Disease-associated prion protein elicits immunoglobulin M responses in vivo

Prion diseases such as Creutzfeldt-Jakob disease are believed to result from the misfolding of a widely expressed normal cellular prion protein, PrPc. The resulting disease-associated isoforms, PrP(Sc), have much higher beta-sheet content, are insoluble in detergents, and acquire relative resistance to proteases. Although known to be highly aggregated and to form amyloid fibrils, the molecular architecture of PrP9Sc) is poorly understood. To date, it has been impossible to elicit antibodies to native PrP(Sc) that are capable of recognizing PrP(Sc) without denaturation, even in Pm-P(o/o) mice that are intolerant of it. Here we demonstrate that antibodies for native PrPc and PrP(Sc) can be produced by immunization of Pm-P(o/o) mice with partially purified PrPc and PrP(Sc) adsorbed to immunomagnetic particles using high-affinity anti-PrP monoclonal antibodies (mAbs). Interestingly, the polyclonal response to PrP(Sc) was predominantly of the immunoglobulin M (IgM) isotype, unlike the immunoglobulin G (IgG) responses elicited by PrP(c) or by recombinant PrP adsorbed or not to immunomagnetic particles, presumably reflecting the polymeric structure of disease-associated prion protein. Although heat-denatured PrP(Sc) elicited more diverse antibodies with the revelation of C-terminal epitopes, remarkably, these were also predominantly IgM suggesting that the increasing immunogenicity, acquisition of protease sensitivity, and reduction in infectivity induced by heat are not associated with dissociation of the PrP molecules in the diseased-associated protein. Adsorbing native proteins to immunomagnetic particles may have general applicability for raising polyclonal or monoclonal antibodies to any native protein, without attempting laborious purification steps that might affect protein conformation.     

Assessment of losses in honey yield due to the chalkbrood disease, with reference to the determination of its economic injury levels in Egypt

In Egypt, the chalk brood (CHB) disease caused by the fungus, Ascosphaera apis Maassen started again infesting the honeybee, Apis melifera L (Hymenoptera: Apidae) colonies after a cessation pause of seven to nine years. For the first time, an attempt has been made to look into the problem of the CHB disease from the view point of assessing losses in both clover (Trifolium alexandrinum) and chinus (Chinus molus) honey yields. In this regard, two techniques were adopted. Under the natural conditions of the experimental apiary (first technique), loss in clover honey was 18.412 +/- 0.663%, on average, whereas the average loss in chinus honey was 18.332 +/- 1.536%. In the second technique of loss appraisal (artificial infection). The tested honeybee colonies were artificially infected with four sources of A. apis infection, I.e. black mummies, white mummies, pollen grains and water, in addition to controls to create the so-called "different levels of infection". The mean percentages of losses in clover honey were 30.06 +/- 1.807, 27.95 +/- 1.062; 21.13 +/- 0.987; 16.96 +/- 0.672 and 0.00 +/- 0.00 for black mummies, white mummies, pollen grains, water and control, respectively. Taking into account the relationship between number of resulted mummies in each source of infection and % loss in clover honey, it could be concluded that as the resulted mummies increased, the corresponding clover honey yield decreased in each source of infection. It was noticed that the % loss in clover honey differed as the used technique differed. For example, the percentage loss in clover honey produced from colonies exposed to the natural conditions was relatively less than that of the artificially infected ones. This has been discussed in the text. However, the causative pathogen of CHB disease resulted in serious decrease in honey production. Loss appraisal is a perquisite step for the determination of the economic injury levels (EILs). By regressing % loss in clover honey yield against the total number of fallen mummies, the linear equation was worked out to be: Y = 0.242 + 0.040 x (r2 = 0.99, F = 62408.865). Depending on the total costs of controlling CHB disease, the EIL values were 31.875 and 11.250 mummies/colony for cedar oil and thymol granules, subsequently. As to the chinus honey the corresponding EILs were 18.940 and 6.683 mummies/colony for cedar oil and thymol granules, in respect, as a result of adopting the regression formula: Y = -6.762 + 0.101 X. As for the clover honey (artificial infection) the EIL values were 39.844 and 14.063 mummies/ colony in case of using cedar oil and thymol grains, subsequently. These values were worked out using the formula Y = 5.871 + 0.032 X. From the practical point of view, apiarists should not use thymol or cedar oil as fallen mummies don't reach the suggested EILs values to minimize control costs as much as possible.     

Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal ankyrinG. In vitro mapping of the interacting domains and association in synaptosomes

Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K(D) value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.     

Measurement of oxidized glutathione by enzymatic recycling coupled to bioluminescent detection

A new method is described for the quantification of oxidized glutathione (GSSG) in tissues by enzymatic recycling coupled to NADPH bioluminescent detection. Tissue samples are treated with metaphosphoric acid. In a first step, after derivatization of GSH with 4-chloro-7-trifluoromethyl-1-methylquinolinium (CFQ), GSSG is recycled in the presence of dithionitrobenzoic acid (DTNB) and NADPH by glutathione reductase. In a second step, the GSSG-dependent NADPH consumption is measured by luminescence with NADPH:FMN oxidoreductase-bacterial luciferase. The coefficient of variation for GSSG measurements on repeated assays (n = 5) is 2 and 3% for standards and tissue samples, respectively. The sensitivity of this method is at the picomole level and is convenient for determination of GSSG physiological concentrations in tissues: GSSG levels measured in rat liver and kidney ranged from 76 to 215 and 52 to 170 nmol/g wet weight, respectively.     

