Virulence exaltation of Clostridium perfringens strains from bovines

Ten out of eighty-nine strains biochemically identified as Clostridium perfringens, isolated from bovine organs, were selected by their different results showed in toxigenicity test on mice. Those and the standard strains, ATCC types A, B, C, and D, had their virulence exalted through serial intramuscular inoculation into guinea pigs. Results showed that, for toxigenic strains (6), one or two passages were enough to cause exaltation, while for the atoxigenic (4), five or six inoculations were needed. Esterase electrophoresis of standard and isolated strains, with and without exaltation, was performed. Electrophoresis analysis permits the following conclusions: strains that do not show any clinical symptoms in mice, when exalted demonstrate decreased esterase activity; on the contrary, it is increased when correlated with animal symptoms

Identification and characterization of the TonB region and its role in transferrin-mediated iron acquisition in Haemophilus parasuis

12 p.Haemophilus parasuis is the causative agent of Glässer's disease, which is responsible for considerable economic losses in the pigrearing industry. The aim of the study reported here was the identification, sequencing and molecular characterization of the TonB region that includes tonB, exbBD, and tbpBA genes in H. parasuis. In addition, two fusion proteins were generated. One of them (pGEX-6P-1-GST-TbpB) contained the first 501 amino acids of H. parasuis TbpB protein, while the second (pBAD-Thio-TbpBV5- His) included the first 102 amino acids of H. parasuis TbpB N-terminus domain. A panel of 14 hybridomas secreting monoclonal antibodies was raised against the two recombinant TbpB fusion proteins. Furthermore, to assess whether the expression of the H. parasuis ExbB, TbpB, and TbpA proteins was upregulated under conditions of restricted availability of iron, a rabbit polyclonal antibody against H. parasuis TbpB-His fusion protein was produced. A rabbit polyclonal antibody against serotype 7 of Actinobacillus pleuropneumoniae ExbB and TbpA proteins was also used for the detection of the homologous proteins in H. parasuis. Overall, the data indicate that H. parasuis, like other members of the Pasteurellaceae family, possesses the genetic elements of the TonB region for iron acquisition and the transferrin-binding proteins encoded under this region are upregulated under restricted iron availabilityS

Measuring Electrical And Mechanical Properties Of Red Blood Cells With A Double Optical Tweezers

The fluid lipid bilayer viscoelastic membrane of red blood cells (RBC) contains antigen glycolproteins and proteins which can interact with antibodies to cause cell agglutination. This is the basis of most of the immunohematologic tests in blood banks and the identification of the antibodies against the erythrocyte antigens is of fundamental importance for transfusional routines. The negative charges of the RBCs creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. The first counterions cloud strongly binded moving together with the RBC is called the compact layer. This report proposes the use of a double optical tweezers for a new procedure for measuring: (1) the apparent membrane viscosity, (2) the cell adhesion, (3) the zeta potential and (4) the compact layer's size of the charges formed around the cell in the electrolytic solution. To measure the membrane viscosity we trapped silica beads strongly attached to agglutinated RBCs and measured the force to slide one RBC over the other as a function of the relative velocity. The RBC adhesion was measured by slowly displacing two RBCs apart until the disagglutination happens. The compact layer's size was measured using the force on the silica bead attached to a single RBC in response to an applied voltage and the zeta potential was obtained by measuring the terminal velocity after releasing the RBC from the optical trap at the last applied voltage. We believe that the methodology here proposed can improve the methods of diagnosis in blood banks.6326Eylar, E.H., Madoff, M.A., Brody, O.V., Oncley, J.L., The contribution of sialic acid to the surface charge of the erythrocyte (1962) J. Biol. Chem., 237, pp. 1992-2000Pollack, W., Reckel, R.P., A reappraisal of the forces involved in Hemagglutination (1977) Int Archs Allergy Appl. Immun., 54, pp. 29-42Ashkin, A., Dziedzic, J.M., Bjorkholm, J.E., Chu, S., Observation of a single-beam gradient force trap for dielectric particles (1986) Opt. Lett., 11, pp. 288-290Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Grier, D.G., A revolution in optical manipulation (2003) Nature, 424, pp. 810-816Zhu, C., Bao, G., Wang, N., Cell Mechanics: Mechanical response, cell adhesion, and molecular deformation (2000) Annu. Rev. Biomed. Eng., 2, pp. 189-226Neuman, K.C., Block, S., Optical trapping (2004) Rev. Sci. Instrum., 75, pp. 2787-2809Saffman, P.G., Delbruck, M., Brownian motion in biological membranes (1975) Proc. Nat. Acad. Sci. USA, 72, pp. 3111-3113Dimova, R., Danov, K., Pouligny, B., Ivanov, I.B., Drag of a solid particle trapped in a thin film or at an interface: Influence of surface viscosity and elasticity (2000) J. Colloid and Interface Science, 226, pp. 35-43Hochmuth, R., Worthy, P., Evans, E., Red cell extensional recovery and the determination of membrane viscosity (1979) Biophys. J., 26, pp. 101-114Sze, A., Erickson, D., Ren, L., Li, D., Zeta-potential measurement using the Smoluchowski equation and the slope of the current-time relationship in electroosmotic flow (2003) J. Colloid and Interface Science, 261, pp. 402-410Hunter, R.J., (1981) Zeta Potential in Colloid Science, , Academic Press, New YorkPollack, W., Hager, H.J., Reckel, R., Toren, D.A., Singher, H.O., A study of the forces involved in the second stage of hemaggltination (1965) Transfusion, 5, pp. 158-183Chelidze, T., Dielectric spectroscopy of blood (2002) J. Non-crystalline Solids, 305, pp. 285-294Hymer, W.C., Barlow, G.H., Blaisdell, S.J., Continuous flow electrophoretic separation of proteins and cells from mammalian tissues (1987) Cell Biophys., 10, pp. 61-85Hashimoto, N., Fujita, S., Yokoyama, T., Cell electrophoretic mobility and glycerol lysis of human erythrocytes in various diseases (1998) Electrophoresis, 19, pp. 1227-123

