Two-Dimensional Turbulence with Local Interactions: Statistics of the Condensate.

Two-dimensional turbulence self-organizes through a process of energy accumulation at large scales, forming a coherent flow termed a condensate. We study the condensate in a model with local dynamics, the large-scale quasigeostrophic equation, observed here for the first time. We obtain analytical results for the mean flow and the two-point, second-order correlation functions, and validate them numerically. The condensate state requires partiy+time-reversal symmetry breaking. We demonstrate distinct universal mechanisms for the even and odd correlators under this symmetry. We find that the model locality is imprinted in the small scale dynamics, which the condensate spatially confines.

Statistics of inhomogeneous turbulence in large-scale quasigeostrophic dynamics.

A remarkable feature of two-dimensional turbulence is the transfer of energy from small to large scales. This process can result in the self-organization of the flow into large, coherent structures due to energy condensation at the largest scales. We investigate the formation of this condensate in a quasigeostropic flow in the limit of small Rossby deformation radius, namely the large-scale quasigeostrophic model. In this model potential energy is transferred up-scale while kinetic energy is transferred down-scale in a direct cascade. We focus on a jet mean flow and carry out a thorough investigation of the second-order statistics for this flow, combining a quasilinear analytical approach with direct numerical simulations. We show that the quasilinear approach applies in regions where jets are strong and is able to capture all second-order correlators in that region, including those related to the kinetic energy. This is a consequence of the blocking of the direct cascade by the mean flow in jet regions, suppressing fluctuation-fluctuation interactions. The suppression of the direct cascade is demonstrated using a local coarse-graining approach allowing us to measure space dependent interscale kinetic energy fluxes, which we show are concentrated in between jets in our simulations. We comment on the possibility of a similar direct cascade arrest in other two-dimensional flows, arguing that it is a special feature of flows in which the fluid element interactions are local in space.

Turbulence Statistics in a Two-Dimensional Vortex Condensate.

Disentangling the evolution of a coherent mean-flow and turbulent fluctuations, interacting through the nonlinearity of the Navier-Stokes equations, is a central issue in fluid mechanics. It affects a wide range of flows, such as planetary atmospheres, plasmas, or wall-bounded flows, and hampers turbulence models. We consider the special case of a two-dimensional flow in a periodic box, for which the mean flow, a pair of box-size vortices called "condensate," emerges from turbulence. As was recently shown, a perturbative closure describes correctly the condensate when turbulence is excited at small scales. In this context, we obtain explicit results for the statistics of turbulence, encoded in the Reynolds stress tensor. We demonstrate that the two components of the Reynolds stress, the momentum flux and the turbulent energy, are determined by different mechanisms. It was suggested previously that the momentum flux is fixed by a balance between forcing and mean-flow advection: using unprecedently long numerical simulations, we provide the first direct evidence supporting this prediction. By contrast, combining analytical computations with numerical simulations, we show that the turbulent energy is determined only by mean-flow advection and obtain for the first time a formula describing its profile in the vortex.

Predictability of escape for a stochastic saddle-node bifurcation: When rare events are typical.

Transitions between multiple stable states of nonlinear systems are ubiquitous in physics, chemistry, and beyond. Two types of behaviors are usually seen as mutually exclusive: unpredictable noise-induced transitions and predictable bifurcations of the underlying vector field. Here, we report a different situation, corresponding to a fluctuating system approaching a bifurcation, where both effects collaborate. We show that the problem can be reduced to a single control parameter governing the competition between deterministic and stochastic effects. Two asymptotic regimes are identified: When the control parameter is small (e.g., small noise), deviations from the deterministic case are well described by the Freidlin-Wentzell theory. In particular, escapes over the potential barrier are very rare events. When the parameter is large (e.g., large noise), such events become typical. Unlike pure noise-induced transitions, the distribution of the escape time is peaked around a value which is asymptotically predicted by an adiabatic approximation. We show that the two regimes are characterized by qualitatively different reacting trajectories with algebraic and exponential divergences, respectively.

