Conditional transgenesis using Dimerizable Cre (DiCre)

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCrexR26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters.     

Tumor necrosis factor alpha and adalimumab differentially regulate CD36 expression in human monocytes

In chronic inflammatory diseases, such as rheumatoid arthritis, inflammation acts as an independent cardiovascular risk factor and the use of anti-inflammatory drugs, such as anti-tumor necrosis factor alpha (anti-TNFα), may decrease this risk. The phagocytosis of oxidized low density lipoproteins (LDLs) accumulated in the subendothelium by mononuclear cells influences atherosclerosis and depends on CD36 expression. We investigated the role of TNFα and adalimumab, a human anti-TNFα monoclonal antibody widely used in human pathology, in CD36 expression in human monocytes. Human monocytes were prepared by adherence from whole-blood buffy-coat fractions from healthy donors. CD36 expression was assessed by RT-PCR and flow cytometry, with various TNFα or adalimumab concentrations. Implication of peroxisome proliferator-activated receptor (PPAR)γ in the regulation of CD36 expression was assessed using specific inhibitor or gel shift assays. The impact of redox signaling was investigated using quantification of reactive oxygen species, antioxidant and a NADPH oxidase inhibitor. The F(ab')2 fragment of adalimumab was isolated and its effect was analyzed. TNFα inhibits both CD36 membrane expression and mRNA expression. This inhibition involves a reduction in PPARγ activation. In contrast, adalimumab increases both CD36 membrane expression and mRNA expression. This induction is independent of the Fc portion of adalimumab and involves redox signaling via NADPH oxidase activation. CD36 expression on human monocytes is inhibited by TNFα and independently increased by adalimumab. These data highlight that pro-inflammatory cytokines and their specific neutralization influence the expression of cellular receptors implicated in atherosclerosis. Further studies are needed to investigate the clinical implications of these results in accelerated atherosclerosis observed in rheumatoid arthritis.     

In vivo noninvasive optical imaging of receptor-mediated RGD internalization using self-quenched Cy5-labeled RAFT-c(-RGDfK-)(4)

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)(4 )presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin alpha(V)beta(3) with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a "stealth" molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)(4)-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)(4)-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)(4)-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)(4)-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)(4)-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization.     

Spontaneous duplications in diploid Saccharomyces cerevisiae cells

The duplication of DNA sequences is a powerful determinant of genomic plasticity and is known to be one of the key factors responsible for evolution. Recent genomic sequence data demonstrate the abundance of duplicated genes in all surveyed organisms. Over the past years, experimental systems were adequately designed to explore the molecular mechanisms involved in their formation in haploid Saccharomyces cerevisiae strains. To obtain a more global and accurate view of the events leading to DNA sequence duplications, we have selected and characterized duplication occurrences in diploid S. cerevisiae cells. The molecular analysis showed that two other predominant ways lead to duplication in this context: formation of extra chimeric chromosomes and non-reciprocal translocation events. Moreover, we demonstrated that these two types of rearrangements are RAD52 independent and therefore that homologous recombination plays no part in their formation. Finally, our results show the multiplicity of mechanisms involved in duplication events and provide the first experimental evidence that these mechanisms might be ploidy dependent.     

Evaluation of the influence of protein precipitation prior to on-line SPE-LC-API/MS procedures using multivariate data analysis

Matrix effects on mass spectrometry (MS) response were investigated with three atmospheric pressure ionization (API) sources after on-line solid-phase extraction (SPE) of human plasma. On-line SPE was evaluated with one restricted access material (RAM), two large particle supports (LPS) and one monolith. A sample protein precipitation (PP) with acetonitrile (2:1) and a direct injection were tested. Principal component analysis (PCA) was performed to simplify data presentation and interpretation. Protein precipitation was found to be mandatory for reducing signal modification. Regarding sensitivity towards matrix effects after PP, atmospheric pressure photoionization (APPI) was globally the least sensitive ionization mode while electrospray ionization ESI was the most sensitive.     

Quantitative metabolic profiles of tomato flesh and seeds during fruit development: complementary analysis with ANN and PCA

Tomato, an essential crop in terms of economic importance and nutritional quality, is also used as a model species for all fleshy fruits and for genomics of Solanaceae. Tomato fruit quality at harvest is a direct function of its metabolite content, which in turn is a result of many physiological changes during fruit development. The aim of the work reported here was to develop a global approach to characterize changes in metabolic profiles in two interdependent tissues from the same tomato fruits. Absolute quantification data of compounds in flesh and seeds from 8 days to 45 days post anthesis (DPA) were obtained through untargeted (proton nuclear magnetic resonance, ¹H-NMR) and targeted metabolic profiling (liquid chromatography with diode array detection (LC-DAD) or gas chromatography with flame ionization detection (GC-FID)). These data were analyzed with chemometric approaches. Kohonen self organizing maps (SOM) analysis of these data allowed us to combine multivariate (distribution of samples on Kohonen SOMs) and univariate information (component plane representation of metabolites) in a single analysis. This strategy confirmed published data and brought new insights on tomato flesh and seed composition, thus demonstrating its potential in metabolomics. The compositional changes were related to physiological processes occurring in each tissue. They pointed to (i) some parallel changes at early stages in relation to cell division and transitory storage of carbon, (ii) metabolites participating in the fleshy trait and (iii) metabolites involved in the specific developmental patterns of the seeds.     

Identification and distribution of the NBS-LRR gene family in the Cassava genome

Background

Plant resistance genes (R genes) exist in large families and usually contain both a nucleotide-binding site domain and a leucine-rich repeat domain, denoted NBS-LRR. The genome sequence of cassava (Manihot esculenta) is a valuable resource for analysing the genomic organization of resistance genes in this crop.

