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Perteguer M.J.,Institute Salud Carlos III Majadahonda | Canavate C.,World Health Organization | Dagger F.,Central University of Venezuela | Garate T.,Institute Salud Carlos III Majadahonda | And 2 more authors.
Cell Stress and Chaperones | Year: 2013

Eukaryotic cells respond to DNA damage by activating damage checkpoint pathways, which arrest cell cycle progression and induce gene expression. We isolated a full-length cDNA encoding a 49-kDa protein from Leishmania major, which exhibited significant deduced amino acid sequence homology with the annotated Leishmania sp. DNA damage-inducible (Ddi1-like) protein, as well as with the Ddi1 protein from Saccharomyces cerevisiae. In contrast to the previously described Ddi1 protein, the protein from L. major displays three domains: (1) an NH2-terminal ubiquitin like; (2) a COOH terminal ubiquitin-associated; (3) a retroviral aspartyl proteinase, containing the typical D[S/T]G signature. The function of the L. major Ddi1-like recombinant protein was investigated after expression in baculovirus/insect cells and biochemical analysis, revealing preferential substrate selectivity for aspartyl proteinase A2 family substrates, with optimal activity in acidic conditions. The proteolytic activity was inhibited by aspartyl proteinase inhibitors. Molecular modeling of the retroviral domain of the Ddi1-like Leishmania protein revealed a dimer structure that contained a double Asp-Ser-Gly-Ala amino acid sequence motif, in an almost identical geometry to the exhibited by the homologous retroviral aspartyl protease domain of yeast Ddi1 protein. Our results indicate that the isolated Ddi1-like protein is a functional aspartyl proteinase in L. major, opening possibility to be considered as a potential target for novel antiparasitic drugs. © 2012 Cell Stress Society International.


PubMed | Institute Salud Carlos Iii Majadahonda, Biotherapix, CSIC - National Institute of Aerospace Technology and CIBER ISCIII
Type: Journal Article | Journal: PloS one | Year: 2016

The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with drug- and multidrug-resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm, which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5-10% of the total quasispecies. The developed genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice.


PubMed | Institute Salud Carlos III Majadahonda, Institute Pasteur Paris and Complutense University of Madrid
Type: | Journal: Frontiers in microbiology | Year: 2015

Cryptococcus neoformans is an opportunistic fungal pathogen that has several well-described virulence determinants. A polysaccharide capsule and the ability to produce melanin are among the most important. Melanization occurs both in vitro, in the presence of catecholamine and indole compounds, and in vivo during the infection. Despite the importance of melanin production for cryptococcal virulence, the component and mechanisms involved in its synthesis have not been fully elucidated. In this work, we describe the role of a G1/S cyclin (Cln1) in the melanization process. Cln1 has evolved specifically with proteins present only in other basidiomycetes. We found that Cln1 is required for the cell wall stability and production of melanin in C. neoformans. Absence of melanization correlated with a defect in the expression of the LAC1 gene. The relation between cell cycle elements and melanization was confirmed by the effect of drugs that cause cell cycle arrest at a specific phase, such as rapamycin. The cln1 mutant was consistently more susceptible to oxidative damage in a medium that induces melanization. Our results strongly suggest a novel and hitherto unrecognized role for C. neoformans Cln1 in the expression of virulence traits.


Cicuendez M.,Complutense University of Madrid | Cicuendez M.,CIBER ISCIII | Portoles P.,Consejo Superior de Investigaciones Cientificas | Portoles P.,Institute Salud Carlos III Majadahonda | And 6 more authors.
Journal of Materials Chemistry B | Year: 2014

The interaction of new nanocomposite mesoporous glass/hydroxyapatite (MGHA) scaffolds with immune cells involved in both innate and acquired immunity has been studied in vitro as an essential aspect of their biocompatibility assessment. Since the immune response can be affected by the degradation products of bioresorbable scaffolds and scaffold surface changes, both processes have been evaluated. No alterations in proliferation and viability of RAW-264.7 macrophage-like cells were detected after culture on MGHA scaffolds which did not induce cell apoptosis. However, a slight cell size decrease and an intracellular calcium content increase were observed after contact of this cell line with MGHA scaffolds or their extracts. Although no changes in the percentages of RAW cells with low and high contents of reactive oxygen species (ROS) are observed by the treatment with 7 day extracts, this study has revealed modifications of these percentages after direct contact with scaffolds and by the treatment with 24 h extracts, related to the high reactivity/bioactivity of this MGHA nanocomposite at initial times. Furthermore, when normal fresh murine spleen cells were used as an experimental model closer to physiological conditions, no significant alterations in the activation of different immune cell subpopulations were detected in the presence of 24 h MGHA extract. MGHA scaffolds did not affect either the spontaneous apoptosis or intracellular cytokine expression (IL-2, IL-10, IFN-γ, and TNF-α) after 24 h treatment. The results obtained in the present study with murine immune cell subpopulations (macrophages, lymphocytes B, lymphocytes T and natural killer cells) support the biocompatibility of the MGHA material and suggest an adequate host tissue response to their scaffolds upon their implantation. This journal is © the Partner Organisations 2014.


