Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 mu g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c(+), CD11b(+) and CD19(+) cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k(2)=21 +/- 1 s(-1)) was much higher than the HNE deacylation step (k(3)=0.57 +/- 0.05 s(-1)). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k(1) 2.4-fold and reducing k(-1) 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k(2) value, whereas the k(3) value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.

Observational evidences on the modulation of the South American Low Level Jet east of the Andes according the ENSO variability

The differences on the phase and wavelength of the quasi-stationary waves over the South America generated by El Nino (EN) and La Nina (LN) events seem to affect the daily evolution of the South American Low Level Jet east of the Andes (SALLJ). For the austral summer period of 1977 2004 the SALLJ episodes detected according to Bonner criterion 1 show normal to above-normal frequency in EN years, and in LN years the episodes show normal to below-normal frequency. During EN and LN years the SALLJ episodes were associated with positive rainfall anomalies over the La Plata Basin, but more intense during LN years. During EN years the increase in the SALLJ cases were associated to intensification of the Subtropical Jet (SJ) around 30 degrees S and positive Sea Level Pressure (SLP) anomalies over the western equatorial Atlantic and tropical South America, particularly over central Brazil. This favored the intensification of the northeasterly trade winds over the northern continent and it channeled by the Andes mountain to the La Plata Basin region where negative SLP are found. The SALLJ cases identified during the LN events were weaker and less frequent when compared to those for EN years. In this case the SJ was weaker than in EN years and the negative SLP anomalies over the tropical continent contributed to the inversion of the northeasterly trade winds. Also a southerly flow anomaly was generated by the geostrophic balance due to the anomalous blocking over southeast Pacific and the intense cyclonic transient over the southern tip of South America. As result the warm tropical air brought by the SALLJ encounters the cold extratropical air from the southerly winds over the La Plata basin. This configuration can increase the conditional instability over the La Plata basin and may explain the more intense positive rainfall anomalies in SALLJ cases during LN years than in EN years.

Optical identification of the transient supersoft X-ray source RX J0527.8-6954, in the LMC

Context. Close binary supersoft X-ray sources (CBSS) are binary systems that contain a white dwarf with stable nuclear burning on its surface. These sources, first discovered in the Magellanic Clouds, have high accretion rates and near-Eddington luminosities (10(37)-10(38) erg s(-1)) with high temperatures (T = 2-7 x 10(5) K). Aims. The total number of known objects in the MC is still small and, in our galaxy, even smaller. We observed the field of the unidentified transient supersoft X-ray source RX J0527.8-6954 in order to identify its optical counterpart. Methods. The observation was made with the IFU-GMOS on the Gemini South telescope with the purpose of identifying stars with possible He II or Balmer emission or else of observing nebular extended jets or ionization cones, features that may be expected in CBSS. Results. The X-ray source is identified with a B5e V star that is associated with subarcsecond extended H alpha emission, possibly bipolar. Conclusions. If the primary star is a white dwarf, as suggested by the supersoft X-ray spectrum, the expected orbital period exceeds 21 h; therefore, we believe that the 9.4 h period found so far is not associated to this system.

CONTRASTING PHYLOGEOGRAPHIC PATTERNS IN MITOCHONDRIAL DNA AND MICROSATELLITES: EVIDENCE OF FEMALE PHILOPATRY AND MALE-BIASED GENE FLOW AMONG REGIONAL POPULATIONS OF THE BLUE-AND-YELLOW MACAW (PSITTACIFORMES: ARA ARARAUNA) IN BRAZIL

Comparing the patterns of population differentiation among genetic markers with different modes of inheritance call provide insights into patterns of sex-biased dispersal and gene flow. The blue-and-yellow Macaw (Ara ararauna) is a Neotropical parrot with a broad geographic distribution ill South America. However, little is known about the natural history and current status Of remaining wild populations, including levels of genetic variability. The progressive decline and possible fragmentation of populations may endanger this species in the near future. We analyzed mitochondrial DNA (mtDNA) control-region sequences and six microsatellite 106 Of Blue-and-yellow Macaws sampled throughout their geographic range ill Brazil to describe population genetic Structure, to make inferences about historical demography and dispersal behavior, and to provide insight for conservation efforts. Analyses of population genetic structure based on mtDNA showed evidence of two major populations ill western and eastern Brazil that share a few low-frequency haplotypes. This phylogeographic pattern seems to have originated by the historical isolation of Blue-and-yellow Macaw populations similar to 374,000 years ago and has been maintained by restricted gene flow and female philopatry. By contrast, variation ill biparentally inherited microsatellites was not structured geographically, Male-biased dispersal and female philopatry best explain the different patterns observed in these two markers. Because females disperse less than males, the two regional populations with well-differentiated mtDNA haplogroups should be considered two different management units for conservation purposes. Received 4 November 2007 accepted 10 December 2008.

PARTITIONED ANALYSIS FOR DIMENSIONALLY-HETEROGENEOUS HYDRAULIC NETWORKS

In this work an iterative strategy is developed to tackle the problem of coupling dimensionally-heterogeneous models in the context of fluid mechanics. The procedure proposed here makes use of a reinterpretation of the original problem as a nonlinear interface problem for which classical nonlinear solvers can be applied. Strong coupling of the partitions is achieved while dealing with different codes for each partition, each code in black-box mode. The main application for which this procedure is envisaged arises when modeling hydraulic networks in which complex and simple subsystems are treated using detailed and simplified models, correspondingly. The potentialities and the performance of the strategy are assessed through several examples involving transient flows and complex network configurations.

Two-dimensional supersonic nonlinear Schrodinger flow past an extended obstacle

Supersonic flow of a superfluid past a slender impenetrable macroscopic obstacle is studied in the framework of the two-dimensional (2D) defocusing nonlinear Schroumldinger (NLS) equation. This problem is of fundamental importance as a dispersive analog of the corresponding classical gas-dynamics problem. Assuming the oncoming flow speed is sufficiently high, we asymptotically reduce the original boundary-value problem for a steady flow past a slender body to the one-dimensional dispersive piston problem described by the nonstationary NLS equation, in which the role of time is played by the stretched x coordinate and the piston motion curve is defined by the spatial body profile. Two steady oblique spatial dispersive shock waves (DSWs) spreading from the pointed ends of the body are generated in both half planes. These are described analytically by constructing appropriate exact solutions of the Whitham modulation equations for the front DSW and by using a generalized Bohr-Sommerfeld quantization rule for the oblique dark soliton fan in the rear DSW. We propose an extension of the traditional modulation description of DSWs to include the linear ""ship-wave"" pattern forming outside the nonlinear modulation region of the front DSW. Our analytic results are supported by direct 2D unsteady numerical simulations and are relevant to recent experiments on Bose-Einstein condensates freely expanding past obstacles.

Elliptic and Hexadecapole Flow of Charged Hadrons in Au plus Au Collisions at root s(NN)=200 GeV

Differential measurements of the elliptic (upsilon(2)) and hexadecapole (upsilon(4)) Fourier flow coefficients are reported for charged hadrons as a function of transverse momentum (p(T)) and collision centrality or number of participant nucleons (N(part)) for Au + Au collisions at root s(NN) = 200 GeV/ The upsilon(2,4) measurements at pseudorapidity vertical bar eta vertical bar <= 0.35, obtained with four separate reaction-plane detectors positioned in the range 1.0 < vertical bar eta vertical bar < 3.9, show good agreement, indicating the absence of significant Delta eta-dependent nonflow correlations. Sizable values for upsilon(4)(p(T)) are observed with a ratio upsilon(4)(p(T), N(part))/upsilon(2)(2)(p(T), N(part)) approximate to 0.8 for 50 less than or similar to N(part) less than or similar to 200, which is compatible with the combined effects of a finite viscosity and initial eccentricity fluctuations. For N(part) greater than or similar to 200 this ratio increases up to 1.7 in the most central collisions.

Systematic studies of elliptic flow measurements in Au plus Au collisions at s(NN)=200 GeV

We present inclusive charged hadron elliptic flow (v(2)) measured over the pseudorapidity range vertical bar eta vertical bar < 0.35 in Au+Au collisions at s(NN)=200 GeV. Results for v(2) are presented over a broad range of transverse momentum (p(T)=0.2-8.0 GeV/c) and centrality (0-60%). To study nonflow effects that are correlations other than collective flow, as well as the fluctuations of v(2), we compare two different analysis methods: (1) the event-plane method from two independent subdetectors at forward (vertical bar eta vertical bar=3.1-3.9) and beam (vertical bar eta vertical bar>6.5) pseudorapidities and (2) the two-particle cumulant method extracted using correlations between particles detected at midrapidity. The two event-plane results are consistent within systematic uncertainties over the measured p(T) and in centrality 0-40%. There is at most a 20% difference in the v(2) between the two event-plane methods in peripheral (40-60%) collisions. The comparisons between the two-particle cumulant results and the standard event-plane measurements are discussed.

Directed flow at midrapidity in event-by-event hydrodynamics

Fluctuations in the initial geometry of a nucleus-nucleus collision have been recently shown to result in a new type of directed flow (v(1)) that, unlike the usual directed flow, is also present at midrapidity. We compute this new v(1) versus transverse momentum and centrality for Au-Au collisions at RHIC using the hydrodynamic code NeXSPheRIO. We find that the event plane of v(1) is correlated with the angle of the initial dipole of the distribution, as predicted, though with a large dispersion. It is uncorrelated with the reaction plane. Our results are in excellent agreement with results inferred from STAR correlation data.

Importance of granular structure in the initial conditions for the elliptic flow

We show the effects of the granular structure of the initial conditions of a hydrodynamic description of high-energy nucleus-nucleus collisions on some observables, especially on the elliptic-flow parameter upsilon(2). Such a structure enhances production of isotropically distributed high-p(T) particles, making upsilon(2) smaller there. Also, it reduces upsilon(2) in the forward and backward regions where the global matter density is smaller and, therefore, where such effects become more efficacious.

Charged and strange hadron elliptic flow in Cu plus Cu collisions at root s(NN)=62.4 and 200 GeV

We present the results of an elliptic flow, v(2), analysis of Cu + Cu collisions recorded with the solenoidal tracker detector (STAR) at the BNL Relativistic Heavy Ion Collider at root s(NN) = 62.4 and 200 GeV. Elliptic flow as a function of transverse momentum, v(2)(p(T)), is reported for different collision centralities for charged hadrons h(+/-) and strangeness-ontaining hadrons K(S)(0), Lambda, Xi, and phi in the midrapidity region vertical bar eta vertical bar < 1.0. Significant reduction in systematic uncertainty of the measurement due to nonflow effects has been achieved by correlating particles at midrapidity, vertical bar eta vertical bar < 1.0, with those at forward rapidity, 2.5 < vertical bar eta vertical bar < 4.0. We also present azimuthal correlations in p + p collisions at root s = 200 GeV to help in estimating nonflow effects. To study the system-size dependence of elliptic flow, we present a detailed comparison with previously published results from Au + Au collisions at root s(NN) = 200 GeV. We observe that v(2)(p(T)) of strange hadrons has similar scaling properties as were first observed in Au + Au collisions, that is, (i) at low transverse momenta, p(T) < 2 GeV/c, v(2) scales with transverse kinetic energy, m(T) - m, and (ii) at intermediate p(T), 2 < p(T) < 4 GeV/c, it scales with the number of constituent quarks, n(q.) We have found that ideal hydrodynamic calculations fail to reproduce the centrality dependence of v(2)(p(T)) for K(S)(0) and Lambda. Eccentricity scaled v(2) values, v(2)/epsilon, are larger in more central collisions, suggesting stronger collective flow develops in more central collisions. The comparison with Au + Au collisions, which go further in density, shows that v(2)/epsilon depends on the system size, that is, the number of participants N(part). This indicates that the ideal hydrodynamic limit is not reached in Cu + Cu collisions, presumably because the assumption of thermalization is not attained.

Chaotic advection in blood flow

In this paper we argue that the effects of irregular chaotic motion of particles transported by blood can play a major role in the development of serious circulatory diseases. Vessel wall irregularities modify the flow field, changing in a nontrivial way the transport and activation of biochemically active particles. We argue that blood particle transport is often chaotic in realistic physiological conditions. We also argue that this chaotic behavior of the flow has crucial consequences for the dynamics of important processes in the blood, such as the activation of platelets which are involved in the thrombus formation.

Flow injection analysis of paracetamol using a biomimetic sensor as a sensitive and selective amperometric detector

This work describes the coupling of a biomimetic sensor to a flow injection system for the sensitive determination of paracetamol. The sensor was prepared as previously described in the literature (M. D. P. T. Sotomayor, A. Sigoli, M. R. V. Lanza, A. A. Tanaka and L. T. Kubota, J. Braz. Chem. Soc., 2008, 19, 734) by modifying a glassy carbon electrode surface with a Nafion (R) membrane doped with iron tetrapyridinoporphyrazine (FeTPyPz), a biomimetic catalyst of the P450 enzyme. The performance of the sensor for paracetamol detection was investigated and optimized in a flow injection system (FIA) using a wall jet electrochemical cell. Under optimized conditions a wide linear response range (1.0 x 10(-5) to 5.0 x 10(-2) mol L(-1)) was obtained, with a sensitivity of 2579 (+/- 129) mu A L mu mol(-1). The detection and quantification limits of the sensor for paracetamol in the FIA system were 1.0 and 3.5 mu mol L(-1), respectively. The analytical frequency was 51 samples h(-1), and over a period of five days (320 determinations) the biosensor maintained practically the same response. The system was successfully applied to paracetamol quantification in seven pharmaceutical formulations and in water samples from six rivers in Sao Paulo State, Brazil.

Direct Solid-Phase Optical Measurements in Flow Systems: A Review

Measurements based on absorption, reflectance, or luminescence of molecular species or complex ions can be carried out directly on a solid support simultaneously to the retention of the analyte. The use of this strategy in flow-based systems is advantageous in view of the reproducible handling of solutions in retention and elution steps of the analyte. This approach can be exploited to increase sensitivity, minimize reagent consumption as well as waste generation, improve selectivity or for simultaneous determination based on selective retention or differences in sorption rates of the analytes. This review focuses on the main characteristics of direct solid-phase measurements in flow systems, including the discussion of advantages and limitations and practical guidelines to the successful implementation of this approach. Selected applications in diverse fields, such as pharmaceutical, food, and environmental analysis are discussed.