Results

With searches for Pfam domains and manual curation of the cassava gene annotations, we identified 228 NBS-LRR type genes and 99 partial NBS genes. These represent almost 1% of the total predicted genes and show high sequence similarity to proteins from other plant species. Furthermore, 34 contained an N-terminal toll/interleukin (TIR)-like domain, and 128 contained an N-terminal coiled-coil (CC) domain. 63% of the 327 R genes occurred in 39 clusters on the chromosomes. These clusters are mostly homogeneous, containing NBS-LRRs derived from a recent common ancestor.

Conclusions

This study provides insight into the evolution of NBS-LRR genes in the cassava genome; the phylogenetic and mapping information may aid efforts to further characterize the function of these predicted R genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1554-9) contains supplementary material, which is available to authorized users.     

A Physiological and Behavioral Mechanism for Leaf Herbivore-Induced Systemic Root Resistance

Indirect plant-mediated interactions between herbivores are important drivers of community composition in terrestrial ecosystems. Among the most striking examples are the strong indirect interactions between spatially separated leaf- and root-feeding insects sharing a host plant. Although leaf feeders generally reduce the performance of root herbivores, little is known about the underlying systemic changes in root physiology and the associated behavioral responses of the root feeders. We investigated the consequences of maize (Zea mays) leaf infestation by Spodoptera littoralis caterpillars for the root-feeding larvae of the beetle Diabrotica virgifera virgifera, a major pest of maize. D. virgifera strongly avoided leaf-infested plants by recognizing systemic changes in soluble root components. The avoidance response occurred within 12 h and was induced by real and mimicked herbivory, but not wounding alone. Roots of leaf-infested plants showed altered patterns in soluble free and soluble conjugated phenolic acids. Biochemical inhibition and genetic manipulation of phenolic acid biosynthesis led to a complete disappearance of the avoidance response of D. virgifera. Furthermore, bioactivity-guided fractionation revealed a direct link between the avoidance response of D. virgifera and changes in soluble conjugated phenolic acids in the roots of leaf-attacked plants. Our study provides a physiological mechanism for a behavioral pattern that explains the negative effect of leaf attack on a root-feeding insect. Furthermore, it opens up the possibility to control D. virgifera in the field by genetically mimicking leaf herbivore-induced changes in root phenylpropanoid patterns.Insect herbivores constantly compete for plants as a primary terrestrial source of organic carbon and nitrogen (Denno et al., 1995). Consequently, resource competition is thought to be a major determinant of the distribution and abundance of insects in natural and agricultural systems (Begon et al., 2006). Recent evidence suggests, however, that in many cases, insect herbivore competition may not follow the traditional theoretical assumptions of direct interference and/or resource exploitation, but may be determined by indirect plant-mediated effects (Kaplan and Denno, 2007; Poelman et al., 2008). Among the most striking examples of indirect plant-mediated interactions is the interplay between root- and leaf-feeding insects (Blossey and Hunt-Joshi, 2003). Despite their nonoverlapping feeding niches, leaf and root herbivores determine each other’s performance through shared host plants (Bezemer and van Dam, 2005). Although root feeders can have positive or negative effects on leaf feeders (van Dam and Heil, 2011), the effect of leaf herbivores on root consumers is predominantly negative (Johnson et al., 2012; Huang et al., 2014).Despite the increasing number of examples demonstrating negative effects of leaf attack on root herbivores (Tindall and Stout, 2001; Blossey and Hunt-Joshi, 2003; Soler et al., 2007; Gill et al., 2011), the mechanisms underlying this form of systemic induced resistance remain poorly understood (Erb et al., 2008; Rasmann and Agrawal, 2008). Pieris brassicae, for instance, was found to increase glucosinolate levels in the roots, which correlated with a reduced survival of the root feeder Delia radicum (Soler et al., 2007). Understanding why root feeders perform worse on leaf-infested plants would allow for more detailed investigations regarding the adaptive and evolutionary context of the phenomenon, and may allow for its exploitation in agriculture (for instance, by triggering root resistance through targeted leaf treatments).A promising system to study the mechanisms and agroecological consequences of plant-mediated interactions between herbivores is maize (Zea mays) and its associated pests. In the field, maize is attacked by a suite of herbivores, including leaf feeders, stem borers, and root feeders. The highly specialized root-feeding larvae of the western corn rootworm Diabrotica virgifera virgifera cause significant plant damage and yield loss in the United States and Eastern Europe. Earlier studies demonstrated that D. virgifera attack increases leaf resistance against Spodoptera spp. by triggering drought stress responses (Erb et al., 2009, 2011b). In the opposite direction, leaf feeding by Spodoptera spp. caterpillars reduces D. virgifera growth and development in a sequence-specific manner in the laboratory and the field (Erb et al., 2011c; Gill et al., 2011). D. virgifera was subsequently demonstrated to avoid leaf-infested plants by detecting and responding to a reduction in root ethylene emissions (Robert et al., 2012). However, it remains unclear whether nonvolatile chemical changes in the roots of leaf-infested maize plants affect D. virgifera foraging and performance. In this study, we explored the hypothesis that leaf infestation by Spodoptera spp. caterpillars triggers a short-range avoidance response in D. virgifera. Through a combination of bioactivity-guided fractionation of root extracts and biochemical and molecular manipulation, we show that systemic changes in soluble phenylpropanoid derivatives trigger a strong avoidance response in D. virgifera. We furthermore demonstrate that this avoidance response is mediated by systemic internal signals and is triggered specifically by herbivory, suggesting that D. virgifera actively and specifically recognizes and avoids leaf-infested plants.