21 results on '"Briones, Marcelo R. S."'
Search Results
2. Mitochondrial genome variants associated with amyotrophic lateral sclerosis and their haplogroup distribution.
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Briones, Marcelo R. S., Campos, João H., Ferreira, Renata C., Schneper, Lisa, Santos, Ilda M., Antoneli, Fernando M., and Broach, James R.
- Abstract
Introduction/Aims: Amyotrophic lateral sclerosis (ALS) may be familial or sporadic, and twin studies have revealed that even sporadic forms have a significant genetic component. Variants in 55 nuclear genes have been associated with ALS and although mitochondrial dysfunction is observed in ALS, variants in mitochondrial genomes (mitogenomes) have not yet been tested for association with ALS. The aim of this study was to determine whether mitogenome variants are associated with ALS. Methods: We conducted a genome‐wide association study (GWAS) in mitogenomes of 1965 ALS patients and 2547 controls. Results: We identified 51 mitogenome variants with p values <10−7, of which 13 had odds ratios (ORs) >1, in genes RNR1, ND1, CO1, CO3, ND5, ND6, and CYB, while 38 variants had OR <1 in genes RNR1, RNA2, ND1, ND2, CO2, ATP8, ATP6, CO3, ND3, ND4, ND5, ND6, and CYB. The frequencies of haplogroups H, U, and L, the most frequent in our ALS data set, were the same in different onset sites (bulbar, limb, spinal, and axial). Also, intra‐haplogroup GWAS revealed unique ALS‐associated variants in haplogroups L and U. Discussion: Our study shows that mitogenome single nucleotide variants (SNVs) are associated with ALS and suggests that these SNVs could be included in routine genetic testing for ALS and that mitochondrial replacement therapy has the potential to serve as a basis for ALS treatment. [ABSTRACT FROM AUTHOR]
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- 2024
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3. The Relationship between HERV, Interleukin, and Transcription Factor Expression in ZIKV Infected versus Uninfected Trophoblastic Cells.
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Costa, Anderson Luís da, Prieto-Oliveira, Paula, Duarte-Barbosa, Márcia, Andreata-Santos, Robert, Peter, Cristina M., Prolo de Brito, Thamires, Antoneli, Fernando, Durães-Carvalho, Ricardo, Briones, Marcelo R. S., Maricato, Juliana T., Zanotto, Paolo M. A., Jacob Machado, Denis, and Janini, Luiz M. R.
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TRANSCRIPTION factors ,HUMAN endogenous retroviruses ,ZIKA virus infections ,IMMUNOLOGICAL tolerance ,PREGNANCY proteins ,TROPHOBLAST - Abstract
Zika virus (ZIKV) is an arbovirus with maternal, sexual, and TORCH-related transmission capabilities. After 2015, Brazil had the highest number of ZIVK-infected pregnant women who lost their babies or delivered them with Congenital ZIKV Syndrome (CZS). ZIKV triggers an immune defense in the placenta. This immune response counts with the participation of interleukins and transcription factors. Additionally, it has the potential involvement of human endogenous retroviruses (HERVS). Interleukins are immune response regulators that aid immune tolerance and support syncytial structure development in the placenta, where syncytin receptors facilitate vital cell-to-cell fusion events. HERVs are remnants of ancient viral infections that integrate into the genome and produce syncytin proteins crucial for placental development. Since ZIKV can infect trophoblast cells, we analyzed the relationship between ZIKV infection, HERV, interleukin, and transcription factor modulations in the placenta. To investigate the impact of ZIKV on trophoblast cells, we examined two cell types (BeWo and HTR8) infected with ZIKV-MR766 (African) and ZIKV-IEC-Paraíba (Asian–Brazilian) using Taqman and RT2 Profiler PCR Array assays. Our results indicate that early ZIKV infection (24–72 h) does not induce differential interleukins, transcription factors, and HERV expression. However, we show that the expression of a few of these host defense genes appears to be linked independently of ZIKV infection. Future studies involving additional trophoblastic cell lineages and extended infection timelines will illuminate the dynamic interplay between ZIKV, HERVs, interleukins, and transcription factors in the placenta. [ABSTRACT FROM AUTHOR]
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- 2024
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4. Reconstructing Prehistoric Viral Genomes from Neanderthal Sequencing Data.
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Ferreira, Renata C., Alves, Gustavo V., Ramon, Marcello, Antoneli, Fernando, and Briones, Marcelo R. S.
