CD8 T-cell induction against vascular endothelial growth factor receptor 2 by Salmonella for vaccination purposes against a murine melanoma.

The Salmonella type III secretion system (T3SS) efficiently translocates heterologous proteins into the cytosol of eukaryotic cells. This leads to an antigen-specific CD8 T-cell induction in mice orally immunized with recombinant Salmonella. Recently, we have used Salmonella's T3SS as a prophylactic and therapeutic intervention against a murine fibrosarcoma. In this study, we constructed a recombinant Salmonella strain translocating the immunogenic H-2D(b)-specific CD8 T-cell epitope VILTNPISM (KDR2) from the murine vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a member of the tyrosine protein kinase family and is upregulated on proliferating endothelial cells of the tumor vasculature. After single orogastric vaccination, we detected significant numbers of KDR2-tetramer-positive CD8 T cells in the spleens of immunized mice. The efficacy of these cytotoxic T cells was evaluated in a prophylactic setting to protect mice from challenges with B16F10 melanoma cells in a flank tumor model, and to reduce dissemination of spontaneous pulmonary melanoma metastases. Vaccinated mice revealed a reduction of angiogenesis by 62% in the solid tumor and consequently a significant decrease of tumor growth as compared to non-immunized mice. Moreover, in the lung metastasis model, immunization with recombinant Salmonella resulted in a reduction of the metastatic melanoma burden by approximately 60%

Kolmogorov condition near hyperbolic singularities of integrable Hamiltonian systems

In this paper we show that, if an integrable Hamiltonian system admits a nondegenerate hyperbolic singularity then it will satisfy the Kolmogorov condegeneracy condition near that singularity (under a mild additional condition, which is trivial if the singularity contains a fixed point)Comment: revised version, 11p, accepted for publication in a sepecial volume in Regular and Chaotic Dynamics in honor of Richard Cushma

KAM \`{a} la R

Recently R\"ussmann proposed a new new variant of KAM theory based on a slowly converging iteration scheme. It is the purpose of this note to make this scheme accessible in an even simpler setting, namely for analytic perturbations of constant vector fields on a torus. As a side effect the result may be the shortest complete KAM proof for perturbations of integrable vector fields available so far.Comment: 11 pages, version 2.

A Computational Procedure to Detect a New Type of High Dimensional Chaotic Saddle and its Application to the 3-D Hill's Problem

A computational procedure that allows the detection of a new type of high-dimensional chaotic saddle in Hamiltonian systems with three degrees of freedom is presented. The chaotic saddle is associated with a so-called normally hyperbolic invariant manifold (NHIM). The procedure allows to compute appropriate homoclinic orbits to the NHIM from which we can infer the existence a chaotic saddle. NHIMs control the phase space transport across an equilibrium point of saddle-centre-...-centre stability type, which is a fundamental mechanism for chemical reactions, capture and escape, scattering, and, more generally, ``transformation'' in many different areas of physics. Consequently, the presented methods and results are of broad interest. The procedure is illustrated for the spatial Hill's problem which is a well known model in celestial mechanics and which gained much interest e.g. in the study of the formation of binaries in the Kuiper belt.Comment: 12 pages, 6 figures, pdflatex, submitted to JPhys

Optimal stability and instability for near-linear Hamiltonians

In this paper, we will prove a very general result of stability for perturbations of linear integrable Hamiltonian systems, and we will construct an example of instability showing that both our result and our example are optimal. Moreover, in the same spirit as the notion of KAM stable integrable Hamiltonians, we will introduce a notion of effectively stable integrable Hamiltonians, conjecture a characterization of these Hamiltonians and show that our result prove this conjecture in the linear case