TiO2 Nanocrystals Synthesized by Laser Pyrolysis for the Up‐Scaling of Efficient Solid‐State Dye‐Sensitized Solar Cells

A crucial issue regarding emerging nanotechnologies remains the up‐scaling of new functional nanostructured materials towards their implementation in high performance applications on a large scale. In this context, we demonstrate high efficiency solid‐state dye‐sensitized solar cells prepared from new porous TiO₂ photoanodes based on laser pyrolysis nanocrystals. This strategy exploits a reduced number of processing steps as well as non‐toxic chemical compounds to demonstrate highly porous TiO₂ films. The possibility to easily tune the TiO₂ nanocrystal physical properties allows us to demonstrate all solid‐state dye‐sensitized devices based on a commercial benchmark materials (organic indoline dye and molecular hole transporter) presenting state‐of‐the‐art performance comparable with reference devices based on a commercial TiO₂ paste. In particular, a drastic improvement in pore infiltration, which is found to balance a relatively lower surface area compared to the reference electrode, is evidenced using laser‐synthesized nanocrystals resulting in an improved short‐circuit current density under full sunlight. Transient photovoltage decay measurements suggest that charge recombination kinetics still limit device performance. However, the proposed strategy emphasizes the potentialities of the laser pyrolysis technique for up‐scaling nanoporous TiO₂ electrodes for various applications, especially for solar energy conversion.     

Detection of a fourth orbivirus non-structural protein

The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1-VP7) and 3 non-structural proteins (NS1-NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4.     

Visualization of pairwise and multilocus linkage disequilibrium structure using latent forests

Linkage disequilibrium study represents a major issue in statistical genetics as it plays a fundamental role in gene mapping and helps us to learn more about human history. The linkage disequilibrium complex structure makes its exploratory data analysis essential yet challenging. Visualization methods, such as the triangular heat map implemented in Haploview, provide simple and useful tools to help understand complex genetic patterns, but remain insufficient to fully describe them. Probabilistic graphical models have been widely recognized as a powerful formalism allowing a concise and accurate modeling of dependences between variables. In this paper, we propose a method for short-range, long-range and chromosome-wide linkage disequilibrium visualization using forests of hierarchical latent class models. Thanks to its hierarchical nature, our method is shown to provide a compact view of both pairwise and multilocus linkage disequilibrium spatial structures for the geneticist. Besides, a multilocus linkage disequilibrium measure has been designed to evaluate linkage disequilibrium in hierarchy clusters. To learn the proposed model, a new scalable algorithm is presented. It constrains the dependence scope, relying on physical positions, and is able to deal with more than one hundred thousand single nucleotide polymorphisms. The proposed algorithm is fast and does not require phase genotypic data.     

Metabolic amplification of insulin secretion by glucose is independent of β-cell microtubules

Glucose-induced insulin secretion (IS) by β-cells is controlled by two pathways. The triggering pathway involves ATP-sensitive potassium (K(ATP)) channel-dependent depolarization, Ca(2+) influx, and rise in the cytosolic Ca(2+) concentration ([Ca(2+)](c)), which triggers exocytosis of insulin granules. The metabolic amplifying pathway augments IS without further increasing [Ca(2+)](c). After exclusion of the contribution of actin microfilaments, we here tested whether amplification implicates microtubule-dependent granule mobilization. Mouse islets were treated with nocodazole or taxol, which completely depolymerized and polymerized tubulin. They were then perifused to measure [Ca(2+)](c) and IS. Metabolic amplification was studied during imposed steady elevation of [Ca(2+)](c) by tolbutamide or KCl or by comparing [Ca(2+)](c) and IS responses to glucose and tolbutamide. Nocodazole did not alter [Ca(2+)](c) or IS changes induced by the three secretagogues, whereas taxol caused a small inhibition of IS that is partly ascribed to a decrease in [Ca(2+)](c). When [Ca(2+)](c) was elevated and controlled by KCl or tolbutamide, the amplifying action of glucose was unaffected by microtubule disruption or stabilization. Both phases of IS were larger in response to glucose than tolbutamide, although triggering [Ca(2+)](c) was lower. This difference, due to amplification, persisted in nocodazole- or taxol-treated islets, even when IS was augmented fourfold by microfilament disruption with cytochalasin B or latrunculin B. In conclusion, metabolic amplification rapidly augments first and second phases of IS independently of insulin granule translocation along microtubules. We therefore extend our previous proposal that it does not implicate the cytoskeleton but corresponds to acceleration of the priming process conferring release competence to insulin granules.     

The proteome and interactome of Streptococcus pneumoniae phage Cp-1

Mass spectrometry analysis of Streptococcus pneumoniae bacteriophage Cp-1 identified a total of 12 proteins, and proteome-wide yeast two-hybrid screens revealed 17 binary interactions mainly among these structural proteins. On the basis of the resulting linkage map, we suggest an improved structural model of the Cp-1 virion.