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NEANDERTHALS ,FOSSIL DNA ,DNA viruses ,VIRAL genomes ,GENE mapping ,DATA mapping - Abstract
DNA viruses that produce persistent infections have been proposed as potential causes for the extinction of Neanderthals, and, therefore, the identification of viral genome remnants in Neanderthal sequence reads is an initial step to address this hypothesis. Here, as proof of concept, we searched for viral remnants in sequence reads of Neanderthal genome data by mapping to adenovirus, herpesvirus and papillomavirus, which are double-stranded DNA viruses that may establish lifelong latency and can produce persistent infections. The reconstructed ancient viral genomes of adenovirus, herpesvirus and papillomavirus revealed conserved segments, with nucleotide identity to extant viral genomes and variable regions in coding regions with substantial divergence to extant close relatives. Sequence reads mapped to extant viral genomes showed deamination patterns of ancient DNA, and these ancient viral genomes showed divergence consistent with the age of these samples (≈50,000 years) and viral evolutionary rates (10
−5 to 10−8 substitutions/site/year). Analysis of random effects showed that the Neanderthal mapping to genomes of extant persistent viruses is above what is expected by random similarities of short reads. Also, negative control with a nonpersistent DNA virus does not yield statistically significant assemblies. This work demonstrates the feasibility of identifying viral genome remnants in archaeological samples with signal-to-noise assessment. [ABSTRACT FROM AUTHOR]- Published
- 2024
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5. Convergent Mutations and Single Nucleotide Variants in Mitochondrial Genomes of Modern Humans and Neanderthals.
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Ferreira, Renata C., Rodrigues, Camila R., Broach, James R., and Briones, Marcelo R. S.
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SINGLE nucleotide polymorphisms ,NEANDERTHALS ,PARKINSON'S disease ,ALZHEIMER'S disease ,HUMAN genome ,GENOMES - Abstract
The genetic contributions of Neanderthals to the modern human genome have been evidenced by the comparison of present-day human genomes with paleogenomes. Neanderthal signatures in extant human genomes are attributed to intercrosses between Neanderthals and archaic anatomically modern humans (AMHs). Although Neanderthal signatures are well documented in the nuclear genome, it has been proposed that there is no contribution of Neanderthal mitochondrial DNA to contemporary human genomes. Here we show that modern human mitochondrial genomes contain 66 potential Neanderthal signatures, or Neanderthal single nucleotide variants (N-SNVs), of which 36 lie in coding regions and 7 result in nonsynonymous changes. Seven N-SNVs are associated with traits such as cycling vomiting syndrome, Alzheimer's disease and Parkinson's disease, and two N-SNVs are associated with intelligence quotient. Based on recombination tests, principal component analysis (PCA) and the complete absence of these N-SNVs in 41 archaic AMH mitogenomes, we conclude that convergent evolution, and not recombination, explains the presence of N-SNVs in present-day human mitogenomes. [ABSTRACT FROM AUTHOR]
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- 2024
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6. The epitranscriptome of Vero cells infected with SARS-CoV-2 assessed by direct RNA sequencing reveals m6A pattern changes and DRACH motif biases in viral and cellular RNAs.
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Campos, João H. C., Alves, Gustavo V., Maricato, Juliana T., Braconi, Carla T., Antoneli, Fernando M., Janini, Luiz Mario R., and Briones, Marcelo R. S.