Approaches targeting the FGF-FGFR system: a review of the recent patent literature and associated advanced therapeutic agents.

Fibroblast growth factor receptors (FGFRs) and associated ligands (FGFs) are a family of well-validated targets for therapeutic interventions notably in cancer diseases in relation to their prominent roles in cell growth, survival, differentiation and angiogenesis. This patent review encompasses all different approaches (modulators of FGF or FGFR expression, anti-FGF antibodies, anti-FGFR antibodies, FGF traps, tyrosine-kinase (TK) inhibitors, allosteric modulators) used to block completely or partially the activities of the FGF-FGFR complexes resulting in clinical drug candidates or tool agents. Comparative analysis of biochemical, pharmacological or clinical data will be discussed for each class of molecules together with some perspectives.

Nonlinear energy transfers and phase diagrams for geostrophically balanced rotating-stratified flows.

Equilibrium statistical mechanics tools have been developed to obtain indications about the natural tendencies of nonlinear energy transfers in two-dimensional and quasi-two-dimensional flows like rotating and stratified flows in geostrophic balance. In this article we consider a simple model of such flows with a nontrivial vertical structure, namely, two-layer quasigeostrophic flows, which remain amenable to analytical study. We obtain the statistical equilibria of the system in the case of a linear vorticity-stream function relation, build the corresponding phase diagram, and discuss the most probable outcome of nonlinear energy transfers, both on the horizontal and on the vertical, in the presence of stratification and rotation.

Restricted partition functions and inverse energy cascades in parity symmetry breaking flows.

When the symmetries of homogenous isotropic turbulent flows are broken, different sets of modes with different physical roles emerge. In particular, choosing a forcing which puts more weight on one or the other of these sets may result in different statistics for the energy transfers. We use the general method of computing a partition function restricted to a portion of phase space to study analytically these different statistics. We illustrate this method in the case of parity symmetry breaking, measured by helicity. It is shown that when helicity is sign-definite at all scales, an inverse cascade is expected for the energy. When sign-definiteness is lost, even for a small set of modes, this cascade disappears and there is a sharp phase transition to the standard helical equipartition spectra.

Automated image analysis of in vitro angiogenesis assay.

Angiogenesis is the biological process of generating new capillary blood vessels. It is a fundamental component of a number of normal (reproduction and wound healing) and pathological processes (diabetic retinopathy, rheumatoid arthritis, tumor growth, and metastasis). In vitro angiogenesis assays provide a platform for evaluating the effects of pro- or antiangiogenic compounds. One of the most informative assays is the endothelial cells capillary tube formation assay performed on a biological matrix. This assay is based on quantification of the stimulatory and inhibitory effects of various agents, which is estimated through the measurement of the pseudo-tubules network length. This standard measurement is usually carried out manually by trained operators but requires time, attention, and dedication to achieve a reasonable degree of accuracy. Moreover, the screening is operator dependent. In this article, we propose an automated procedure to evaluate the pseudo-tubule network lengths. We propose a series of image analysis procedures developed using a freely available image analysis software library. More than 800 images from 12 experiments were analyzed automatically and manually, and their results were compared to improve and validate the proposed image analysis procedure. The resulting image analysis software is currently running on a dedicated server, with comparable accuracy to manual measurements. Using this new automated procedure, we are able to treat 540 images, or three complete assays per hour.

Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties.

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.

Molecular mechanism of SSR128129E, an extracellularly acting, small-molecule, allosteric inhibitor of FGF receptor signaling.

The fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling network plays an important role in cell growth, survival, differentiation, and angiogenesis. Deregulation of FGFR signaling can lead to cancer development. Here, we report an FGFR inhibitor, SSR128129E (SSR), that binds to the extracellular part of the receptor. SSR does not compete with FGF for binding to FGFR but inhibits FGF-induced signaling linked to FGFR internalization in an allosteric manner, as shown by crystallography studies, nuclear magnetic resonance, Fourier transform infrared spectroscopy, molecular dynamics simulations, free energy calculations, structure-activity relationship analysis, and FGFR mutagenesis. Overall, SSR is a small molecule allosteric inhibitor of FGF/FGFR signaling, acting via binding to the extracellular part of the FGFR.

