dependant manner. Treg and Th17 cells from ACS patients were significantly more susceptible to ox-LDL-mediated alterations. Th17/Treg numerical and functional imbalance exists in ACS patients, and ox-LDL has a direct effect on Th17/Treg imbalance which may contribute to the occurrence of ACS. [Copyright &y& Elsevier]