Browsing by Author "Melo Ledermann, Francisco Javier"
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- ItemA new multiplex PCR assay for the simultaneous detection of vancomycin-resistant enterococci from rectal swabs(2010) Benadof, D.; San Martín, M.; Aguirre, J.; Paredes, L.; Malig, R.; Melo Ledermann, Francisco Javier; Lehouque, G.Objectives This study describes the diagnostic performance of a recently available multiplex PCR-based kit for the simultaneous detection and identification of Enterococcus faecium, Enterococcus faecalis, vanA, vanB, vanC1 and vanC2/C3 genes, directly from rectal swabs constituting the most complete existing molecular assay currently available. Methods The diagnostic performance of this assay was evaluated by a multicenter study involving three independent public hospitals and consisted in the analysis of 187 rectal swabs from patients at high risk for vancomycin-resistant enterococci colonization. Results When bacteria culture was used as the gold standard, the sensitivity, specificity, positive and negative predicted values for the assay were 96.8%, 76.0%, 67.7% and 97.9%, respectively. When a composite reference standard consisting of culture and DNA sequencing of PCR products was used as the gold standard, the sensitivity, specificity, positive and negative predicted values for the PCR-based assay were 97.8%, 96.9%, 96.7% and 97.9%, respectively. Conclusions Based on these results, we conclude that this assay is considerably more sensitive than traditional microbiological methods for detecting vancomycin-resistant enterococci from rectal swabs. It is also much faster than culture. We believe that the implementation of this assay in routine clinical laboratories could help to reduce hospital-acquired vancomycin-resistant enterococci infections.
- ItemA novel extracellular multicopper oxidase from Phanerochaete chrysosporium with ferroxidase activity(2003) Larrondo Castro, Luis Fernando; Salas, L.; Melo Ledermann, Francisco Javier; Vicuña, Rafael; Cullen, D.Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe2+, with a Km close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.
- ItemAssessment of Mutations Associated With Genomic Variants of SARS-CoV-2: RT-qPCR as a Rapid and Affordable Tool to Monitoring Known Circulating Variants in Chile, 2021(2022) Angulo, J.; Martinez Valdebenito, C.; Pardo Roa, C.; Almonacid, L. I.; Fuentes Luppichini, E.; Contreras, A. M.; Maldonado, C.; Le Corre, N.; Melo Ledermann, Francisco Javier; Medina, R. A.; Ferrés, M.Since the first report of SARS-CoV-2 infection in humans, the virus has mutated to develop new viral variants with higher infection rates and more resistance to neutralization by antibodies elicited after natural SARS-CoV-2 infection or by vaccines. Therefore, rapid identification of viral variants circulating in the population is crucial for epidemiological assessment and efforts to contain the resurgence of the pandemic. Between January and November 2021, we performed a large variant RT-qPCR-based screening of mutations in the spike protein of 1851 SARS-CoV-2-positive samples derived from outpatients from the UC-Christus Health Network in Chile. In a portion of samples (n = 636), we validated our RT-qPCR-pipeline by WGS, obtaining a 99.2% concordance. Our results indicate that from January to March 2021 there was a dominance of non-identifiable variants by the RT-qPCR-based screening; however, throughout WGS we were able to identify the Lambda (C.37) variant of interest (VOI). From March to July, we observed the rapid emergence of mutations associated with the Gamma variant (P.1), which was quickly replaced by the appearance of a combination of samples harboring mutations associated with the Delta variant (B.1.617.2), which predominated until the end of the study. Our results highlight the applicability of cost-effective RT-qPCR-based screening of mutations associated with known variants of concern (VOC), VOI and variants under monitoring (VUM) of SARS-CoV-2, being a rapid and reliable tool that complements WGS-based surveillance.
- ItemEvolutionary potentials: structure specific knowledge-based potentials exploiting the evolutionary record of sequence homologs(2008) Panjkovich, A.; Melo Ledermann, Francisco Javier; Marti Renom, M. A.We introduce a new type of knowledge-based potentials for protein structure prediction, called 'evolutionary potentials', which are derived using a single experimental protein structure and all three-dimensional models of its homologous sequences. The new potentials have been benchmarked against other knowledge-based potentials, resulting in a significant increase in accuracy for model assessment. In contrast to standard knowledge-based potentials, we propose that evolutionary potentials capture key determinants of thermodynamic stability and specific sequence constraints required for fast folding.