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RNA sequencing ,RNA modification & restriction ,SARS-CoV-2 ,RNA methylation ,CERCOPITHECUS aethiops ,SHOTGUN sequencing - Abstract
The epitranscriptomics of the SARS-CoV-2 infected cell reveals its response to viral replication. Among various types of RNA nucleotide modifications, the m6A is the most common and is involved in several crucial processes of RNA intracellular location, maturation, half-life and translatability. This epitranscriptome contains a mixture of viral RNAs and cellular transcripts. In a previous study we presented the analysis of the SARS-CoV-2 RNA m6A methylation based on direct RNA sequencing and characterized DRACH motif mutations in different viral lineages. Here we present the analysis of the m6A transcript methylation of Vero cells (derived from African Green Monkeys) and Calu-3 cells (human) upon infection by SARS-CoV-2 using direct RNA sequencing data. Analysis of these data by nonparametric statistics and two computational methods (m6anet and EpiNano) show that m6A levels are higher in RNAs of infected cells. Functional enrichment analysis reveals increased m6A methylation of transcripts involved in translation, peptide and amine metabolism. This analysis allowed the identification of differentially methylated transcripts and m6A unique sites in the infected cell transcripts. Results here presented indicate that the cell response to viral infection not only changes the levels of mRNAs, as previously shown, but also its epitranscriptional pattern. Also, transcriptome-wide analysis shows strong nucleotide biases in DRACH motifs of cellular transcripts, both in Vero and Calu-3 cells, which use the signature GGACU whereas in viral RNAs the signature is GAACU. We hypothesize that the differences of DRACH motif biases, might force the convergent evolution of the viral genome resulting in better adaptation to target sequence preferences of writer, reader and eraser enzymes. To our knowledge, this is the first report on m6A epitranscriptome of the SARS-CoV-2 infected Vero cells by direct RNA sequencing, which is the sensu stricto RNA-seq. [ABSTRACT FROM AUTHOR]
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- 2022
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7. Trans-sialidase genes expressed in mammalian forms of Trypanosoma cruzi evolved from ancestor genes expressed in insect forms of the parasite
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Briones, Marcelo R. S., Egima, Claudia M., Eichinger, Daniel, and Schenkman, Sergio
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- 1995
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8. Evolutionary distances and identification of Candida species in clinical isolates by Randomly Amplified Polymorphic DNA (RAPD)
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Melo, Analy S. A., de Almeida, Leila P., Colombo, Arnaldo L., and Briones, Marcelo R. S.
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- 1998
9. Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags
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de Souza, Sandro J., Camargo, Anamaria A., Briones, Marcelo R. S., Costa, Fernando F., Nagai, Maria Aparecida, Verjovski-Almeida, Sergio, Zago, Marco A., Andrade, Luis Eduardo C., Carrer, Helaine, El-Dorry, Hamza F. A., Espreafico, Enilza M., Habr-Gama, Angelita, Giannella-Neto, Daniel, Goldman, Gustavo H., Gruber, Arthur, Hackel, Christine, Kimura, Edna T., Maciel, Rui M. B., Marie, Suely K. N., Martins, Elizabeth A. L., Nobrega, Marina P., Paco-Larson, Maria Luisa, Pardini, Maria Ines M. C., Pereira, Goncalo G., Pesquero, Joao Bosco, Rodrigues, Vanderlei, Rogatto, Silvia R., da Silva, Ismael D. C. G., Sogayar, Mari C., de Fatima, Sonati Maria, Tajara, Eloiza H., Valentini, Sandro R., Acencio, Marcio, Alberto, Fernando L., Amaral, Maria Elisabete J., Aneas, Ivy, Bengtson, Mario Henrique, Carraro, Dirce M., Carvalho, Alex F., Carvalho, Lucia Helena, Cerutti, Janete M., Correa, Maria Lucia C., Costa, Maria Cristina R., Curcio, Cyntia, Gushiken, Tsieko, Ho, Paulo L., Kimura, Elza, Leite, Luciana C. C., Maia, Gustavo, Majumder, Paromita, Marins, Mozart, Matsukuma, Adriana, Melo, Analy S. A., Mestriner, Carlos Alberto, Miracca, Elisabete C., Miranda, Daniela C., Nascimento, Ana Lucia T. O., Nobrega, Francisco G., Ojopi, Elida P. B., Pandolfi, Jose Rodrigo C., Pessoa, Luciana Gilbert, Rahal, Paula, Rainho, Claudia A., da Ro's, Nancy, de Sa, Renata G., Sales, Magaly M., da Silva, Neusa P., Silva, Tereza C., da Silva, Wilson Jr., Simao, Daniel F., Sousa, Josane F., Stecconi, Daniella, Tsukumo, Fernando, Valente, Valeria, Zalcberg, Heloisa, Brentani, Ricardo R., Reis, Luis F. L., Dias-Neto, Emmanuel, and Simpson, Andrew J. G.
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Genomes -- Analysis ,Nucleotide sequence -- Design and construction ,Genetic transcription -- Analysis ,Chromosomes -- Analysis ,Science and technology - Abstract
Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by GENSCAN. (http://genes.mit.edu/GENSCAN.html).