PubMed | Institute Salud Carlos III Majadahonda, São Paulo State University, University of Medellín and University of Sao Paulo
Type: | Journal: Frontiers in microbiology | Year: 2016

[This corrects the article on p. 153 in vol. 7, PMID: 26909069.].


Calero M.,Institute Salud Carlos III Majadahonda | Gasset M.,CSIC - Institute of Physical Chemistry "Rocasolano"
Methods in Molecular Biology | Year: 2012

Amyloids are fibrillar aggregates of proteins characterized by a basic scaffold consisting of cross β-sheet structure that can exert physiological or pathological effects. Both far-UV circular dichroism and Fourier transform infrared (FTIR) spectroscopies are techniques used for the fast analysis of protein secondary structure. Both techniques are complementary and preferentially used depending on the physical state of the analyte, the major secondary structure element and the relative abundance of given amino acids. Although there are special setups for working with films, circular dichroism is best suited for ideal diluted solutions of polypeptides exhibiting α-helix as major structural element and low content of aromatic residues. During the last decade, a related technique, linear dichroism, has been applied to study the orientation of protein subunits within amyloid oligomers or fibrils in solution. Alternatively, FTIR works best with concentrated solutions, solids and films, and resolves with accuracy the β-sheet composition, but it is affected by contributions of amide groups. The advent of new infrared techniques based on correlation analysis of time-dependent variations induced by external perturbations that generates two-dimensional IR maps has enabled to greatly increase spectral resolution and to extend its applicability to protein secondary structure characterization in a variety of physical environments. Within the amyloid field, conjunction of both spectroscopies has provided the first filter step for amyloid detection and has contributed to decipher the structural aspects of the amyloid formation mechanism. © 2012 Springer Science+Business Media, LLC.


Moreira R.N.,New University of Lisbon | Domingues S.,New University of Lisbon | Viegas S.C.,New University of Lisbon | Amblar M.,Institute Salud Carlos Iii Majadahonda | Arraiano C.M.,New University of Lisbon
BMC Microbiology | Year: 2012

Background: Ribonuclease R (RNase R) is an exoribonuclease that recognizes and degrades a wide range of RNA molecules. It is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. RNase R has also been implicated in the trans-translation process. Transfer-messenger RNA (tmRNA/SsrA RNA) and SmpB are the main effectors of trans-translation, an RNA and protein quality control system that resolves challenges associated with stalled ribosomes on non-stop mRNAs. Trans-translation has also been associated with deficiencies in stress-response mechanisms and pathogenicity. Results: In this work we study the expression of RNase R in the human pathogen Streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery (SmpB and tmRNA/SsrA). We show that RNase R is induced after a 37°C to 15°C temperature downshift and that its levels are dependent on SmpB. On the other hand, our results revealed a strong accumulation of the smpB transcript in the absence of RNase R at 15°C. Transcriptional analysis of the S. pneumoniae rnr gene demonstrated that it is co-transcribed with the flanking genes, secG and smpB. Transcription of these genes is driven from a promoter upstream of secG and the transcript is processed to yield mature independent mRNAs. This genetic organization seems to be a common feature of Gram positive bacteria, and the biological significance of this gene cluster is further discussed. Conclusions: This study unravels an additional contribution of RNase R to the trans-translation system by demonstrating that smpB is regulated by this exoribonuclease. RNase R in turn, is shown to be under the control of SmpB. These proteins are therefore mutually dependent and cross-regulated. The data presented here shed light on the interactions between RNase R, trans-translation and cold-shock response in an important human pathogen. © 2012 Moreira et al.; licensee BioMed Central Ltd.


Calero M.,Institute Salud Carlos III Majadahonda | Rostagno A.,New York University | Ghiso J.,New York University
Methods in Molecular Biology | Year: 2012

'Amyloid binging proteins' is a generic term used to designate proteins that interact with different forms of amyloidogenic peptides or proteins and that, as a result, may modulate their physiological and pathological functions by altering solubility, transport, clearance, degradation, and fibril formation. We describe a simple affinity chromatography protocol to isolate and characterize amyloid-binding proteins based on the use of sequential elution steps that may provide further information on the type of binding interaction. As an example, we depict the application of this protocol to the study of Alzheimer's amyloid β (Aβ) peptide-binding proteins derived from human plasma. Biochemical analysis of the proteins eluted under different conditions identified serum amyloid P component (SAP) and apolipoprotein J (clusterin) as the main plasma Aβ-binding proteins while various apolipoproteins (apoA-IV, apoE, and apoA-I), as well as albumin (HSA) and fibulin were identified as minor contributors. © 2012 Springer Science+Business Media, LLC.


PubMed | Institute Salud Carlos III Majadahonda, Technical University of Madrid, University of Bern and Hospital Universitario Severo Ochoa Avda Orellana s n
Type: Journal Article | Journal: New microbes and new infections | Year: 2014

Human diphyllobothriasis is sporadically detected in Spain. Diphyllobothrium latum and Diplogonoporus balaenopterae have been identified. In the study, four cases of presumably imported diphyllobothriasis in Spanish patients were appraised. Molecular diagnosis allowed us to identify exotic fish tapeworms such as Diplogonoporus balaenopterae in one patient and Diphyllobothrium pacificum in the others.

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