Extreme value and cluster analysis of European daily temperature series

Time series of daily mean temperature obtained from the European Climate Assessment data set is analyzed with respect to their extremal properties. A time-series clustering approach which combines Bayesian methodology, extreme value theory and classification techniques is adopted for the analysis of the regional variability of temperature extremes. The daily mean temperature records are clustered on the basis of their corresponding predictive distributions for 25-, 50- and 100-year return values. The results of the cluster analysis showa clear distinction between the highest altitude stations, for which the return values are lowest, and the remaining stations. Furthermore, a clear distinction is also found between the northernmost stations in Scandinavia and the stations in central and southern Europe. This spatial structure of the return period distributions for 25-, 50- and 100-years seems to be consistent with projected changes in the variability of temperature extremes over Europe pointing to a different behavior in central Europe than in northern Europe and the Mediterranean area, possibly related to the effect of soil moisture and land-atmosphere coupling.‘Acções Integradas Luso-Espanholas’ under the grants E-83/09 and HP2008- 008

Mechanical Properties Of Stored Red Blood Cells Using Optical Tweezers

We have developed a method for measuring the red blood cell (RBC) membrane overall elasticity μ by measuring the deformation of the cells when dragged at a constant velocity through a plasma fluid by an optical tweezers. The deformability of erythrocytes is a critical determinant of blood flow in the microcirculation. We tested our method and hydrodynamic models, which included the presence of two walls, by measuring the RBC deformation as a function of drag velocity and of the distance to the walls. The capability and sensitivity of this method can be evaluated by its application to a variety of studies, such as, the measurement of RBC elasticity of sickle cell anemia patients comparing homozygous (HbSS), including patients taking hydroxyrea (HU) and heterozygous (HbAS) with normal donors and the RBC elasticity measurement of gamma irradiated stored blood for transfusion to immunosupressed patients as a function of time and dose. These studies show that the technique has the sensitivity to discriminate heterozygous and homozygous sickle cell anemia patients from normal donors and even follow the course of HU treatment of Homozygous patients. The gamma irradiation studies show that there is no significant change in RBC elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not, but there was a huge change in the measured elasticity for the RBC units stored for more than 21 days after irradiation. These finds are important for the assessment of stored irradiated RBC viability for transfusion purposes because the present protocol consider 28 storage days after irradiation as the limit for the RBC usage.593016Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Barjas-Castro, M.L., Brandão, M.M., Fontes, A., Costa, F.F., Cesar, C.L., Saad, S.T.O., Elastic properties of irradiated red blood cell units measured by optical tweezer (2002) Transfusion, 42, pp. 1196-1199Brandão, M.M., Fontes, A., Barjas-Castro, M.L., Barbosa, L.C., Costa, F.F., Cesar, C.L., Saad, S.T.O., Optical tweezers for measuring red blood cell elasticity: Application to the study of drug response in sickle cell disease (2003) European Journal of Haematology, 70, pp. 207-211Williamson, L.M., Warwick, R.M., Transfusion-associated graft-versus-host disease and its prevention (1995) Blood Rev., 9, pp. 251-261Button, L.N., Dewolf, W.C., Newburger, P.E., The effecr of irradiation on blood components (1981) Transfusion, 21, pp. 419-426Platt, O.S., The sickle syndrome (1995) Blood: Principles and Practice of Hematology, , R. I Hadlin, S. E. Lux, T. P. Stossel, J. B. Lippincott, PhiladelphiaBallas, S.K., Dover, G.J., Charache, S., Effect of hydroxyurea on the rheological properties of sickle erythrocytes in vivo (1989) Am. J. Hematol, 32, pp. 104-111Groner, W., Mohandas, N., Bessis, M., New optical technique for measuring erythrocyte deformability with the ektacytometer (1980) Clin. Chem., 26, pp. 1435-1442De Franceschi, L., Bachir, D., Galacteros, F., Tchernia, G., Cynober, T., Alper, S., Platt, O., Brugnara, C., Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease (1997) J Clin Invest, 100, pp. 1847-1852Hochmuth, R.M., Worthy, P.R., Evans, E.A., Red cell extensional recovery and the determination of membrane viscosity (1979) Biophys. J., 26, pp. 101-114Evans, E.A., La Celle, P.L., Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation (1975) Blood, 45, pp. 29-43Itoh, T., Chien, S., Usami, S., Effects of hemoglobin concentration on deformability of individual sickle cells after deoxygenation (1995) Blood, 85, pp. 2245-2253Evans, E.A., Mohandas, N., Membrane-associated sickle hemoglobin: A major determinant of sickle erythrocyte rigidity (1987) Blood, 70, pp. 1443-1449Dong, C., Chadwick, R.S., Schechter, A.N., Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation (1992) Biophys. J., 63, pp. 774-783Suzuki, Y., Tateishi, N., Cicha, I., Decreased deformability of the X-ray irradiated red blood cells stored in manitol-adenine-phosphate medium (2000) Clin. Hemorheol. Micro-cire., 22, pp. 131-14