- Published
- 2000
10. Shotgun sequencing of the human transcriptome with ORF expressed sequence tags
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Neto, Emmanuel Dias, Correa, Ricardo Garcia, Verjovski-Almeida, Sergio, Briones, Marcelo R. S., Nagai, Maria Aparecida, da Silva, Wilson Jr., Zago, Marco Antonio, Bordin, Silvana, Costa, Fernando Ferreira, Goldman, Gustavo Henrique, Carvalho, Alex F., Matsukuma, Adriana, Baia, Gilson S., Simpson, David H., Brunstein, Adriana, de Oliveira, Paulo S. L., Bucher, Philipp, Jongeneel, C. Victor, O'Hare, Michael J., Soares, Fernando, Brentani, Ricardo R., Reis, Luis F. L., de Souza, Sandro J., and Simpson, Andrew J. G.
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Reverse transcriptase -- Usage ,Nucleotide sequence -- Information management ,Databases ,Genetic code -- Identification and classification ,Science and technology - Abstract
Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.
- Published
- 2000
11. Evidence for Mitochondrial Genome Methylation in the Yeast Candida albicans: A Potential Novel Epigenetic Mechanism Affecting Adaptation and Pathogenicity?
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Bartelli, Thais F., Bruno, Danielle C. F., and Briones, Marcelo R. S.
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CANDIDA albicans genetics ,MITOCHONDRIAL physiology ,DNA methylation - Abstract
The commensal yeast Candida albicans is an opportunistic pathogen. In order to successfully colonize or infect the human body, the fungus must adapt to the host's environmental conditions, such as low oxygen tension (hypoxia), temperature (37°C), and the different carbon sources available. Previous studies demonstrated the adaptive importance of C. albicans genetic variability for its pathogenicity, although the contributions of epigenetic and the influence of environmental factors are not fully understood. Mitochondria play important roles in fungal energetic metabolism, regulation of nuclear epigenetic mechanisms and pathogenicity. However, the specific impact of inter-strain mitochondrial genome variability and mitochondrial epigenetics in pathogenicity is unclear. Here, we draw attention to this relevant organelle and its potential role in C. albicans pathogenicity and provide preliminary evidence, for the first time, for methylation of the yeast mitochondrial genome. Our results indicate that environmental conditions, such as continuous exposure for 12 weeks to hypoxia and 37°C, decrease the mitochondrial genome methylation in strains SC5314 and L757. However, the methylation decrease is quantitatively different in specific genome positions when strains SC5314 and L757 are compared. We hypothesize that this phenomenon can be promising for future research to understand how physical factors of the host affect the C. albicans mitochondrial genome and its possible impact on adaptation and pathogenicity. [ABSTRACT FROM AUTHOR]
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- 2018
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12. A Possible role for Platelet-Activating Factor receptor in Amyotrophic Lateral sclerosis treatment.
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Briones, Marcelo R. S., Snyder, Amanda M., Ferreira, Renata C., Neely, Elizabeth B., Connor, James R., and Broach, James R.
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AMYOTROPHIC lateral sclerosis treatment ,MOTOR neurons ,INTERLEUKIN-18 - Abstract
Amyotrophic lateral sclerosis (ALS) is the third most prevalent neurodegenerative disease affecting upper and lower motor neurons. An important pathway that may lead to motor neuron degeneration is neuroinflammation. Cerebrospinal Fluids of ALS patients have increased levels of the inflammatory cytokine IL-18. Because IL-18 is produced by dendritic cells stimulated by the platelet-activating factor (PAF), a major neuroinflammatory mediator, it is expected that PAF is involved in ALS. Here we show pilot experimental data on amplification of PAF receptor (PAFR) mRNA by RT-PCR. PAFR is overexpressed, as compared to age matched controls, in the spinal cords of transgenic ALS SOD1-G93A mice, suggesting PAF mediation. Although anti-inflammatory drugs have been tested for ALS before, no clinical trial has been conducted using PAFR specific inhibitors. Therefore, we hypothesize that administration of PAFR inhibitors, such as Ginkgolide B, PCA 4248 and WEB 2086, have potential to function as a novel therapy for ALS, particularly in SOD1 familial ALS forms. Because currently there are only two approved drugs with modest effectiveness for ALS therapy, a search for novel drugs and targets is essential. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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13. A Kolmogorov-Smirnov test for the molecular clock based on Bayesian ensembles of phylogenies.
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Antoneli, Fernando, Passos, Fernando M., Lopes, Luciano R., and Briones, Marcelo R. S.
