Empresa edificaciones centroamericanas S.A EDICASA

Presenta el desarrollo de conocimientos, aptitudes y habilidades, previamente adquiridas en el plan de estudios de arquitectura y poner en práctica las competencias y la capacidad para diseñar las tareas y roles que se esperan de un arquitecto

flexiMAP: a regression-based method for discovering differential alternative polyadenylation events in standard RNA-seq data

Motivation: We present flexible Modeling of Alternative PolyAdenylation (flexiMAP), a newbeta-regression-based method implemented in R, for discovering differential alternative polyadenylation events in standard RNA-seq data. Results: We show, using both simulated and real data, that flexiMAP exhibits a good balance between specificity and sensitivity and compares favourably to existing methods, especially at low fold changes. In addition, the tests on simulated data reveal some hitherto unrecognized caveats of existing methods. Importantly, flexiMAP allows modeling of multiple known covariates that often confound the results of RNA-seq data analysis. Availability and implementation: The flexiMAPR package is available at:https://github.com/kszkop/flexiMAP. Scripts and data to reproduce the analysis in this paper are available at: https://doi.org/10.5281/zenodo.3689788

Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion

The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection

DNA Polymerase Conformational Dynamics and the Role of Fidelity-Conferring Residues: Insights from Computational Simulations

Herein we investigate the molecular bases of DNA polymerase I conformational dynamics that underlie the replication fidelity of the enzyme. Such fidelity is determined by conformational changes that promote the rejection of incorrect nucleotides before the chemical ligation step. We report a comprehensive atomic resolution study of wild type and mutant enzymes in different bound states and starting from different crystal structures, using extensive molecular dynamics (MD) simulations that cover a total timespan of ~5 ms. The resulting trajectories are examined via a combination of novel methods of internal dynamics and energetics analysis, aimed to reveal the principal molecular determinants for the (de)stabilization of a certain conformational state. Our results show that the presence of fidelity-decreasing mutations or the binding of incorrect nucleotides in ternary complexes tend to favor transitions from closed toward open structures, passing through an ensemble of semi-closed intermediates. The latter ensemble includes the experimentally observed ajar conformation which, consistent with previous experimental observations, emerges as a molecular checkpoint for the selection of the correct nucleotide to incorporate. We discuss the implications of our results for the understanding of the relationships between the structure, dynamics, and function of DNA polymerase I at the atomistic level

Relative Abundance of Transcripts (RATs):Identifying differential isoform abundance from RNA-seq [version 1; referees: 1 approved, 2 approved with reservations]

The biological importance of changes in RNA expression is reflected by the wide variety of tools available to characterise these changes from RNA-seq data. Several tools exist for detecting differential transcript isoform usage (DTU) from aligned or assembled RNA-seq data, but few exist for DTU detection from alignment-free RNA-seq quantifications. We present the RATs, an R package that identifies DTU transcriptome-wide directly from transcript abundance estimates. RATs is unique in applying bootstrapping to estimate the reliability of detected DTU events and shows good performance at all replication levels (median false positive fraction < 0.05). We compare RATs to two existing DTU tools, DRIM-Seq & SUPPA2, using two publicly available simulated RNA-seq datasets and a published human RNA-seq dataset, in which 248 genes have been previously identified as displaying significant DTU. RATs with default threshold values on the simulated Human data has a sensitivity of 0.55, a Matthews correlation coefficient of 0.71 and a false discovery rate (FDR) of 0.04, outperforming both other tools. Applying the same thresholds for SUPPA2 results in a higher sensitivity (0.61) but poorer FDR performance (0.33). RATs and DRIM-seq use different methods for measuring DTU effect-sizes complicating the comparison of results between these tools, however, for a likelihood-ratio threshold of 30, DRIM-Seq has similar FDR performance to RATs (0.06), but worse sensitivity (0.47). These differences persist for the simulated drosophila dataset. On the published human RNA-seq dataset the greatest agreement between the tools tested is 53%, observed between RATs and SUPPA2. The bootstrapping quality filter in RATs is responsible for removing the majority of DTU events called by SUPPA2 that are not reported by RATs. All methods, including the previously published qRT-PCR of three of the 248 detected DTU events, were found to be sensitive to annotation differences between Ensembl v60 and v87

