Rhizomania is an economically important disease of sugar beet, which is caused by Beet necrotic yellow vein virus (BNYVV). As previously shown, RNA silencing mechanism effectively inhibit the viral propagation in transgenic sugar beet plants. To investigate possible proteomic changes induced by gene insertion and/or RNA silencing mechanism, the root protein profiles of wild type sugar beet genotype 9597, as a control, and transgenic events named 6018-T3:S6-44 (S6) and 219-T3:S3-13.2 (S3) were compared by two-dimensional gel electrophoresis. The accumulation levels of 25 and 24 proteins were differentially regulated in S3 and S6 plants, respectively. The accumulation of 15 spots were increased or decreased more than 2-fold. Additionally, 10 spots repressed or induced in both, while seven spots showed variable results in two events. All the differentially expressed spots were analyzed by MALDI-TOF-TOF mass spectrometry. The functional analysis of differentially accumulated proteins showed that most of them are related to the metabolism and defense/stress response. None of these recognized proteins were allergens or toxic proteins except for a spot identified as phenylcoumaran benzylic ether reductase, Pyrc5, which was decreased in the genetically modified S6 plant. These data are in favor of substantial equivalence of the transgenic plants in comparison to their related wild type cultivar since the proteomic profile of sugar beet root was not remarkably affected by gene transfer and activation RNA silencing mechanism.

Quercetin (Qu), as an essential flavonoid in plants with antioxidant properties, scavenges environmental stress-induced ROS. Quercetin-based nanocomposites (QNCs) with the same and adequate properties were designed and synthesized for effective Qu delivery in Ocimum basilicum. QNCs were synthesized using the coacervation method, and their effect on the growth, physiological, biochemical, and phytochemical traits of O. basilicum under salinity stress was investigated. Various treatments, including selected concentrations of Qu (0.01 mg/mL) and QNCs (0.01 mg/mL), and four concentrations of NaCl (0, 50, 100, 150 mM) at the vegetative stages, were applied. Results showed that stress markers (Electrolyte leakage, malondialdehyde, hydrogen peroxide) increased with increasing salinity levels. Conversely, salinized plants showed a reduction in plant growth parameters (seed germination, root and shoot length, fresh and dry weight of shoot and root, and plant height) and physiological and photosynthetic parameters (Relative water content, photosynthesis rate, stomatal conductance, photosynthetic pigments, and chlorophyll fluorescence), while application of Qu and QNCs increased these critical parameters. Furthermore, Qu and QNCs enhanced O. basilicum’s tolerance to salinity by increasing compatible solutes content such as glycine betaine, proline, total free amino acids, and soluble carbohydrates; increasing antioxidant enzyme activity; increasing antioxidants content like anthocyanins, tannins, phenols, and flavonoids; and decreasing proteins content and stress markers in plant tissues. Our study suggests that treatment with Qu and QNCs is an effective strategy that can be used to enhance the salt tolerance of O. basilicum plants, and QNCs treatment had a better effect than treatment with Qu.