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KOLMOGOROV complexity ,MOLECULAR clock ,BAYESIAN analysis ,BIOLOGICAL evolution ,BIOLOGICAL divergence ,GOODNESS-of-fit tests - Abstract
Divergence date estimates are central to understand evolutionary processes and depend, in the case of molecular phylogenies, on tests of molecular clocks. Here we propose two non-parametric tests of strict and relaxed molecular clocks built upon a framework that uses the empirical cumulative distribution (ECD) of branch lengths obtained from an ensemble of Bayesian trees and well known non-parametric (one-sample and two-sample) Kolmogorov-Smirnov (KS) goodness-of-fit test. In the strict clock case, the method consists in using the one-sample Kolmogorov-Smirnov (KS) test to directly test if the phylogeny is clock-like, in other words, if it follows a Poisson law. The ECD is computed from the discretized branch lengths and the parameter λ of the expected Poisson distribution is calculated as the average branch length over the ensemble of trees. To compensate for the auto-correlation in the ensemble of trees and pseudo-replication we take advantage of thinning and effective sample size, two features provided by Bayesian inference MCMC samplers. Finally, it is observed that tree topologies with very long or very short branches lead to Poisson mixtures and in this case we propose the use of the two-sample KS test with samples from two continuous branch length distributions, one obtained from an ensemble of clock-constrained trees and the other from an ensemble of unconstrained trees. Moreover, in this second form the test can also be applied to test for relaxed clock models. The use of a statistically equivalent ensemble of phylogenies to obtain the branch lengths ECD, instead of one consensus tree, yields considerable reduction of the effects of small sample size and provides a gain of power. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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14. MIA: Mutual Information Analyzer, a graphic user interface program that calculates entropy, vertical and horizontal mutual information of molecular sequence sets.
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Lichtenstein, Flavio, Antoneli Jr, Fernando, and Briones, Marcelo R. S.
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GENOMICS ,JENSEN-Shannon divergence ,PROBABILITY theory ,SYSTEMS theory ,STATISTICAL physics - Abstract
Background: Short and long range correlations in biological sequences are central in genomic studies of covariation. These correlations can be studied using mutual information because it measures the amount of information one random variable contains about the other. Here we present MIA (Mutual Information Analyzer) a user friendly graphic interface pipeline that calculates spectra of vertical entropy (VH), vertical mutual information (VMI) and horizontal mutual information (HMI), since currently there is no user friendly integrated platform that in a single package perform all these calculations. MIA also calculates Jensen-Shannon Divergence (JSD) between pair of different species spectra, herein called informational distances. Thus, the resulting distance matrices can be presented by distance histograms and informational dendrograms, giving support to discrimination of closely related species. Results: In order to test MIA we analyzed sequences from Drosophila Adh locus, because the taxonomy and evolutionary patterns of different Drosophila species are well established and the gene Adh is extensively studied. The search retrieved 959 sequences of 291 species. From the total, 450 sequences of 17 species were selected. With this dataset MIA performed all tasks in less than three hours: gathering, storing and aligning fasta files; calculating VH, VMI and HMI spectra; and calculating JSD between pair of different species spectra. For each task MIA saved tables and graphics in the local disk, easily accessible for future analysis. Conclusions: Our tests revealed that the "informational model free" spectra may represent species signatures. Since JSD applied to Horizontal Mutual Information spectra resulted in statistically significant distances between species, we could calculate respective hierarchical clusters, herein called Informational Dendrograms (ID). When compared to phylogenetic trees all Informational Dendrograms presented similar taxonomy and species clusterization. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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15. Extracellular enolase of Candida albicans is involved in colonization of mammalian intestinal epithelium.
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Silva, Richard C., Padovan, Ana Carolina B., Pimenta, Daniel C., Ferreira, Renata C., da Silva, Claudio V., and Briones, Marcelo R. S.
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CANDIDA albicans ,ENOLASE ,CELL adhesion -- Molecular aspects ,INFECTION ,INTESTINAL diseases ,HYDRATASES ,GENETICS ,PHYSIOLOGY - Abstract
Enolase is secreted by Candida albicans and is present in its biofilms although its extracellular function is unknown. Here we show that extracellular enolase mediates the colonization of small intestine mucosa by C. albicans. Assays using intestinal mucosa disks show that C. albicans adhesion is inhibited, in a dose dependent mode, either by pretreatment of intestinal epithelium mucosa disks with recombinant C. albicans enolase (70% at 0.5 mg/ml enolase) or by pretreatment of C. albicansyeasts with anti-enolase antibodies (48% with 20μg antiserum). Also using flow cytometry, immunoblots of conditioned media and confocal microscopy we demonstrate that enolase is present in biofilms and that the extracellular enolase is not an artifact due to cell lysis, but must represent functional secretion of a stable form. This is the first direct evidence that C. albicans' extracellular enolase mediates colonization on its primary translocation site. Also, because enolase is encoded by a single locus in C. albicans, its dual role peptide, as glycolytic enzyme and extracellular peptide, is a remarkable example of gene sharing in fungi. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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16. A quick and low-cost PCR-based assay for Candida spp. identification in positive blood culture bottles.