Holomorphic linearization of commuting germs of holomorphic maps

Let $f_1, ..., f_h$ be $h\ge 2$ germs of biholomorphisms of \C^n fixing the origin. We investigate the shape a (formal) simultaneous linearization of the given germs can have, and we prove that if $f_1, ..., f_h$ commute and their linear parts are almost simultaneously Jordanizable then they are simultaneously formally linearizable. We next introduce a simultaneous Brjuno-type condition and prove that, in case the linear terms of the germs are diagonalizable, if the germs commutes and our Brjuno-type condition holds, then they are holomorphically simultaneously linerizable. This answers to a multi-dimensional version of a problem raised by Moser.Comment: 24 pages; final version with erratum (My original paper failed to cite the work of L. Stolovitch [ArXiv:math/0506052v2]); J. Geom. Anal. 201

Direct Injection of Functional Single-Domain Antibodies from E. coli into Human Cells

Intracellular proteins have a great potential as targets for therapeutic antibodies (Abs) but the plasma membrane prevents access to these antigens. Ab fragments and IgGs are selected and engineered in E. coli and this microorganism may be also an ideal vector for their intracellular delivery. In this work we demonstrate that single-domain Ab (sdAbs) can be engineered to be injected into human cells by E. coli bacteria carrying molecular syringes assembled by a type III protein secretion system (T3SS). The injected sdAbs accumulate in the cytoplasm of HeLa cells at levels ca. 105–106 molecules per cell and their functionality is shown by the isolation of sdAb-antigen complexes. Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. These results are proof-of-principle for the capacity of E. coli bacteria to directly deliver intracellular sdAbs (intrabodies) into human cells for analytical and therapeutic purposes

Time quasi-periodic gravity water waves in finite depth

We prove the existence and the linear stability of Cantor families of small amplitude time quasi-periodic standing water wave solutions\u2014namely periodic and even in the space variable x\u2014of a bi-dimensional ocean with finite depth under the action of pure gravity. Such a result holds for all the values of the depth parameter in a Borel set of asymptotically full measure. This is a small divisor problem. The main difficulties are the fully nonlinear nature of the gravity water waves equations\u2014the highest order x-derivative appears in the nonlinear term but not in the linearization at the origin\u2014and the fact that the linear frequencies grow just in a sublinear way at infinity. We overcome these problems by first reducing the linearized operators, obtained at each approximate quasi-periodic solution along a Nash\u2013Moser iterative scheme, to constant coefficients up to smoothing operators, using pseudo-differential changes of variables that are quasi-periodic in time. Then we apply a KAM reducibility scheme which requires very weak Melnikov non-resonance conditions which lose derivatives both in time and space. Despite the fact that the depth parameter moves the linear frequencies by just exponentially small quantities, we are able to verify such non-resonance conditions for most values of the depth, extending degenerate KAM theory

Functional and Computational Analysis of Amino Acid Patterns Predictive of Type III Secretion System Substrates in Pseudomonas syringae

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant pathogen Pseudomonas syringae pathovar tomato strain DC3000 possess characteristic patterns, including (i) greater than 10% serine within the first 50 amino acids, (ii) an aliphatic residue or proline at position 3 or 4, and (iii) a lack of acidic amino acids within the first 12 residues. Here, the functional significance of the P. syringae T3SS substrate compositional patterns was tested. A mutant AvrPto effector protein lacking all three patterns was secreted into culture and translocated into plant cells, suggesting that the compositional characteristics are not absolutely required for T3SS targeting and that other recognition mechanisms exist. To further analyze the unique properties of T3SS targeting signals, we developed a computational algorithm called TEREE (Type III Effector Relative Entropy Evaluation) that distinguishes DC3000 T3SS substrates from other proteins with a high sensitivity and specificity. Although TEREE did not efficiently identify T3SS substrates in Salmonella enterica, it was effective in another P. syringae strain and Ralstonia solanacearum. Thus, the TEREE algorithm may be a useful tool for identifying new effector genes in plant pathogens. The nature of T3SS targeting signals was additionally investigated by analyzing the N-terminus of FtsX, a putative membrane protein that was classified as a T3SS substrate by TEREE. Although the first 50 amino acids of FtsX were unable to target a reporter protein to the T3SS, an AvrPto protein substituted with the first 12 amino acids of FtsX was translocated into plant cells. These results show that the T3SS targeting signals are highly mutable and that secretion may be directed by multiple features of substrates