Background Activating RAS mutations drive around 30% of pre-B cell acute lymphoblastic leukemias (pre-B ALL) and are particularly common in relapsed ALL with a consecutively poor outcome. Recently published data demonstrated the critical role of the unfolded protein response (UPR) network, namely its IRE1α-XBP1 axis, for the survival of pre-B ALL cells: High expression of XBP1 confers a poor prognosis in pre B-ALL. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. In this study, we aimed at identifying the molecular mechanism underlying pro-survival IRE1α-XBP1 signaling in RAS mutated pre-B ALL. Methods For a TET-ON inducible NRASG12D model in conditional Xbp1 knockout mice, we used interleukin 7 (IL-7)-dependent murine Mx1-Cre;Xbp1fl/fl pre-B cells transduced with a TET-ON inducible NRASG12D. We performed in vitro cell cycle and apoptosis assays with propidium iodide (PI) and annexin-V/PI. Furthermore, Western Blot and RT-qPCR were applied to analyze target gene expression. In a second approach, we focused on the signaling events following the blockade of RAS downstream targets using the MEK inhibitor PD0325901 and the dual PI3K/mTOR inhibitor BEZ235. We then assessed the efficacy of small molecule inhibition of IRE1α by MKC-8866 on XBP1 inactivation in RAS-mutated pre-B ALL cells either as a single treatment and in combination with the above mentioned drugs. Results We found the expression of Xbp1 significantly increased at the mRNA level with induction of NRASG12D. To determine the significance of Xbp1 in NRASG12D-driven pre-B ALL, we genetically deleted the IRE1α target Xbp1 using Cre-mediated deletion of Xbp1fl/fl in our mouse model of pre-B ALL. Genetic loss of Xbp1 significantly induced apoptosis (2.0-fold, p<0.0001) and caused cell cycle arrest (induction of G0/1, 1.7-fold, p=0.0003) along with an increase in the expression of CDK inhibitors, p21CIP1 and p27KIP1 at the protein level. Genetic ablation of Xbp1 abrogated IL-7 receptor (IL-7R) signaling by reducing the phosphorylation levels of STAT5-Y694 and JAK1-Y1022/Y1023. In an additional approach, we revealed that IL-7-deprived pre-B ALL cells reduce the mRNA expression of Xbp1s, indicating that Xbp1 acts as a downstream linchpin of the IL-7 receptor signaling pathway. Both IL-7-deprivation and genetic loss of Xbp1 increased the phosphorylation levels of ERK1/2-T202/Y204, AKT-S473 and the protein levels of NRASG12D and MAPK negative regulator DUSP6. Pharmacological inhibition of XBP1 activation using MKC-8866 resulted in similar effects on the expression of RAS downstream targets. We therefore tested MKC-8866 in combination with MEK inhibition by PD0325901 as a potential therapeutic strategy against pre-B ALL, which proved non-efficient. As a second option with therapeutic implications, we focused on the PI3K pathway which acts downstream of both the IL-7R and RAS signaling pathways. Strikingly, we observed that genetic ablation of Xbp1 (viable cells after 72 h, BEZ: 71.9 ± 9.0 vs BEZ+ Mx1-Cre;Xbp1fl/f: 10.0 ± 4.9) or pharmacological inhibition of its production with MKC-8866 (viable cells after five days, BEZ: 58.0 ± 6.8 vs BEZ+ MKC-8866: 13.3 ± 7.4) sensitizes pre-B ALL to dual inhibition of PI3K/mTOR with BEZ235. By applying the Bliss formula, we were able to show that BEZ235 in combination with MKC-8866 synergistically reduces the viability of RAS-mutated pre-B ALL cells. Gene expression analysis indicated that BEZ235 in combination with MKC-8866 fully blocked IL-7R signaling and caused an aberrant activation of Ras-Erk signaling. Targeting PI3K/mTOR signaling along with XBP1 inactivation increased expression of NRASG12D and its target DUSP6. In addition, we showed that combined therapy increased expression levels of p19Arf in RAS-mutated pre-B ALL, implicating cell senescence mediated by activated RAS signaling. Conclusion Our work strongly supports the hypothesis that XBP1 induces its positive effects on progression of pre-B ALL cells through the IL-7R signaling pathway. IL-7R signaling through its downstream effector XBP1 counteracts the RAS signaling pathway to promote leukemogenesis in pre-B ALL cells. Active XBP1 prevents the cytotoxic effects of BEZ235 in pre-B ALL cells, and hence targeting XBP1 in combination with dual PI3K/mTOR inhibition by BEZ235 appears as a promising targeted strategy against the "undruggable" driver RAS in NRASG12D-mutated pre-B ALL. Disclosures Brümmendorf: Janssen: Consultancy; Merck: Consultancy; Novartis: Consultancy, Other: travel, accommodation, expenses, Research Funding; Takeda: Consultancy; Pfizer: Consultancy, Honoraria, Other: Travel, Accommodation, Expenses, Research Funding.