- Author
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Xafranski, Hemílio, Melo, Analy S. A., Machado, Antonia M., Briones, Marcelo R. S., and Colombo, Arnaldo L.
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Background: Differences in the susceptibility of Candida species to antifungal drugs make identification to the species level important for clinical management of candidemia. Molecular tests are not yet standardized or available in most clinical laboratories, although such tests can reduce the time required for species identification, as compared to the conventional culture-based methods. To decrease laboratory costs and improve diagnostic accuracy, different molecular methods have been proposed, including DNA extraction protocols to produce pure DNA free of PCR inhibitors. The objective of this study was to validate a new format of molecular method, based on the internal transcribed spacer (ITS) of the rDNA gene amplification followed by sequencing, to identify common and cryptic Candida species causing candidemia by analyzing DNA in blood culture bottles positive for yeasts. Methods: For DNA extraction, an "in-house" protocol based on organic solvent extraction was tested. Additional steps of liquid nitrogen incubation followed by mechanical disruption ensured complete cell lysis, and highly pure DNA. One hundred sixty blood culture bottles positive for yeasts were processed. PCR assays amplified the ITS region. The DNA fragments of 152 samples were sequenced and these sequences were identified using the GenBank database (NCBI). Molecular yeast identification was compared to results attained by conventional method. Results: The organic solvent extraction protocol showed high reproducibility in regards to DNA quantity, as well as high PCR sensitivity (10 pg of C. albicans DNA and 95% amplification on PCR). The identification of species at the molecular level showed 97% concordance with the conventional culturing method. The molecular method tested in the present study also allowed identification of species not commonly implicated in human infections. Conclusions: This study demonstrated that our molecular method presents significant advantages over the conventional yeast culture identification method by providing accurate results within 24 hours, in contrast to at least 72 hours required by the automated conventional culture method. Additionally, our molecular method allowed the identification of mixed infections, as well as infections due to emergent fungal pathogens. This economical DNA extraction method developed in our laboratory provided high-quality DNA and 60% cost savings compared to commercial methods. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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17. Differential Infectivity by the Oral Route of Trypanosoma cruzi Lineages Derived from Y Strain.
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Cortez, Cristian, Martins, Rafael M., Alves, Renan M., Silva, Richard C., Bilches, Luciana C., Macedo, Silene, Atayde, Vanessa D., Kawashita, Silvia Y., Briones, Marcelo R. S., and Yoshida, Nobuko
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TRYPANOSOMA cruzi ,CHAGAS' disease ,SYMPTOMS ,DIFFERENTIAL evolution ,HAPLOTYPES ,EPITHELIAL cells ,PARACOCCIDIOIDOMYCOSIS - Abstract
Background: Diversity of T. cruzi strains is a central problem in Chagas disease research because of its correlation with the wide range of clinical manifestations and the biogeographical parasite distribution. The role played by parasite microdiversity in Chagas disease epidemiology is still debatable. Also awaits clarification whether such diversity is associated with the outcome of oral T. cruzi infection, responsible for frequent outbreaks of acute Chagas disease. Methods and Findings: We addressed the impact of microdiversity in oral T. cruzi infection, by comparative analysis of two strains, Y30 and Y82, both derived from Y strain, a widely used experimental model. Network genealogies of four nuclear genes (SSU rDNA, actin, DHFR-TS, EF1α) revealed that Y30 is closely related to Discrete Typing Unit TcII while Y82 is more closely related to TcVI, a group containing hybrid strains. Nevertheless, excepting one A-G transition at position 1463, Y30 and Y82 SSU rDNAs were identical. Y82 strain, expressing the surface molecule gp82, infected mice orally more efficiently than Y30, which expresses a related gp30 molecule. Both molecules are involved in lysosome