Untranslated parts of genes interpreted: making heads or tails of high-throughput transcriptomic data via computational methods

The fate of eukaryotic transcripts is closely linked to their untranslated regions, which are determined by where transcription starts and ends on a genomic locus. The extent of alternative transcription start and alternative poly-adenylation has been revealed by sequencing methods focused on the ends of transcripts, but the application of these methods is not yet widely adopted by the community. In this review we highlight the importance of defining the untranslated parts of transcripts and suggest that computational methods applied to standard high-throughput technologies are a useful alternative to the expertise-demanding 5’ and 3’ sequencing. We present a number of computational approaches for the discovery and quantification of alternative transcription start and poly-adenylation events, focusing on technical challenges and arguing for the need to include better normalization of the data and more appropriate statistical models of the expected variation in the signal

The effects of blend ratio and storage time on thermoplastic starch/poly(butylene adipate-co-terephthalate) films

The objective of this work was to investigate blend ratio and storage time effects on the morphological, mechanical, and thermal properties of thermoplastic starch/poly(butylene adipate-co-terephthalate) (TPS/PBAT) films. TPS was prepared from plasticized cassava starch using a twin-screw extruder. TPS was subsequently melt-blended with PBAT with varied weight ratios (i.e., 20/80, 40/60 and 60/40) and blown to form TPS/PBAT films. It was found that increasing the TPS/PBAT ratio to 40/60 led to improved distributions of polymeric components and increased PBAT crystallization temperatures while reducing TPS melting transitions and tensile properties of TPS/PBAT films.After three months of storage at 30 °C, the tensile strength and secant modulus at 2% strain of TPS/PBAT films increased due to recrystallization of both TPS and PBAT. Blend ratios were the primary determinant for changes in TPS/PBAT film elongation at break with this storage time. Elongation at break decreased at low TPS:PBAT ratios (i.e., 20/80) and increased at high blend ratios (i.e., 60/40). The recrystallization of both TPS and PBAT components were observed from XRD and DSC analyses. Results obtained from both techniques confirmed the formation of additional crystalline structures of individual components during storage. The recrystallization phenomena also affected thermal transition temperatures of blend components. The crystallization temperature of PBAT-rich phase increased as starch could act as nucleating sites for PBAT. Using DMA, the tan δ curve of TPS/PBAT film exhibited two sharp individual peaks corresponding to the glass transitions of PBAT-rich and starch-rich phases. The tan δ of TPS-rich phase shifted to higher temperature due to recrystallization of TPS-rich phase

Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing.

The ribosome small subunit is expressed in all living cells. It performs numerous essential functions during translation, including formation of the initiation complex and proofreading of base-pairs between mRNA codons and tRNA anticodons. The core constituent of the small ribosomal subunit is a ~1.5 kb RNA strand in prokaryotes (16S rRNA) and a homologous ~1.8 kb RNA strand in eukaryotes (18S rRNA). Traditional sequencing-by-synthesis (SBS) of rRNA genes or rRNA cDNA copies has achieved wide use as a 'molecular chronometer' for phylogenetic studies, and as a tool for identifying infectious organisms in the clinic. However, epigenetic modifications on rRNA are erased by SBS methods. Here we describe direct MinION nanopore sequencing of individual, full-length 16S rRNA absent reverse transcription or amplification. As little as 5 picograms (~10 attomole) of purified E. coli 16S rRNA was detected in 4.5 micrograms of total human RNA. Nanopore ionic current traces that deviated from canonical patterns revealed conserved E. coli 16S rRNA 7-methylguanosine and pseudouridine modifications, and a 7-methylguanosine modification that confers aminoglycoside resistance to some pathological E. coli strains