Molecular characterization of Haemophilus parasuis ferric hydroxamate uptake (fhu) genes and constitutive expression of the FhuA receptor

P. 49-59Bacteria have evolved a set of highly specialized proteins to capture iron in irondepleted environments. The acquisition and uptake of iron present in the extracellular milieu of eukaryotic organisms is indispensable for the growth and survival of microbial pathogens in the course of infection. Haemophilus parasuis is the causative agent of Glässer disease, which is responsible for considerable financial losses in pig-rearing worldwide. To gain insight into the mechanisms involved in siderophore-mediated iron uptake in H. parasuis, genes in the H. parasuis ferric hydroxamate uptake (Fhu) region were amplified in the work being reported here. As has been described in A. pleuropneumoniae, an Fhu genomic region was also present in H. parasuis, being composed of four potential consecutive open reading frames (ORF) designated as fhuC, fhuD, fhuB, and fhuA, respectively. By immunoblotting, using a cross-reactive polyclonal antibody raised against Actinobacillus pleuropneumoniae FhuA protein, it was demonstrated that this protein was constitutively expressed in H. parasuis and its level of expression was not modified under conditions of restricted iron availability. This is the first report describing the presence of the fhu genes in H. parasuis. Our results indicate that FhuA protein expression is not affected under ironrestricted conditions, however, it is one of the targets of the humoral immune responseS

Immunotherapeutic targeting of LIGHT/LTβR/HVEM pathway fully recapitulates the reduced cytotoxic phenotype of LIGHT-deficient T cells.

Tumor necrosis factor (TNF)/TNF receptor (TNFR) superfamily members play essential roles in the development of the different phases of the immune response. Mouse LIGHT (TNFSF14) is a type II transmembrane protein with a C-terminus extracellular TNF homology domain (THD) that assembles in homotrimers and regulates the course of the immune responses by signaling through 2 receptors, the herpes virus entry mediator (HVEM, TNFSFR14) and the lymphotoxin β receptor (LTβR, TNFSFR3). LIGHT is a membrane-bound protein transiently expressed on activated T cells, natural killer (NK) cells and immature dendritic cells that can be proteolytically cleaved by a metalloprotease and released to the extracellular milieu. The immunotherapeutic potential of LIGHT blockade was evaluated in vivo. Administration of an antagonist of LIGHT interaction with its receptors attenuated the course of graft-versus-host reaction and recapitulated the reduced cytotoxic activity of LIGHT-deficient T cells adoptively transferred into non-irradiated semiallogeneic recipients. The lack of LIGHT expression on donor T cells or blockade of LIGHT interaction with its receptors slowed down the rate of T cell proliferation and decreased the frequency of precursor alloreactive T cells, retarding T cell differentiation toward effector T cells. The blockade of LIGHT/LTβR/HVEM pathway was associated with delayed downregulation of interleukin-7Rα and delayed upregulation of inducible costimulatory molecule expression on donor alloreactive CD8 T cells that are typical features of impaired T cell differentiation. These results expose the relevance of LIGHT/LTβR/HVEM interaction for the potential therapeutic control of the allogeneic immune responses mediated by alloreactive CD8 T cells that can contribute to prolong allograft survival