exocytosis-dependent host cell invasion, but exhibit differential gastric mucin-binding capacity, a property critical for parasite migration toward the gastric mucosal epithelium. Upon oral infection of mice, the number of Y30 and Y82 parasites in gastric epithelial cells differed widely. Conclusions: We conclude that metacyclic forms of gp82-expressing Y82 strain, closely related to TcVI, are better adapted than Y30 strain (TcII) to traverse the stomach mucous layer and establish oral route infection. The efficiency to infect target cell is the same because gp82 and gp30 strains have similar invasion-promoting properties. Unknown is whether differences in Y30 and Y82 are natural parasite adaptations or a product of lab-induced evolution by differential selection along the 60 years elapsed since the Y strain isolation. Author Summary: Globalization of Chagas disease, from Latin America toward non endemic countries, has become a world health problem. In endemic countries, acute cases of Chagas disease transmitted by oral Trypanosoma cruzi infection, have been frequently reported in recent years. The diverse clinical manifestations of the disease are mainly attributed to the highly complex population structure of the parasite. We aimed in this study to investigate the impact of T. cruzi microdiversity in oral infection, by comparative analysis of Y30 and Y82 strains, both derived from Y strain, a widely used experimental model. Network phylogenies were inferred to determine their haplotype distribution and classification. Y30 and Y82 were closely related to Discrete Typing Unit TcII and TcVI, respectively. Y82, expressing the surface molecule gp82, was more efficient than Y30, expressing a related gp30 molecule, in establishing infection in mice by oral route. Both molecules are involved in host cell invasion, but exhibit differential gastric mucin-binding capacity, which is critical for parasite migration toward the gastric mucosal epithelium. The number of Y30 and Y82 parasites in gastric epithelial cells differed widely. Our results indicate that gp82-expressing strains are better adapted than gp30-expressing to traverse the stomach mucous layer and establish oral route infection. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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18. A novel allele of HWP1, isolated from a clinical strain of Candida albicans with defective hyphal growth and biofilm formation, has deletions of Gln/Pro and Ser/Thr repeats involved in cellular adhesion.
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Padovan, Ana Carolina B., Chaves, Guilherme M., Colombo, Arnaldo L., and Briones, Marcelo R. S.
- Abstract
Gene HWP1 encodes a major Candida albicans hyphae cell wall protein which is a substrate of mammalian transglutaminases, promoting the cross-link of the fungus to epithelial cells. Here, we describe a novel HWP1 allele, isolated from C. albicans blood isolates. Analysis of the translated sequence shows that three important regions are absent in the novel allele, HWP1-2, relative to the previously described allele, HWP1-1. Regions 1 and 2 consist of 10 amino acid repeats important for functional conformation of peptide chains and attachment of C. albicans cells to the mammalian epithelia. Region 3 consists of 34 amino acid residues rich in threonine and serine, with O-glycosylation sites that promote the cross-linking with other proteins on C. albicans surface. The HWP1-2 homozygous strain L757 and the heterozygous strain L296 ( HWP1-1/HWP1-2) have significantly lower levels of HWP1 expression during hyphal growth and biofilm formation compared to strain SC5314 ( HWP1-1/HWP1-1). However, strain L296 properly forms hyphae and biofilms in vitro while strain L757 has reduced hyphal growth (40.4%) and biofilm formation (90.8%). Our results indicate that the HWP1 locus has biofilm specific allelic differential expression and suggest that the HWP1-2 encoded protein is less efficient to maintain cell-to-cell and cell-to-surface adhesion during biofilm formation. This is the first report of a natural variant of HWP1. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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19. Experimental Phylogeny of Neutrally Evolving DNA Sequences Generated by a Bifurcate Series of Nested Polymerase Chain Reactions.
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Sanson, Gerdine F. O., Kawashita, Silvia Y., Brunstein, Adriana, and Briones, Marcelo R. S.