Influence of heparin mimetics on assembly of the FGF.FGFR4 signaling complex.

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF.FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM(6)), octasaccharide (HM(8)), and decasaccharide (HM(10)), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM(8) and HM(10) are significantly more potent than HM(6) in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1.FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2.FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1.FGFR4 interaction site and a direct FGFR4.FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF.FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM(8) relative to HM(6) in stimulating FGF2.FGFR4 signaling correlates with the higher affinity of HM(8) to bind and dimerize FGF2. Notably FGF2.HM(8) exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF.FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.

Transgenic sequences are frequently lost in Phytophthora parasitica transformants without reversion of the transgene-induced silenced state.

Little data exist on the mechanism and stability of transformation in Phytophthora parasitica, a major oomycete parasite of plants. Here, we studied the stability of drug-resistant protoplast transformants by analyzing single-zoospore derivatives. We show that the transgenic sequences are not stably integrated into the chromosomes, resulting in the loss of drug resistance in single-zoospore derivatives. However, in strains where the P. parasitica gene encoding the CBEL elicitor was silenced by transformation with sense or antisense constructs, silencing is not reversed when the transgenic sequences are lost. This suggests that instability of P. parasitica transformants is not an obstacle for loss-of-function studies in this organism.

Regulation and role of a STE12-like transcription factor from the plant pathogen Colletotrichum lindemuthianum.

In phytopathogenic fungi, STE12-like genes encode transcription factors essential for appressorium-mediated host penetration. However, their regulation and downstream targets are still unknown. In the present study, a STE12-like gene (CLSTE12) from Colletotrichum lindemuthianum was isolated. We identified a spliced variant whose expression was negatively regulated during early stages of pathogenesis, whereas the correctly spliced mRNA remained expressed up to the penetration step, suggesting distinct roles for these two transcripts. Indeed, the full-length sequence was able to complement a yeast STE12 mutant, whereas overexpression of the transcript variant had a dominant-negative effect on yeast invasive growth and C. lindemuthianum pathogenicity. To further investigate the downstream genes that could be regulated by CLSTE12, disruption mutants were generated. Phenotypic analyses of the mutants revealed reduced pectinase activity and conidial adhesion to polystyrene. Analysis of cell surface proteins allowed the identification of a major protein, Clsp1p, which was absent from the mutants. Clsp1p belongs to a new family of wall-associated proteins only found in euascomycetous fungi. Overall, these results suggest that the activity of CLSTE12 can be modulated by a regulated alternative splicing mechanism and that this factor is involved in the production of cell surface proteins and host cell wall degrading enzymes.

Functional analysis of CLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogen Colletotrichum lindemuthianum.

In eukaryotic cells, Rab/GTPases are major regulators of vesicular trafficking and are involved in essential processes including exocytosis, endocytosis and cellular differentiation. To investigate the role of these proteins in fungal pathogenicity, a dominant-negative mutant allele of CLPT1, a Rab/GTPase of the bean pathogen Colletotrichum lindemuthianum, was expressed in transgenic strains. This mutated gene encodes the amino-acid substitution N123I analogous to the N133I substitution in a known trans-dominant inhibitor of the Sec4 Rab/GTPase from Saccharomyces cerevisiae. A pectinase gene promoter was used to drive the CLPT1(N123I) allele in C. lindemuthianum, allowing the expression of the foreign gene on pectin medium and during pathogenesis, but not on glucose. The same strategy was used to overexpress the wild-type CLPT1 allele. During growth on pectin medium, production of extracellular pectinases was strongly impaired only in CLPT1(N123I)-expressing strains. Cytological analysis revealed that CLPT1(N123I) strains accumulated intracellular aggregates only on pectin, resulting from the fusion of vesicles containing polysaccharides or glycoproteins. Moreover, these strains showed a severe reduction of pathogenesis and were unable to penetrate the host cells. These results indicated that the Rab/GTPase CLPT1 is essential for fungal pathogenesis by regulating the intracellular transport of secretory vesicles involved in the delivery of proteins to the extracellular medium and differentiation of infectious structures.