- ItemExtracellular Matrix Is Required for Skeletal Muscle Differentiation But Not Myogenin Expression(1996) Melo Ledermann, Francisco Javier; Brandan, Enrique
- ItemProNA2020 predicts protein-DNA, protein-RNA, and protein-protein binding proteins and residues from sequence(2020) Qiu, J.; Bernhofer, M.; Heinzinger, M.; Kemper, S.; Norambuena, T.; Melo Ledermann, Francisco Javier; Rost, B.The intricate details of how proteins bind to proteins, DNA, and RNA are crucial for the understanding of almost all biological processes. Disease-causing sequence variants often affect binding residues. Here, we described a new, comprehensive system of in silico methods that take only protein sequence as input to predict binding of protein to DNA, RNA, and other proteins. Firstly, we needed to develop several new methods to predict whether or not proteins bind (per-protein prediction). Secondly, we developed independent methods that predict which residues bind (per-residue). Not requiring three-dimensional information, the system can predict the actual binding residue. The system combined homology-based inference with machine learning and motif-based profile-kernel approaches with word-based (ProtVec) solutions to machine learning protein level predictions. This achieved an overall non-exclusive three-state accuracy of 77% ± 1% (±one standard error) corresponding to a 1.8 fold improvement over random (best classification for protein–protein with F1 = 91 ± 0.8%). Standard neural networks for per-residue binding residue predictions appeared best for DNA-binding (Q2 = 81 ± 0.9%) followed by RNA-binding (Q2 = 80 ± 1%) and worst for protein–protein binding (Q2 = 69 ± 0.8%). The new method, dubbed ProNA2020, is available as code through github (https://github.com/Rostlab/ProNA2020.git) and through PredictProtein (www.predictprotein.org
- ItemScoring functions for protein structure prediction(2008) Melo Ledermann, Francisco Javier; Feytmans, E.; Schwede, Torsten; Peitsch, ManuelThe following sections are included: Introduction Structure and Components of Scoring Functions Contact Scoring Functions Distance-dependent Scoring Functions Accessible Surface Scoring Functions Combined Scoring Functions How is a Scoring Function Derived? Selection of Source Experimental Data Reference Systems How is a Scoring Function Used? Classical Overall Score Calculation Sequence space reference frame Structure space reference frame Classical Detailed Score Calculation Variations on Score Calculations Typical Applications of Scoring Functions in Protein Structure Prediction Fold Assessment Model Ranking Error Detection Folding and Molecular Simulations Other Applications of Scoring Functions Future Outlook Reference Systems and Atom Type Definitions Solvation Models Evolutionary Information Multivariate Scoring Functions Acknowledgements References
- ItemSignificantly reduced expression of the proteoglycan decorin in Alzheimer's disease fibroblasts(1996) Brandan, Enrique; Melo Ledermann, Francisco Javier; García, M.; Contreras, M.Aims—To investigate whether proteoglycan synthesis is altered in skin fibroblasts in patients with Alzheimer's disease compared with normal subjects. Methods—Cell lines obtained from donors with Alzheimer's disease and healthy controls were incubated with radioactive sulphate. The proteoglycans synthesised were determined and analysed by chromatographic, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and glycosaminoglycans-lyase treatment. The amount of decorin synthesised by each cell line was quantified using western blot analysis. Transcripts for human decorin were determined using northern blot analysis. Results—No significant changes in total sulphate incorporation and glycos-aminoglycan (GAG) composition were detected in the incubation media of these cells. However, chromatographic and SDS-PAGE analysis of the proteoglycans secreted by the cell lines showed that a dermatan sulphate proteoglycan of 150-125 kilodaltons was substantially reduced in Alzheimer's disease fibroblasts. The molecular characteristics of this proteoglycan correspond to decorin. Western blot analysis indicated that decorin was reduced in Alzheimer's disease incubation medium compared with normal medium. Northern blotting indicated that in Alzheimer's disease fibroblasts decorin transcripts were significantly reduced compared with normal fibroblasts. Glypican concentrations, a cell surface heparan sulphate proteoglycan, remained the same. Conclusions—These results strongly suggest that the expression and synthesis of decorin is affected in Alzheimer's disease skin fibroblasts.
- ItemStatistical potentials for fold assessment(2002) Melo Ledermann, Francisco Javier; Sánchez, R.; Sali, A.A protein structure model generally needs to be evaluated to assess whether or not it has the correct fold. To improve fold assessment, four types of a residue-level statistical potential were optimized, including distance-dependent, contact, ϕ/Ψ dihedral angle, and accessible surface statistical potentials. Approximately 10,000 test models with the correct and incorrect folds were built by automated comparative modeling of protein sequences of known structure. The criterion used to discriminate between the correct and incorrect models was the Z-score of the model energy. The performance of a Z-score was determined as a function of many variables in the derivation and use of the corresponding statistical potential. The performance was measured by the fractions of the correctly and incorrectly assessed test models. The most discriminating combination of any one of the four tested potentials is the sum of the normalized distance-dependent and accessible surface potentials. The distance-dependent potential that is optimal for assessing models of all sizes uses both Cα and Cβ atoms as interaction centers, distinguishes between all 20 standard residue types, has the distance range of 30 Å, and is derived and used by taking into account the sequence separation of the interacting atom pairs. The terms for the sequentially local interactions are significantly less informative than those for the sequentially nonlocal interactions. The accessible surface potential that is optimal for assessing models of all sizes uses Cβ atoms as interaction centers and distinguishes between all 20 standard residue types. The performance of the tested statistical potentials is not likely to improve significantly with an increase in the number of known protein structures used in their derivation. The parameters of fold assessment whose optimal values vary significantly with model size include the size of the known protein structures used to derive the potential and the distance range of the accessible surface potential. Fold assessment by statistical potentials is most difficult for the very small models. This difficulty presents a challenge to fold assessment in large-scale comparative modeling, which produces many small and incomplete models. The results described in this study provide a basis for an optimal use of statistical potentials in fold assessment.
- ItemThe internal region leucine-rich repeat 6 of decorin interacts with low density lipoprotein receptor-related protein-1, modulates transforming growth factor (TGF)-β-dependent signaling, and inhibits TGF-β-dependent fibrotic response in skeletal muscles(2012) Cabello Verrugio, C.; Santander, C.; Cofré, C.; Acuña, M. J.; Melo Ledermann, Francisco Javier; Brandan, EnriqueDecorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury.