A Track Geometry Measuring System Based on Multibody Kinematics, Inertial Sensors and Computer Vision.

This paper describes the kinematics used for the calculation of track geometric irregularities of a new Track Geometry Measuring System (TGMS) to be installed in railway vehicles. The TGMS includes a computer for data acquisition and process, a set of sensors including an inertial measuring unit (IMU, 3D gyroscope and 3D accelerometer), two video cameras and an encoder. The kinematic description, that is borrowed from the multibody dynamics analysis of railway vehicles used in computer simulation codes, is used to calculate the relative motion between the vehicle and the track, and also for the computer vision system and its calibration. The multibody framework is thus used to find the formulas that are needed to calculate the track irregularities (gauge, cross-level, alignment and vertical profile) as a function of sensor data. The TGMS has been experimentally tested in a 1:10 scaled vehicle and track specifically designed for this investigation. The geometric irregularities of a 90 m-scale track have been measured with an alternative and accurate method and the results are compared with the results of the TGMS. Results show a good agreement between both methods of calculation of the geometric irregularities.

Application and Experimental Validation of a Multibody Model with Weakly Coupled Lateral and Vertical Dynamics to a Scaled Railway Vehicle.

In this paper, a multibody dynamic model of a railway vehicle that assumes that vertical and lateral dynamics are weakly coupled, has been experimentally validated using an instrumented scaled vehicle running on a 5-inch-wide experimental track. The proposed linearised model treats the vertical and lateral dynamics of the multibody system almost independently, being coupled exclusively by the suspension forces. Several experiments have been carried out at the scaled railroad facilities at the University of Seville in order to test and validate the simulation model under different working conditions. The scaled vehicle used in the experiments is a bogie instrumented with various sensors that register the accelerations and angular velocities of the vehicle, its forward velocity, its position along the track, and the wheel-rail contact forces in the front wheelset. The obtained results demonstrate how the proposed computational model correctly reproduces the dynamics of the real mechanical system in an efficient computational manner.

The inositol-1,2-cyclic phosphate moiety of the cross-reacting determinant, carbohydrate chains, and proteinaceous components are all responsible for the cross-reactivity of trypanosome variant surface glycoproteins.

Salivarian trypanosomes evade the host immune system by continually swapping their protective variant surface glycoprotein (VSG) coat. Given that VSGs from various trypanosome stocks exhibited cross-reactivity (Camargo et al., Vet. Parasitol. 207, 17-33, 2015), we analyzed here which components are the antigenic determinants for this cross-reaction. Soluble forms of VSGs were purified from four Venezuelan animal trypanosome isolates: TeAp-N/D1, TeAp-ElFrio01, TeAp-Mantecal01, and TeGu-Terecay323. By using the VSG soluble form from TeAp-N/D1, we found that neither the inositol-1,2-cyclic phosphate moiety of the cross-reacting determinant nor the carbohydrate chains were exclusively responsible for its cross-reactivity. Then, all four purified glycoproteins were digested with papain and the resulting peptides were separated by high-performance liquid chromatography. Dot blot evaluation of the fractions using sera from trypanosome-infected animals yielded peptides that possessed cross-reaction activity, demonstrating for the first time that proteinaceous epitopes are also responsible for the cross-reactivity of trypanosome VSGs.

Identification of potential protein partners that bind to the variant surface glycoprotein in Trypanosoma equiperdum.

Trypanosoma equiperdum possesses a dense coat of a variant surface glycoprotein (VSG) that is used to evade the host immune response by a process known as antigenic variation. Soluble and membrane forms of the predominant VSG from the Venezuelan T. equiperdum TeAp-N/D1 strain (sVSG and mVSG, respectively) were purified to homogeneity; and antibodies against sVSG and mVSG were raised, isolated, and employed to produce anti-idiotypic antibodies that structurally mimic the VSG surface. Prospective VSG-binding partners were initially detected by far-Western blots, and then by immunoblots using the generated anti-idiotypic antibodies. Polypeptides of ~80 and 55 kDa were isolated when anti-idiotypic antibodies-Sepharose affinity matrixes were used as baits. Mass spectrometry sequencing yielded hits with various proteins from Trypanosoma brucei such as heat-shock protein 70, tryparedoxin peroxidase, VSG variants, expression site associated gene product 6, and two hypothetical proteins. In addition, a possible interaction with a protein homologous to the glutamic acid/alanine-rich protein from Trypanosoma congolense was also found. These results indicate that the corresponding orthologous gene products are candidates for VSG-interacting proteins in T. equiperdum.

Immunological identification of a cAMP-dependent protein kinase regulatory subunit-like protein from the Trypanosoma equiperdum TeAp-N/D1 isolate.

Polyclonal immunoglobulin Y (IgY) antibodies were produced in chicken eggs against the purified R(II)-subunit of the cAMP-dependent protein kinase (PKA) from pig heart, which corresponds to the Sus scrofa R(II)α isoform. In order to evaluate whether Trypanosoma equiperdum possessed PKA R-like proteins, parasites from the Venezuelan TeAp-N/D1 strain were examined using the generated anti-R(II) IgY antibodies. Western blot experiments revealed a 57-kDa polypeptide band that was distinctively recognized by these antibodies. Likewise, polyclonal antibodies raised in mice ascites against the recombinant T. equiperdum PKA R-like protein recognized the PKA R(II)-subunit purified from porcine heart and the recombinant human PKA R(I)ß-subunit by immunoblotting. However, a partially purified fraction of the parasite PKA R-like protein was not capable of binding cAMP, implying that this protein is not a direct downstream cAMP effector in T. equiperdum. Although the function of the S. scrofa PKA R(II)α and the T. equiperdum PKA R-like protein appear to be different, their cross-reactivity together with results obtained by bioinformatics techniques corroborated the high level of homology exhibited by both proteins. Moreover, its presence in other trypanosomatids suggests an important cellular role of PKA R-like proteins in parasite physiology.

