C4 Photosynthesis evolved in grasses via parallel adaptive genetic changes.

Phenotypic convergence is a widespread and well-recognized evolutionary phenomenon. However, the responsible molecular mechanisms remain often unknown mainly because the genes involved are not identified. A well-known example of physiological convergence is the C4 photosynthetic pathway, which evolved independently more than 45 times [1]. Here, we address the question of the molecular bases of the C4 convergent phenotypes in grasses (Poaceae) by reconstructing the evolutionary history of genes encoding a C4 key enzyme, the phosphoenolpyruvate carboxylase (PEPC). PEPC genes belong to a multigene family encoding distinct isoforms of which only one is involved in C4 photosynthesis [2]. By using phylogenetic analyses, we showed that grass C4 PEPCs appeared at least eight times independently from the same non-C4 PEPC. Twenty-one amino acids evolved under positive selection and converged to similar or identical amino acids in most of the grass C4 PEPC lineages. This is the first record of such a high level of molecular convergent evolution, illustrating the repeatability of evolution. These amino acids were responsible for a strong phylogenetic bias grouping all C4 PEPCs together. The C4-specific amino acids detected must be essential for C4 PEPC enzymatic characteristics, and their identification opens new avenues for the engineering of the C4 pathway in crops.

Tumor sensitizing function and mechanism of action of a RasGAP derived peptide

Cancer is the second cause of death after cardio-vascular diseases in economically developed countries. Two of the most commonly used anti-cancer therapies are chemo and radiotherapy. Despite the remarkable advances made in term of delivery and specificity of these two anti-tumor regimens, their toxicity towards healthy tissue remains a limitation. A promising approach to overcome this obstacle would be the utilization of therapeutic peptides that specifically augment the sensitivity of tumoral cells to treatments. Lower therapeutical doses would then be required to kill malignant cells, limiting toxic effects on healthy tissues. It was previously shown in our laboratory that the caspase-3 generated fragment N2 of RasGAP is able to potentiate the genotoxin-induced apoptosis selectively in cancer cells. In this work we show that fragment N2 strictly requires a cytoplasmic localization to deliver its pro-apoptotic effect in genotoxin-treated cancer cells. The tumor sensitizing capacity of fragment N2 was found to reside within the 10 amino acid sequence 317-326. Our laboratory earlier demonstrated that a peptide corresponding to amino acids 317 to 326 of RasGAP fused to the TAT cell permeable moiety, called TAT-RasGAP317.326, is able to sensitize cancer cells, but not normal cells, to genotoxin-induced apoptosis. In the present study we describe the capacity of TAT-RasGAP 317.326 to sensitize tumors to both chemo and radiotherapy in an in vivo mouse model. The molecular mechanism underlying the TAT-RasGAP 317.326-mediated sensitization starts now to be elucidated. We demonstrate that G3BP1, an endoribonuclease binding to amino acids 317-326 of RasGAP, is not involved in the sensitization mechanism. We also provide evidence showing that TAT-RasGAP3 17-326 potentiates the genotoxin-mediated activation of Bax in a tBid-dependent manner. Altogether our results show that TAT-RasGAP 317.326 could be potentially used in cancer therapy as sensitizer, in order to improve the efficacy of chemo and radiotherapy and prolong the life expectancy of cancer patients. Moreover, the understanding of the TAT-RasGAP317.326 mode of action might help to unravel the mechanisms by which cancer cells resist to chemo and radiotherapy and therefore to design more targeted and efficient anti-tumoral strategies.

Coronary artery anomalies and variants: technical feasibility of assessment with coronary MR angiography at 3 T.

The purpose of this study was to prospectively use a whole-heart three-dimensional (3D) coronary magnetic resonance (MR) angiography technique specifically adapted for use at 3 T and a parallel imaging technique (sensitivity encoding) to evaluate coronary arterial anomalies and variants (CAAV). This HIPAA-compliant study was approved by the local institutional review board, and informed consent was obtained from all participants. Twenty-two participants (11 men, 11 women; age range, 18-62 years) were included. Ten participants were healthy volunteers, whereas 12 participants were patients suspected of having CAAV. Coronary MR angiography was performed with a 3-T MR imager. A 3D free-breathing navigator-gated and vector electrocardiographically-gated segmented k-space gradient-echo sequence with adiabatic T2 preparation pulse and parallel imaging (sensitivity encoding) was used. Whole-heart acquisitions (repetition time msec/echo time msec, 4/1.35; 20 degrees flip angle; 1 x 1 x 2-mm acquired voxel size) lasted 10-12 minutes. Mean examination time was 41 minutes +/- 14 (standard deviation). Findings included aneurysms, ectasia, arteriovenous fistulas, and anomalous origins. The 3D whole-heart acquisitions developed for use with 3 T are feasible for use in the assessment of CAAV.

