Biomedical subjects

Carlos Briones

Publications and source records attributed to Carlos Briones.

9 recordsLinked to original sources

PubMed2006

Modular evolution and increase of functional complexity in replicating RNA molecules.

At early stages of biochemical evolution, the complexity of replicating molecules was limited by unavoidably high mutation rates. In an RNA world, prior to the appearance of cellular life, an increase in molecular length, and thus in functional complexity, could have been mediated by modular evolution. We describe here a scenario in which short, replicating RNA sequences are selected to perform a simple function. Molecular function is represented through the secondary structure corresponding to each sequence, and a given target secondary structure yields the optimal function in the environment where the population evolves. The combination of independently evolved populations may have facilitated the emergence of larger molecules able to perform more complex functions (including RNA replication) that could arise as a combination of simpler ones. We quantitatively show that modular evolution has relevant advantages with respect to the direct evolution of large functional molecules, among them the allowance of higher mutation rates, the shortening of evolutionary times, and the very possibility of finding complex structures that could not be otherwise directly selected.

Computational Biology↗

PubMed2006

Minority memory genomes can influence the evolution of HIV-1 quasispecies in vivo.

One of the consequences of viral quasispecies dynamics is the presence, in the mutant spectrum, of minority memory genomes that reflect those variants that were dominant at an earlier phase of the same evolutionary lineage. Replicative and cellular (or anatomical) contributions to quasispecies memory were previously defined during intrahost evolution of human immunodeficiency virus type 1 (HIV-1) [Briones, C., Domingo, E., Molina-París, C., 2003. Memory in retroviral quasispecies: experimental evidence and theoretical model for human immunodeficiency virus. J. Mol. Biol. 331, 213-229.]. However, the effects of replicative memory regarding virus evolution in vivo have not been investigated. Here we document that a multidrug-resistant (MDR) HIV-1, present at memory level, determined the ensuing evolution of the virus in an infected patient. Nucleotide sequencing and detailed phylogenetic analyses of sequential viral populations and individual molecular clones evidenced that the progeny of a minority MDR genome during a treatment interruption contributed the dominant genomes when an antiretroviral treatment was restored. An extension of a mathematical model of establishment and maintenance of memory, based on quasispecies theory, supports the experimental data. Therefore a replicative memory subpopulation, not detectable in a consensus nucleotide sequence, affected decisively subsequent states of viral evolution in vivo.

Anti-HIV Agents↗

PubMed2006

Microarray-based identification of antigenic variants of foot-and-mouth disease virus: a bioinformatics quality assessment.

BACKGROUND: The evolution of viral quasispecies can influence viral pathogenesis and the response to antiviral treatments. Mutant clouds in infected organisms represent the first stage in the genetic and antigenic diversification of RNA viruses, such as foot and mouth disease virus (FMDV), an important animal pathogen. Antigenic variants of FMDV have been classically diagnosed by immunological or RT-PCR-based methods. DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Recently, a FMDV microarray was described to detect simultaneously the seven FMDV serotypes. These results encourage the development of new oligonucleotide microarrays to probe the fine genetic and antigenic composition of FMDV for diagnosis, vaccine design, and to gain insight into the molecular epidemiology of this pathogen. RESULTS: A FMDV microarray was designed and optimized to detect SNPs at a major antigenic site of the virus. A screening of point mutants of the genomic region encoding antigenic site A of FMDV C-S8c1 was achieved. The hybridization pattern of a mutant includes specific positive and negative signals as well as crosshybridization signals, which are of different intensity depending on the thermodynamic stability of each probe-target pair. Moreover, an array bioinformatic classification method was developed to evaluate the hybridization signals. This statistical analysis shows that the procedure allows a very accurate classification per variant genome. CONCLUSION: A specific approach based on a microarray platform aimed at distinguishing point mutants within an important determinant of antigenicity and host cell tropism, namely the G-H loop of capsid protein VP1, was developed. The procedure is of general applicability as a test for specificity and discriminatory power of microarray-based diagnostic procedures using multiple oligonucleotide probes.

Animals↗

PubMed2005

STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of the LEE pathogenicity island of human and animal Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are important emerging pathogens that can cause a severe and sometimes fatal illness. Differentiation of eae, tir, espA, espD, and espB gene variants of the locus of enterocyte effacement (LEE) pathogenicity island represents an important tool for typing in routine diagnostics as well as in pathogenesis, epidemiologic, clonal, and immunologic studies. METHODS: Type-specific oligonucleotide microarrays and a PCR scheme were designed and constructed for the detection and typing of genetic variants of the LEE genes. Oligonucleotide probes were tested for their specificity against the corresponding type strain by microarray hybridization using fluorescent DNA, either PCR-amplified (single, multiplex, long-range), chromosomal, or amplified chromosomal DNA. RESULTS: The PCR scheme and the oligonucleotide microarray allowed us to distinguish 16 variants (alpha1, alpha2, beta1, beta2, gamma1, gamma2/theta, delta/kappa, epsilon, zeta, eta, iota, lambda, mu, nu, xi, omicron) of the eae gene, 4 variants (alpha1, beta1, gamma1, gamma2/theta) of the tir gene, 4 variants (alpha1, beta1, beta2, gamma1) of the espA gene, 3 variants (alpha1, beta1, gamma1) of the espB gene, and 3 variants (alpha1, beta1, gamma1) of the espD gene. We found a total of 12 different combinations of tir, espA, espB, and espD genes among the 25 typed strains. CONCLUSIONS: The PCR scheme and the oligonucleotide microarray described are effective tools to rapidly screen multiple virulence genes and their variants in E. coli strains isolated from human and animal infections. The results demonstrate the great genetic diversity among LEE genes of human and animal STEC and EPEC strains.