Lipase-Catalyzed Synthesis and Biological Evaluation of N-Picolineamides as Trypanosoma cruzi Antiproliferative Agents.

In our search for new safe antiparasitic agents, an enzymatic pathway was applied to synthesize a series of N-pyridinylmethyl amides derived from structurally different carboxylic acids. Thirty derivatives, including 11 new compounds, were prepared through lipase-catalyzed acylation in excellent yields. In order to optimize the synthetic methodology, the impact of different reaction parameters was analyzed. Some compounds were evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible for American trypanosomiasis (Chagas' disease). Some of them showed significant activity as parasite proliferation inhibitors. Amides derived from 2-aminopicoline and stearic and elaidic acids were as potent as nifurtimox against the amastigote form of T. cruzi, the clinically relevant form of the parasite. Even more, a powerful synergism between nifurtimox and N-(pyridin-2-ylmethyl)stereamide was observed, almost completely inhibiting the proliferation of the parasite. Besides, the obtained compounds showed no toxicity in Vero cells, making them excellent potential candidates as lead drugs.

Design and manufacture of a scaled railway track with mechanically variable geometry.

The objective of this article is to present the design and manufacture of a scaled railroad track to be used as a laboratory track for the study of different railway applications. It could be a guideline for future laboratory railroad tracks. The ideal concept was based on possible future studies and, according to them, design requirements have been specified. The main characteristic of the track is that its geometry can be mechanically modified and irregularities can be introduced under controlled conditions in any kind of track sections: straight, curved and transition ones. Finally, the current installed track is shown and the performed quality controls are described.

Reversible phosphorylation of a protein from Trypanosoma equiperdum that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases.

Homologous proteins of the cAMP-dependent protein kinase (PKA) regulatory and catalytic subunits have been identified in Trypanosoma equiperdum (TeqR-like and TeqC-like, respectively). Partially purified TeqR-like from parasites isolated in the presence of glucose migrated as an apparent 55 kDa/57 kDa polypeptide doublet when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a single polypeptide of 57 kDa was obtained when parasites were deprived of glucose, a condition that has been shown to activate a TeqC-like enzyme. As revealed by immunoblots using anti-phospothreonine antibodies, the 57 kDa band corresponded to a form of TeqR-like that was phosphorylated in threonine residues. TeqR-like phosphorylation was reversible since the level of phospho-TeqR-like decreased once glucose was readded to glucose starved-parasites. Dephospho- and phospho-TeqR-like proteins are monomers with native molecular masses of 54.93-57.41 kDa, Stokes radii of 3.42-3.37 nm, and slightly asymmetric shapes (frictional ratio f/fo = 1.36-1.32). A protein kinase of ∼40 kDa was also partially purified from glucose deprived-trypanosomes, which corresponded to the TeqC-like enzyme by its ability to phosphorylate kemptide, its inhibition by PKA-specific inhibitors, and its immunorecognition by anti-PKA catalytic subunit antibodies. TeqR-like and TeqC-like did not coelute following anion-exchange chromatography, revealing that these proteins are not associated forming a complex in T. equiperdum. Yet, when TeqR-like was incubated in vitro with TeqC-like in the presence of Mg2+ and ATP, the 55 kDa dephospho form of the 55kDa/57 kDa polypeptide doublet of TeqR-like was converted into the 57 kDa phospho form, demonstrating that TeqR-like is a substrate for TeqC-like.

Variant surface glycoproteins from Venezuelan trypanosome isolates are recognized by sera from animals infected with either Trypanosoma evansi or Trypanosoma vivax.

Salivarian trypanosomes sequentially express only one variant surface glycoprotein (VSG) on their cell surface from a large repertoire of VSG genes. Seven cryopreserved animal trypanosome isolates known as TeAp-ElFrio01, TEVA1 (or TeAp-N/D1), TeGu-N/D1, TeAp-Mantecal01, TeGu-TerecayTrino, TeGu-Terecay03 and TeGu-Terecay323, which had been isolated from different hosts identified in several geographical areas of Venezuela were expanded using adult albino rats. Soluble forms of predominant VSGs expressed during the early infection stages were purified and corresponded to concanavalin A-binding proteins with molecular masses of 48-67 kDa by sodium dodecyl sulfate-polyacrylamide gel electropohoresis, and pI values between 6.1 and 7.5. The biochemical characterization of all purified soluble VSGs revealed that they were dimers in their native form and represented different gene products. Sequencing of some of these proteins yielded peptides homologous to VSGs from Trypanosoma (Trypanozoon) brucei and Trypanosoma (Trypanozoon) evansi and established that they most likely are mosaics generated by homologous recombination. Western blot analysis showed that all purified VSGs were cross-reacting antigens that were recognized by sera from animals infected with either T. evansi or Trypanosoma (Dutonella) vivax. The VSG glycosyl-phosphatidylinositol cross-reacting determinant epitope was only partially responsible for the cross-reactivity of the purified proteins, and antibodies appeared to recognize cross-reacting conformational epitopes from the various soluble VSGs. ELISA experiments were performed using infected bovine sera collected from cattle in a Venezuelan trypanosome-endemic area. In particular, soluble VSGs from two trypanosome isolates, TeGu-N/D1 and TeGu-TeracayTrino, were recognized by 93.38% and 73.55% of naturally T. vivax-infected bovine sera, respectively. However, approximately 70% of the sera samples did not recognize all seven purified proteins. Hence, the use of a combination of various VSGs for the diagnosis of animal trypanosomosis is recommended.