- Abstract
A known phylogeny was generated using a four-step serial bifurcate PCR method. The ancestor sequence (SSU rDNA) evolved in vitro for 280 nested PCR cycles, and the resulting 15 ancestor and 16 terminal sequences (2,238 bp each) were determined. Parsimony, distance, and maximum likelihood analysis of the terminal sequences reconstructed the topology of the real phylogeny and branch lengths accurately. Divergence dates and ancestor sequences were estimated with very small error, particularly at the base of the phylogeny, mostly due to insertion and deletion changes. The substitution patterns along the known phylogeny are not described by reversible models, and accordingly, the probability substitution matrix, based on the observed substitutions from ancestor to terminal nodes along the known phylogeny, was calculated. This approach is an extension of previous studies using bacteriophage serial propagation, because here mutations were allowed to occur neutrally rather than by addition of a mutagenic agent, which produced biased mutational changes. These results provide for the first time biochemical experimental support for phylogenies, divergence date estimates, and an irreversible substitution model based on neutrally evolving DNA sequences. The substitution preferences observed here (A to G and T to C) are consistent with the high G+C content of the Thermus aquaticus genome. This suggests, at least in part, that the method here described, which explores the high Taq DNA polymerase error rate, simulates the evolution of a DNA segment in a thermophilic organism. These organisms include the bacterial rod T. aquaticus and several Archaea, and thus, the method and data set described here may well contribute new insights about the genome evolution of these organisms. [ABSTRACT FROM PUBLISHER]
- Published
- 2002
- Full Text
- View/download PDF
20. Maximum-Likelihood Divergence Date Estimates Based on rRNA Gene Sequences Suggest Two Scenarios of Trypanosoma cruzi Intraspecific Evolution.
- Author
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Kawashita, Silvia Y., Sanson, Gerdine F. O., Fernandes, Octavio, Zingales, Bianca, and Briones, Marcelo R. S.
- Abstract
The phylogenetic relationships of Trypanosoma cruzi strains were inferred using maximum-likelihood from complete 18S rDNA sequences and D7-24Sα rDNA regions from 20 representative strains of T. cruzi. For this we sequenced the 18S rDNA of 14 strains and the D7-24Sα rDNA of four strains and aligned them to previously published sequences. Phylogenies inferred from these data sets identified four groups, named Riboclades 1, 2, 3, and 4, and a basal dichotomy that separated Riboclade 1 from Riboclades 2, 3, and 4. Substitution models and other parameters were optimized by hierarchical likelihood tests, and our analysis of the 18S rDNA molecular clock by the likelihood ratio test suggests that a taxa subset encompassing all 2,150 positions in the alignment supports rate constancy among lineages. The present analysis supports the notion that divergence dates of T. cruzi Riboclades can be estimated from 18S rDNA sequences and therefore, we present alternative evolutionary scenarios based on two different views of T. cruzi intraspecific divergence. The first assumes a faster evolutionary rate, which suggests that the divergence between T. cruzi I and II and the extant strains occurred in the Tertiary period (37–18 MYA). The other, which supports the hypothesis that the divergence between T. cruzi I and II occurred in the Cretaceous period (144–65 MYA) and the divergence of the extant strains occurred in the Tertiary period of the Cenozoic era (65–1.8 MYA), is consistent with our previously proposed hypothesis of divergence by geographical isolation and mammalian host coevolution. [ABSTRACT FROM PUBLISHER]
- Published
- 2001
- Full Text
- View/download PDF
21. Histone Deacetylase 1 Is Essential for Rod Photoreceptor Differentiation by Regulating Acetylation at Histone H3 Lysine 9 and Histone H4 Lysine 12 in the Mouse Retina.
- Author
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Ferreira, Renata C., Popova, Evgenya Y., James, Jessica, Briones, Marcelo R. S., Zhang, Samuel S., and Barnstable, Colin J.
- Subjects
- *
LYSINE , *ACETYLATION , *PHOTORECEPTORS , *CELL differentiation , *HISTONE deacetylase , *LABORATORY mice - Abstract
Histone acetylation has a regulatory role in gene expression and is necessary for proper tissue development. To investigate the specific roles of histone deacetylases (HDACs) in rod differentiation in neonatal mouse retinas, we used a pharmacological approach that showed that inhibition of class I but not class IIa HDACs caused the same phenotypic changes seen with broad spectrum HDAC inhibitors, most notably a block in the differentiation of rod photoreceptors. Inhibition of HDAC1 resulted in increase of acetylation of lysine 9 of histone 3 (H3K9) and lysine 12 of histone 4 (H4K12) but not lysine 27 of histone 3 (H3K27) and led to maintained expression of progenitor-specific genes such as Vsx2 and Hes1 with concomitant block of expression of rod-specific genes. ChiP experiments confirmed these changes in the promoters of a group of progenitor genes. Based on our results, we suggest that HDAC1-specific inhibition prevents progenitor cells of the retina from exiting the cell cycle and differentiating. HDAC1 may be an essential epigenetic regulator of the transition from progenitor cells to terminally differentiated photoreceptors. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
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