Rapid Phenotype-Driven Gene Sequencing with the NeoSeq Panel: A Diagnostic Tool for Critically Ill Newborns with Suspected Genetic Disease

New genomic sequencing techniques have shown considerable promise in the field of neonatology, increasing the diagnostic rate and reducing time to diagnosis. However, several obstacles have hindered the incorporation of this technology into routine clinical practice. We prospectively evaluated the diagnostic rate and diagnostic turnaround time achieved in newborns with suspected genetic diseases using a rapid phenotype-driven gene panel (NeoSeq) containing 1870 genes implicated in congenital malformations and neurological and metabolic disorders of early onset (<2 months of age). Of the 33 newborns recruited, a genomic diagnosis was established for 13 (39.4%) patients (median diagnostic turnaround time, 7.5 days), resulting in clinical management changes in 10 (76.9%) patients. An analysis of 12 previous prospective massive sequencing studies (whole genome (WGS), whole exome (WES), and clinical exome (CES) sequencing) in newborns admitted to neonatal intensive care units (NICUs) with suspected genetic disorders revealed a comparable median diagnostic rate (37.2%), but a higher median diagnostic turnaround time (22.3 days) than that obtained with NeoSeq. Our phenotype-driven gene panel, which is specific for genetic diseases in critically ill newborns is an affordable alternative to WGS and WES that offers comparable diagnostic efficacy, supporting its implementation as a first-tier genetic test in NICUs

Studying Red Blood Cell Agglutination By Measuring Membrane Viscosity With Optical Tweezers

The red blood cell (RBC) viscoelastic membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that are responsible for cell agglutination. Manipulating RBCs rouleaux with a double optical tweezers, we observed that the cells slide easily one over the others but are strongly connected by their edges. An explanation for this behavior could be the fact that when the cells slide one over the others, proteins are dragged through the membrane. It confers to the movement a viscous characteristic that is dependent of the velocity between the RBCs and justifies why is so easy to slide them apart. Therefore, in a first step of this work, by measuring the force as a function of the relative velocity between two cells, we confirmed this assumption and used this viscous characteristic of the RBC rouleaux to determine the apparent membrane viscosity of the cell. As this behavior is related to the proteins interactions, we can use the apparent membrane viscosity to obtain a better understanding about cell agglutination. Methods related to cell agglutination induced by antigen-antibody interactions are the basis of most of tests used in transfusion centers. Then, in a second step of this work, we measured the apparent membrane viscosity using antibodies. We observed that this methodology is sensitive to different kinds of bindings between RBCs. Better comprehension of the forces and bindings between RBCs could improve the sensibility and specificity of the hemagglutination reactions and also guides the development of new potentiator substances.6644Fontes, A., Fernandes, H.P., Barjas-Castro, M.L., Thomaz, A.A., Pozzo, L., Barbosa, L.C., Cesar, C.L., Red blood cell membrane viscoelasticity, agglutination and zeta potential measurements with double optical tweezers (2006) Proceedings of SPIE, 6088, pp. 296-305Eylar, E.H., Madoff, M.A., Brody, O.V., Oncley, J.L., The contribution of sialic acid to the surface charge of the erythrocyte (1962) J. Biol. Chem, 237, pp. 1992-2000Pollack, W., Reckel, R.P., A reappraisal of the forces involved in Hemagglutination (1977) Int Archs Allergy Appl. Immun, 54, pp. 29-42Fontes, A., Giorgio, S., de Castro Jr., A.B., Neto, V.M., Pozzo, L.Y., Marques, G.P., Barbosa, L.C., Cesar, C.L., Determination of femto Newton forces and fluid viscosity using optical tweezers: Application to Leishmania amazonensis (2005) Proceedings of SPIE, 5699, pp. 419-425Saffman, P.G., Delbruck, M., Brownian motion in biological membranes (1975) Proc. Nat. Acad. Sci. USA, 72, pp. 3111-3113Dimova, R., Danov, K., Pouligny, B., Ivanov, I.B., Drag of a solid particle trapped in a thin film or at an interface: Influence of surface viscosity and elasticity (2000) J. Colloid and Interface Science, 226, pp. 35-4

Spatial antibunching of photons with parametric down-conversion

The theoretical framework behind a recent experiment by Nogueira et. al. [Phys. Rev. Lett. 86}, 4009 (2001)] of spatial antibunching in a two-photon state generated by collinear type II parametric down-conversion and a birefringent double-slit is presented. The fourth-order quantum correlation function is evaluated and shown to violate the classical Schwarz-type inequality, ensuring that the field does not have a classical analog. We expect these results to be useful in the rapidly growing fields of quantum imaging and quantum information.Comment: 5 pages, 3 figures. Minor changes made, accepted for publication in PR