A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions.

The ability of a Colletotrichum sp., originally isolated from Brassica campestris, to infect Arabidopsis thaliana was examined. Sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal (r)DNA showed the pathogen to be Colletotrichum destructivum. The host range was broad, including many cruciferous plants and some legumes. At 25 degrees C, all A. thaliana accessions tested were susceptible to the Brassica isolates of C. destructivum; however, at 15 degrees C, the accession Ws-2 showed a temperature-dependant resistance, in which single epidermal cells underwent a rapid hypersensitive response. Legume isolates of C. destructivum were unable to infect A. thaliana and induced deposition of callose papillae at sites of attempted penetration. In compatible interactions, C. destructivum showed a two-stage, hemibiotrophic infection process. The initial biotrophic phase was associated with large, intracellular primary hyphae and was confined to one epidermal cell; whereas, in the subsequent necrotrophic phase, narrow secondary hyphae extensively colonized the tissue and conidia were produced in acervuli. An efficient transformation system was established for C. destructivum, using Agrobacterium-mediated transfer of DNA. The ability to genetically manipulate both partners in the interaction is an important advantage, and the Arabidopsis-Colletotrichum pathosystem should provide a valuable new model for dissecting plant-fungal interactions.

Production of a cell wall-associated endopolygalacturonase by Colletotrichum lindemuthianum and pectin degradation during bean infection.

The bean pathogen Colletotrichum lindemuthianum expresses two endopolygalacturonase genes, CLPG1 and CLPG2, during interaction with its host plant. However, only CLPG1 was found to be secreted to the extracellular medium during saprophytic growth of the fungus on pectin. To localize CLPG2, a FLAG epitope sequence was inserted in the C-terminal sequence of CLPG2 and the modified gene was introduced into C. lindemuthianum. Western blot analysis using a FLAG monoclonal antibody allowed the detection of CLPG2 in intracellular protein extracts and in the cell wall fraction, but not in the culture medium. Indirect immunofluorescence microscopy was performed to detect CLPG2 during saprophytic or parasitic growth. According to the expression pattern of CLPG2, it was found that CLPG2 accumulates in the fungal cell wall during growth on pectin medium and during appressorium formation, both in vitro and during interaction with the plant. Pectin degradation was not detected around the infection peg using the monoclonal antibody JIM7, specific for methyl-esterified galacturonan. However, extensive pectin dissolution was observed during the development of secondary hyphae.

A cis-acting sequence homologous to the yeast filamentation and invasion response element regulates expression of a pectinase gene from the bean pathogen Colletotrichum lindemuthianum.

Phytopathogenic fungi secrete hydrolytic enzymes that degrade plant cell walls, notably pectinases. The signaling pathway(s) that control pectinase gene expression are currently unknown in filamentous fungi. Recently, the green fluorescent protein coding sequence was used as a reporter gene to study the expression of CLPG2, a gene encoding an endopolygalacturonase of the bean pathogen Colletotrichum lindemuthianum. CLPG2 is transcriptionally induced by pectin in the axenic culture of the fungus and during formation of the appressorium, an infection structure specialized in plant tissue penetration. In the present study, promoter deletion and mutagenesis, as well as gel shift mobility assays, allowed for the first time identification of cis-acting elements that bind protein factors and are essential for the regulation of a pectinase gene. We found that two different adjacent DNA motifs are combined to form an active element that shows a strong sequence homology with the yeast filamentation and invasion response element. The same element is required for the transcriptional activation of CLPG2 by pectin and during appressorium development. This study strongly suggests that the control of virulence genes of fungal plant pathogens, such as pectinases, involves the formation of a complex of transcriptional activators similar to those regulating the invasive growth in yeast.