Correlations of glycogen synthase and phosphorylase activities with glycogen concentration in human muscle biopsies. Evidence for a double-feedback mechanism regulating glycogen synthesis and breakdown.

The purpose of this study was to verify in man the relationships of muscle glycogen synthase and phosphorylase activities with glycogen concentration that were reported in animal studies. The upper level of glycogen concentration in muscle is known to be tightly controlled, and glycogen concentration was reported to have an inhibitory effect on synthase activity and a stimulatory effect on phosphorylase activity. Glycogen synthase and phosphorylase activity and glycogen concentration were measured in muscle biopsies in a group of nine normal subjects after stimulating an increase of their muscle glycogen concentration through either an intravenous glucose-insulin infusion to stimulate glycogen synthesis, or an Intralipid (Vitrum, Stockholm, Sweden) infusion in the basal state to inhibit glycogen mobilization by favoring lipid oxidation at the expense of glucose oxidation. Phosphorylase activity increased from 71.3 +/- 21.0 to 152.8 +/- 20.0 nmol/min/mg protein (P < .005) after the glucose-insulin infusion. Phosphorylase activity was positively correlated with glycogen concentration (P = .005 and P = .0001) after the glucose-insulin and Intralipid infusions, respectively. Insulin-stimulated glycogen synthase activity was significantly negatively correlated with glycogen concentration at the end of the Intralipid infusion (P < .005). In conclusion, by demonstrating a negative correlation of glycogen concentration with glycogen synthase and a positive correlation with phosphorylase, this study might confirm in man the double-feedback mechanism by which changes in glycogen concentration regulate glycogen synthase and phosphorylase activities. It suggests that this mechanism might play an important role in the regulation of glucose storage.

Identification and mutational analyses of phosphorylation sites of the calcineurin-binding protein CbpA and the identification of domains required for calcineurin binding in Aspergillus fumigatus.

Calcineurin is a key protein phosphatase required for hyphal growth and virulence in Aspergillus fumigatus, making it an attractive antifungal target. However, currently available calcineurin inhibitors, FK506 and cyclosporine A, are immunosuppressive, limiting usage in the treatment of patients with invasive aspergillosis. Therefore, the identification of endogenous inhibitors of calcineurin belonging to the calcipressin family is an important parallel strategy. We previously identified the gene cbpA as the A. fumigatus calcipressin member and showed its involvement in hyphal growth and calcium homeostasis. However, the mechanism of its activation/inhibition through phosphorylation and its interaction with calcineurin remains unknown. Here we show that A. fumigatus CbpA is phosphorylated at three distinct domains, including the conserved SP repeat motif (phosphorylated domain-I; PD-I), a filamentous fungal-specific domain (PD-II), and the C-terminal CIC motif (Calcipressin Inhibitor of Calcineurin; PD-III). While mutation of three phosphorylated residues (Ser208, Ser217, Ser223) in the PD-II did not affect CbpA function in vivo, mutation of the two phosphorylated serines (Ser156, Ser160) in the SP repeat motif caused reduced hyphal growth and sensitivity to oxidative stress. Mutational analysis in the key domains in calcineurin A (CnaA) and proteomic interaction studies confirmed the requirement of PxIxIT motif-binding residues (352-NIR-354) and the calcineurin B (CnaB)-binding helix residue (V371) for the binding of CbpA to CnaA. Additionally, while the calmodulin-binding residues (442-RVF-444) did not affect CbpA binding to CnaA, three mutations (T359P, H361L, and L365S) clustered between the CnaA catalytic and the CnaB-binding helix were also required for CbpA binding. This is the first study to analyze the phosphorylation status of calcipressin in filamentous fungi and identify the domains required for binding to calcineurin.

Parallel feedback loops control the basal activity of the HOG MAPK signaling cascade.

