Virus-Induced Gene Silencing as a Tool for Comparative Functional Studies in Thalictrum

Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group

The relationship between early neural responses to emotional faces at age 3 and later autism and anxiety symptoms in adolescents with autism

Both autism spectrum (ASD) and anxiety disorders are associated with atypical neural and attentional responses to emotional faces, differing in affective face processing from typically developing peers. Within a longitudinal study of children with ASD (23 male, 3 female), we hypothesized that early ERPs to emotional faces would predict concurrent and later ASD and anxiety symptoms. Greater response amplitude to fearful faces corresponded to greater social communication difficulties at age 3, and less improvement by age 14. Faster ERPs to neutral faces predicted greater ASD symptom improvement over time, lower ASD severity in adolescence, and lower anxiety in adolescence. Early individual differences in processing of emotional stimuli likely reflect a unique predictive contribution from social brain circuitry early in life

Acquisition of Chemoresistance in Gliomas Is Associated with Increased Mitochondrial Coupling and Decreased ROS Production

Temozolomide (TMZ) is an alkylating agent used for treating gliomas. Chemoresistance is a severe limitation to TMZ therapy; there is a critical need to understand the underlying mechanisms that determine tumor response to TMZ. We recently reported that chemoresistance to TMZ is related to a remodeling of the entire electron transport chain, with significant increases in the activity of complexes II/III and cytochrome c oxidase (CcO). Moreover, pharmacologic and genetic manipulation of CcO reverses chemoresistance. Therefore, to test the hypothesis that TMZ-resistance arises from tighter mitochondrial coupling and decreased production of reactive oxygen species (ROS), we have assessed mitochondrial function in TMZ-sensitive and -resistant glioma cells, and in TMZ-resistant glioblastoma multiform (GBM) xenograft lines (xenolines). Maximum ADP-stimulated (state 3) rates of mitochondrial oxygen consumption were greater in TMZ-resistant cells and xenolines, and basal respiration (state 2), proton leak (state 4), and mitochondrial ROS production were significantly lower in TMZ-resistant cells. Furthermore, TMZ-resistant cells consumed less glucose and produced less lactic acid. Chemoresistant cells were insensitive to the oxidative stress induced by TMZ and hydrogen peroxide challenges, but treatment with the oxidant L-buthionine-S,R-sulfoximine increased TMZ-dependent ROS generation and reversed chemoresistance. Importantly, treatment with the antioxidant N-acetyl-cysteine inhibited TMZ-dependent ROS generation in chemosensitive cells, preventing TMZ toxicity. Finally, we found that mitochondrial DNA-depleted cells (ρ°) were resistant to TMZ and had lower intracellular ROS levels after TMZ exposure compared with parental cells. Repopulation of ρ° cells with mitochondria restored ROS production and sensitivity to TMZ. Taken together, our results indicate that chemoresistance to TMZ is linked to tighter mitochondrial coupling and low ROS production, and suggest a novel mitochondrial ROS-dependent mechanism underlying TMZ-chemoresistance in glioma. Thus, perturbation of mitochondrial functions and changes in redox status might constitute a novel strategy for sensitizing glioma cells to therapeutic approaches

Allergenicity assessment of genetically modified crops—what makes sense?

GM crops have great potential to improve food quality, increase harvest yields and decrease dependency on certain chemical pesticides. Before entering the market their safety needs to be scrutinized. This includes a detailed analysis of allergenic risks, as the safety of allergic consumers has high priority. However, not all tests currently being applied to assessing allergenicity have a sound scientific basis. Recent events with transgenic crops reveal the fallacy of applying such tests to GM crops

Non-irradiation-derived reactive oxygen species (ROS) and cancer: therapeutic implications

Owing to their chemical reactivity, radicals have cytocidal properties. Destruction of cells by irradiation-induced radical formation is one of the most frequent interventions in cancer therapy. An alternative to irradiation-induced radical formation is in principle drug-induced formation of radicals, and the formation of toxic metabolites by enzyme catalysed reactions. Although these developments are currently still in their infancy, they nevertheless deserve consideration. There are now numerous examples known of conventional anti-cancer drugs that may at least in part exert cytotoxicity by induction of radical formation. Some drugs, such as arsenic trioxide and 2-methoxy-estradiol, were shown to induce programmed cell death due to radical formation. Enzyme-catalysed radical formation has the advantage that cytotoxic products are produced continuously over an extended period of time in the vicinity of tumour cells. Up to now the enzymatic formation of toxic metabolites has nearly exclusively been investigated using bovine serum amine oxidase (BSAO), and spermine as substrate. The metabolites of this reaction, hydrogen peroxide and aldehydes are cytotoxic. The combination of BSAO and spermine is not only able to prevent tumour cell growth, but prevents also tumour growth, particularly well if the enzyme has been conjugated with a biocompatible gel. Since the tumour cells release substrates of BSAO, the administration of spermine is not required. Combination with cytotoxic drugs, and elevation of temperature improves the cytocidal effect of spermine metabolites. The fact that multidrug resistant cells are more sensitive to spermine metabolites than their wild type counterparts makes this new approach especially attractive, since the development of multidrug resistance is one of the major problems of conventional cancer therapy