Virus-Induced Gene Silencing in Diverse Maize Lines Using the Brome Mosaic Virus-based silencing vector

Virus-induced gene silencing (VIGS) is a widely used tool for gene function studies in many plant species, though its use in cereals has been limited. In addition, within cereal species the varieties that best respond during VIGS screens are often not known. Using a Brome mosaic virus (BMV) vector designed to silence the maize phytoene desaturase (PDS) gene, a genetically diverse set of maize inbred lines was screened for development of gene silencing after inoculation of seeds through the novel use of a vascular puncture inoculation technique. In addition to Va35, which previously was shown to support silencing, maize lines NC300, Ki11, Oh7b, M162W and CML52 displayed significant visible photobleaching when challenged with the BMV-PDS. In these plants, targeted PDS mRNA expression was decreased 50-80% relative to levels in plants that were inoculated with BMV containing a fragment of the GUS gene or were mock-inoculated

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Multigene families encode the major enzymes of antioxidant metabolism in Eucalyptus grandis L

Antioxidant metabolism protects cells from oxidative damage caused by reactive oxygen species (ROS). In plants, several enzymes act jointly to maintain redox homeostasis. Moreover, isoform diversity contributes to the fine tuning necessary for plant responses to both exogenous and endogenous signals influencing antioxidant metabolism. This study aimed to provide a comprehensive view of the major classes of antioxidant enzymes in the woody species Eucalyptus grandis. A careful survey of the FORESTs data bank revealed 36 clusters as encoding antioxidant enzymes: six clusters encoding ascorbate peroxidase (APx) isozymes, three catalase (CAT) proteins, three dehydroascorbate reductase (DHAR), two glutathione reductase (GR) isozymes, four monodehydroascorbate reductase (MDHAR), six phospholipid hydroperoxide glutathione peroxidases (PhGPx), and 12 encoding superoxide dismutases (SOD) isozymes. Phylogenetic analysis demonstrated that all clusters (identified herein) grouped with previously characterized antioxidant enzymes, corroborating the analysis performed. With respect to enzymes involved in the ascorbate-glutathione cycle, both cytosolic and chloroplastic isoforms were putatively identified. These sequences were widely distributed among the different ESTs libraries indicating a broad gene expression pattern. Overall, the data indicate the importance of antioxidant metabolism in eucalyptus