Tight regulation of the MAP kinase Hog1 is crucial for survival under changing osmotic conditions. Interestingly, we found that Hog1 phosphorylates multiple upstream components, implying feedback regulation within the signaling cascade. Taking advantage of an unexpected link between glucose availability and Hog1 activity, we used quantitative single cell measurements and computational modeling to unravel feedback regulation operating in addition to the well-known adaptation feedback triggered by glycerol accumulation. Indeed, we found that Hog1 phosphorylates its activating kinase Ssk2 on several sites, and cells expressing a non-phosphorylatable Ssk2 mutant are partially defective for feedback regulation and proper control of basal Hog1 activity. Together, our data suggest that Hog1 activity is controlled by intertwined regulatory mechanisms operating with varying kinetics, which together tune the Hog1 response to balance basal Hog1 activity and its steady-state level after adaptation to high osmolarity.

Diploid males support a two-step mechanism of endosymbiont-induced thelytoky in a parasitoid wasp.

BACKGROUND: Haplodiploidy, where females develop from diploid, fertilized eggs and males from haploid, unfertilized eggs, is abundant in some insect lineages. Some species in these lineages reproduce by thelytoky that is caused by infection with endosymbionts: infected females lay haploid eggs that undergo diploidization and develop into females, while males are very rare or absent. It is generally assumed that in thelytokous wasps, endosymbionts merely diploidize the unfertilized eggs, which would then trigger female development. RESULTS: We found that females in the parasitoid wasp Asobara japonica infected with thelytoky-inducing Wolbachia produce 0.7-1.2 % male offspring. Seven to 39 % of these males are diploid, indicating that diploidization and female development can be uncoupled in A. japonica. Wolbachia titer in adults was correlated with their ploidy and sex: diploids carried much higher Wolbachia titers than haploids, and diploid females carried more Wolbachia than diploid males. Data from introgression lines indicated that the development of diploid individuals into males instead of females is not caused by malfunction-mutations in the host genome but that diploid males are most likely produced when the endosymbiont fails to activate the female sex determination pathway. Our data therefore support a two-step mechanism by which endosymbionts induce thelytoky in A. japonica: diploidization of the unfertilized egg is followed by feminization, whereby each step correlates with a threshold of endosymbiont titer during wasp development. CONCLUSIONS: Our new model of endosymbiont-induced thelytoky overthrows the view that certain sex determination mechanisms constrain the evolution of endosymbiont-induced thelytoky in hymenopteran insects. Endosymbionts can cause parthenogenesis through feminization, even in groups in which endosymbiont-diploidized eggs would develop into males following the hosts' sex determination mechanism. In addition, our model broadens our understanding of the mechanisms by which endosymbionts induce thelytoky to enhance their transmission to the next generation. Importantly, it also provides a novel window to study the yet-poorly known haplodiploid sex determination mechanisms in haplodiploid insects.

Biological variation of established and novel biomarkers for atherosclerosis: Results from a prospective, parallel-group cohort study.

BACKGROUND: Biomarkers are a promising tool for the management of patients with atherosclerosis, but their variation is largely unknown. We assessed within-subject and between-subject biological variation of biomarkers in peripheral artery disease (PAD) patients and healthy controls, and defined which biomarkers have a favorable variation profile for future studies. METHODS: Prospective, parallel-group cohort study, including 62 patients with stable PAD (79% men, 65±7years) and 18 healthy control subjects (44% men, 57±7years). Blood samples were taken at baseline, and after 3-, 6-, and 12-months. We calculated within-subject (CVI) and between-subject (CVG) coefficients of variation and intra-class correlation coefficient (ICC). RESULTS: Mean levels of D-dimer, hs-CRP, IL-6, IL-8, MMP-9, MMP-3, S100A8/A9, PAI-1, sICAM-1, and sP-selectin levels were higher in PAD patients than in healthy controls (P≤.05 for all). CVI and CVG of the different biomarkers varied considerably in both groups. An ICC≥0.5 (indicating moderate-to-good reliability) was found for hs-CRP, D-Dimer, E-selectin, IL-10, MCP-1, MMP-3, oxLDL, sICAM-1 and sP-selectin in both groups, for sVCAM in healthy controls and for MMP-9, PAI-1 and sCD40L in PAD patients. CONCLUSIONS: Single biomarker measurements are of limited utility due to large within-subject variation, both in PAD patients and healthy subjects. D-dimer, hs-CRP, MMP-9, MMP-3, PAI-1, sP-selectin and sICAM-1 are biomarkers with both higher mean levels in PAD patients and a favorable variation profile making them most suitable for future studies.