Dynamics of pneumococcal nasopharyngeal carriage in healthy children attending a day care center in northern Spain. Influence of detection techniques on the results

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A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background: Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrPSc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion. Case presentation: We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrPSc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrPSc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine. Conclusions: A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

Insights into mechanism kinematics for protein motion simulation

Background: The high demanding computational requirements necessary to carry out protein motion simulations make it difficult to obtain information related to protein motion. On the one hand, molecular dynamics simulation requires huge computational resources to achieve satisfactory motion simulations. On the other hand, less accurate procedures such as interpolation methods, do not generate realistic morphs from the kinematic point of view. Analyzing a protein's movement is very similar to serial robots; thus, it is possible to treat the protein chain as a serial mechanism composed of rotational degrees of freedom. Recently, based on this hypothesis, new methodologies have arisen, based on mechanism and robot kinematics, to simulate protein motion. Probabilistic roadmap method, which discretizes the protein configurational space against a scoring function, or the kinetostatic compliance method that minimizes the torques that appear in bonds, aim to simulate protein motion with a reduced computational cost. Results: In this paper a new viewpoint for protein motion simulation, based on mechanism kinematics is presented. The paper describes a set of methodologies, combining different techniques such as structure normalization normalization processes, simulation algorithms and secondary structure detection procedures. The combination of all these procedures allows to obtain kinematic morphs of proteins achieving a very good computational cost-error rate, while maintaining the biological meaning of the obtained structures and the kinematic viability of the obtained motion. Conclusions: The procedure presented in this paper, implements different modules to perform the simulation of the conformational change suffered by a protein when exerting its function. The combination of a main simulation procedure assisted by a secondary structure process, and a side chain orientation strategy, allows to obtain a fast and reliable simulations of protein motion.

Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells

Background Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo. Methodology/Principal Findings Using an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination. Conclusions/Significance These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.