Differential roles of glucosinolates and camalexin at different stages of Agrobacterium‐mediated transformation

Agrobacterium tumefaciens is the causal agent of crown gall disease in a wide range of plants via a unique interkingdom DNA transfer from bacterial cells into the plant genome. Agrobacterium tumefaciens is capable of transferring its T‐DNA into different plant parts at different developmental stages for transient and stable transformation. However, the plant genes and mechanisms involved in these transformation processes are not well understood. We used Arabidopsis thaliana Col‐0 seedlings to reveal the gene expression profiles at early time points during Agrobacterium infection. Common and differentially expressed genes were found in shoots and roots. A gene ontology analysis showed that the glucosinolate (GS) biosynthesis pathway was an enriched common response. Strikingly, several genes involved in indole glucosinolate (iGS) modification and the camalexin biosynthesis pathway were up‐regulated, whereas genes in aliphatic glucosinolate (aGS) biosynthesis were generally down‐regulated, on Agrobacterium infection. Thus, we evaluated the impacts of GSs and camalexin during different stages of Agrobacterium‐mediated transformation combining Arabidopsis mutant studies, metabolite profiling and exogenous applications of various GS hydrolysis products or camalexin. The results suggest that the iGS hydrolysis pathway plays an inhibitory role on transformation efficiency in Arabidopsis seedlings at the early infection stage. Later in the Agrobacterium infection process, the accumulation of camalexin is a key factor inhibiting tumour development on Arabidopsis inflorescence stalks. In conclusion, this study reveals the differential roles of GSs and camalexin at different stages of Agrobacterium‐mediated transformation and provides new insights into crown gall disease control and improvement of plant transformation.     

Improvement of the Fungal Biocontrol Agent Trichoderma atroviride To Enhance both Antagonism and Induction of Plant Systemic Disease Resistance

Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of Trichoderma atroviride, which expresses the Aspergillus niger glucose oxidase-encoding gene, goxA, under a homologous chitinase (nag1) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less N-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of Botrytis cinerea. The transgenic strain also more quickly overgrew and lysed the plant pathogens Rhizoctonia solani and Pythium ultimum. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic Trichoderma strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen B. cinerea. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as Trichoderma spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens.     

Wheat sprout extract-induced apoptosis in human cancer cells by proteasomes modulation

Natural occurring modulators of proteasome functionality are extensively investigated for their implication in cancer therapy. On the basis of our previous evidences both on proteasomal inhibition by monomeric polyphenols, and on the characterization of wheat sprout hydroalcoholic extract, herein we thoroughly report on a comparative study of the effect of wheat sprout extract on both normal and tumour cells. Treatment of isolated 20S proteasomes with wheat sprout extracts induced a gradual inhibition of all proteasome activities. Next, two wheat sprout extract components were separated: a polyphenol and a protein fraction. Both components exerted an in vitro inhibitory effect on proteasome activity. HeLa tumour cells and FHs 74 Int normal cells were exposed to both fractions, resulting in different rates of proteasome inhibition, with tumour cells showing a significantly higher degree of proteasome impairment and apoptosis induction. Furthermore, a decrease in proteasome activities and in cell survival of the human plasmacytoma RPMI 8226 cell line, upon the same treatments, was observed. Collectively, our results provide additional evidences supporting the possible use of natural extracts as coadjuvants in cancer treatments.     

Exploring the HME and HAE1 efflux systems in the genus <Emphasis Type="Italic">Burkholderia</Emphasis>

Background  

The genus Burkholderia includes a variety of species with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role could be played by multidrug efflux pumps belonging to Resistance Nodulation Cell-Division (RND) family, which allow bacterial cells to extrude a wide range of different substrates, including antibiotics. This study aims to i) identify rnd genes in the 21 available completely sequenced Burkholderia genomes, ii) analyze their phylogenetic distribution, iii) define the putative function(s) that RND proteins perform within the Burkholderia genus and iv) try tracing the evolutionary history of some of these genes in Burkholderia.     

A sea urchin in vivo model to evaluate Epithelial‐Mesenchymal Transition

Epithelial‐mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity of the sea urchin embryo to anti‐EMT drugs and pinpoint the sea urchin embryo as a valuable in vivo model system for studying EMT and the screening of anti‐EMT candidates.     

Nanovesicles Are Secreted during Pollen Germination and Pollen Tube Growth： A Possible Role in Fertilization

Dear Editor, During recent decades, a novel mechanism of secretion has been identified in a wide range of mammalian cells. It involves the release of bioactive membrane nanovesicles （30-100 nm）, termed exosomes, upon the fusion of multivesicular bodies with the plasma membrane （Thery et al., 2009）. Exosomes are implicated in diverse functions, such as scavenging of archaic proteins, intercellular messengers delivering cell-specific signals, and vehicles for transmissible pathogens. Exosomes have also been described in other organisms such as bacte- ria, Drosophila, and fungi.     

Structural Integrity of the Contralesional Hemisphere Predicts Cognitive Impairment in Ischemic Stroke at Three Months

After stroke, white matter integrity can be affected both locally and distally to the primary lesion location. It has been shown that tract disruption in mirror’s regions of the contralateral hemisphere is associated with degree of functional impairment. Fourteen patients suffering right hemispheric focal stroke (S) and eighteen healthy controls (HC) underwent Diffusion Weighted Imaging (DWI) and neuropsychological assessment. The stroke patient group was divided into poor (SP; n = 8) and good (SG; n = 6) cognitive recovery groups according to their cognitive improvement from the acute phase (72 hours after stroke) to the subacute phase (3 months post-stroke). Whole-brain DWI data analysis was performed by computing Diffusion Tensor Imaging (DTI) followed by Tract Based Spatial Statistics (TBSS). Assessment of effects was obtained computing the correlation of the projections on TBSS skeleton of Fractional Anisotropy (FA) and Radial Diffusivity (RD) with cognitive test results. Significant decrease of FA was found only in right brain anatomical areas for the S group when compared to the HC group. Analyzed separately, stroke patients with poor cognitive recovery showed additional significant FA decrease in several left hemisphere regions; whereas SG patients showed significant decrease only in the left genu of corpus callosum when compared to the HC. For the SG group, whole brain analysis revealed significant correlation between the performance in the Semantic Fluency test and the FA in the right hemisphere as well as between the performance in the Grooved Pegboard Test (GPT) and theTrail Making Test-part A and the FA in the left hemisphere. For the SP group, correlation analysis revealed significant correlation between the performance in the GPT and the FA in the right hemisphere.     

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.Subject terms: Cancer metabolism, CNS cancer