Animals↗

PubMed2005

Protein evolution in viral quasispecies under selective pressure: a thermodynamic and phylogenetic analysis.

The evolution of RNA viruses under antiviral pressure is characterized by high mutation rates and strong selective forces that induce extremely rapid changes of protein sequences. This makes the course of molecular evolution directly observable on time scales of months. Here we study the interplay between selection for drug resistance and selection for thermodynamic stability in the protease (PR) and the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) clones extracted from two patients with complex treatment histories. This analysis shows that folding thermodynamic properties may fluctuate very strongly in the course of quasispecies evolution under selective pressure. For the first case, our data suggest that folding efficiency of the RT is sacrificed at the advantage of drug resistance, while the corresponding PR seems to undergo selection for thermodynamic stability in the absence of substitutions associated to resistance. The PR of the second case is not submitted to antiviral pressure during the period analyzed and seems to initiate random fluctuations that lead to the accidental increase of its folding efficiency. In summary, joint consideration of sequence evolution and thermodynamic parameters can represent a more comprehensive approach for the study of the evolution of RNA viruses.

Anti-HIV Agents↗

PubMed2004

Reconstructing evolutionary relationships from functional data: a consistent classification of organisms based on translation inhibition response.

The last two decades have witnessed an unsurpassed effort aimed at reconstructing the history of life from the genetic information contained in extant organisms. The availability of many sequenced genomes has allowed the reconstruction of phylogenies from gene families and its comparison with traditional single-gene trees. However, the appearance of major discrepancies between both approaches questions whether horizontal gene transfer (HGT) has played a prominent role in shaping the topology of the Tree of Life. Recent attempts at solving this controversy and reaching a consensus tree combine molecular data with additional phylogenetic markers. Translation is a universal cellular function that involves a meaningful, highly conserved set of genes: both rRNA and r-protein operons have an undisputed phylogenetic value and rarely undergo HGT. Ribosomal function reflects the concerted expression of that genetic network and consequently yields information about the evolutionary paths followed by the organisms. Here we report on tree reconstruction using a measure of the performance of the ribosome: antibiotic sensitivity of protein synthesis. A large database has been used where 33 ribosomal systems belonging to the three major cellular lineages were probed against 38 protein synthesis inhibitors. Different definitions of distance between pairs of organisms have been explored, and the classical algorithm of bootstrap evaluation has been adapted to quantify the reliability of the reconstructions obtained. Our analysis returns a consistent phylogeny, where archaea are systematically affiliated to eukarya, in agreement with recent reconstructions which used information-processing systems. The integration of the information derived from relevant functional markers into current phylogenetic reconstructions might facilitate achieving a consensus Tree of Life.

Archaea↗

PubMed2004

Structural analysis of hepatitis C RNA genome using DNA microarrays.

Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5' non-coding region (5'NCR), the first three-quarters of the core region, the E2-NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions.

Conserved Sequence↗

PubMed2003

Memory in retroviral quasispecies: experimental evidence and theoretical model for human immunodeficiency virus.

Viral quasispecies may possess a molecular memory of their past evolutionary history, imprinted on minority components of the mutant spectrum. Here we report experimental evidence and a theoretical model for memory in retroviral quasispecies in vivo. Apart from replicative memory associated with quasispecies dynamics, retroviruses may harbour a "cellular" or "anatomical" memory derived from their integrative cycle and the presence of viral reservoirs in body compartments. Three independent sets of data exemplify the two kinds of memory in human immunodeficiency virus type 1 (HIV-1). The data provide evidence of re-emergence of sequences that were hidden in cellular or anatomical compartments for extended periods of infection, and recovery of a quasispecies from pre-existing genomes. We develop a three-component model that incorporates the essential features of the quasispecies dynamics of retroviruses exposed to selective pressures. Significantly, a numerical study based on this model is in agreement with the experimental data, further supporting the existence of both replicative and reservoir memory in retroviral quasispecies.

Adaptation, Physiological↗

PubMed2002

Duration and fitness dependence of quasispecies memory.

The duration and fitness dependence of memory in viral quasispecies evolving in cell culture have been investigated using two genetic markers of foot-and-mouth disease virus (FMDV). In lineages of antigenic variant FMDV RED, which reverted to FMDV RGD, memory FMDV RED genomes were detected after 50 infectious cycles, and memory level was fitness dependent. In growth-competition experiments between a reference FMDV RGD and two different FMDV RED populations, a 7.6-fold higher fitness of the initial FMDV RED population resulted in 30 to 100-fold higher memory level. In lineages of low-fitness clones containing an elongated internal polyadenylate tract, revertants lacking excess adenylate residues became dominant by passage 20. However, genomes including a larger number of adenylate residues were detected as memory genomes after at least 150 infectious cycles. Thus, quasispecies memory can be durable and is fitness dependent, as predicted from the growth competition of two mutant forms of a genome. An understanding of factors influencing quasispecies memory levels and duration may have implications for the extended diagnosis of viruses based on the quantification of minority genomes.

Animals↗