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1.
bioRxiv ; 2024 May 12.
Article in English | MEDLINE | ID: mdl-38765958

ABSTRACT

Small extracellular vesicles (sEVs) are heterogenous lipid membrane particles typically less than 200 nm in size and secreted by most cell types either constitutively or upon activation signals. sEVs isolated from biofluids contain RNAs, including small non-coding RNAs (ncRNAs), that can be either encapsulated within the EV lumen or bound to the EV surface. EV-associated microRNAs (miRNAs) are, despite a relatively low abundance, extensively investigated for their selective incorporation and their role in cell-cell communication. In contrast, the sorting of highly-structured ncRNA species is understudied, mainly due to technical limitations of traditional small RNA sequencing protocols. Here, we adapted ALL-tRNAseq to profile the relative abundance of highly structured and potentially methylated small ncRNA species, including transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and Y RNAs in bulk EV preparations. We determined that full-length tRNAs, typically 75 to 90 nucleotides in length, were the dominant small ncRNA species (>60% of all reads in the 18-120 nucleotides size-range) in all cell culture-derived EVs, as well as in human plasma-derived EV samples, vastly outnumbering 21 nucleotides-long miRNAs. Nearly all EV-associated tRNAs were protected from external RNAse treatment, indicating a location within the EV lumen. Strikingly, the vast majority of luminal-sorted, full-length, nucleobase modification-containing EV-tRNA sequences, harbored a dysfunctional 3' CCA tail, 1 to 3 nucleotides truncated, rendering them incompetent for amino acid loading. In contrast, in non-EV associated extracellular particle fractions (NVEPs), tRNAs appeared almost exclusively fragmented or 'nicked' into tRNA-derived small RNAs (tsRNAs) with lengths between 18 to 35 nucleotides. We propose that in mammalian cells, tRNAs that lack a functional 3' CCA tail are selectively sorted into EVs and shuttled out of the producing cell, offering a new perspective into the physiological role of secreted EVs and luminal cargo-selection.

2.
Heliyon ; 10(6): e27360, 2024 Mar 30.
Article in English | MEDLINE | ID: mdl-38515664

ABSTRACT

Liquid biopsy-derived RNA sequencing (lbRNA-seq) exhibits significant promise for clinic-oriented cancer diagnostics due to its non-invasiveness and ease of repeatability. Despite substantial advancements, obstacles like technical artefacts and process standardisation impede seamless clinical integration. Alongside addressing technical aspects such as normalising fluctuating low-input material and establishing a standardised clinical workflow, the lack of result validation using independent datasets remains a critical factor contributing to the often low reproducibility of liquid biopsy-detected biomarkers. Considering the outlined drawbacks, our objective was to establish a workflow/methodology characterised by: 1. Harness the rich diversity of biological features accessible through lbRNA-seq data, encompassing a holistic range of molecular and functional attributes. These components are seamlessly integrated via a Machine Learning-based Ensemble Classification framework, enabling a unified and comprehensive analysis of the intricate information encoded within the data. 2. Implementing and rigorously benchmarking intra-sample normalisation methods to heighten their relevance within clinical settings. 3. Thoroughly assessing its efficacy across independent test sets to ascertain its robustness and potential utility. Using ten datasets from several studies comprising three different sources of biological material, we first show that while the best-performing normalisation methods depend strongly on the dataset and coupled Machine Learning method, the rather simple Counts Per Million method is generally very robust, showing comparable performance to cross-sample methods. Subsequently, we demonstrate that the innovative biofeature types introduced in this study, such as the Fraction of Canonical Transcript, harbour complementary information. Consequently, their inclusion consistently enhances prediction power compared to models relying solely on gene expression-based biofeatures. Finally, we demonstrate that the workflow is robust on completely independent datasets, generally from different labs and/or different protocols. Taken together, the workflow presented here outperforms generally employed methods in prediction accuracy and may hold potential for clinical diagnostics application due to its specific design.

3.
Biology (Basel) ; 12(6)2023 Jun 13.
Article in English | MEDLINE | ID: mdl-37372134

ABSTRACT

As the genome carries the historical information of a species' biotic and environmental interactions, analyzing changes in genome structure over time by using powerful statistical physics methods (such as entropic segmentation algorithms, fluctuation analysis in DNA walks, or measures of compositional complexity) provides valuable insights into genome evolution. Nucleotide frequencies tend to vary along the DNA chain, resulting in a hierarchically patchy chromosome structure with heterogeneities at different length scales that range from a few nucleotides to tens of millions of them. Fluctuation analysis reveals that these compositional structures can be classified into three main categories: (1) short-range heterogeneities (below a few kilobase pairs (Kbp)) primarily attributed to the alternation of coding and noncoding regions, interspersed or tandem repeats densities, etc.; (2) isochores, spanning tens to hundreds of tens of Kbp; and (3) superstructures, reaching sizes of tens of megabase pairs (Mbp) or even larger. The obtained isochore and superstructure coordinates in the first complete T2T human sequence are now shared in a public database. In this way, interested researchers can use T2T isochore data, as well as the annotations for different genome elements, to check a specific hypothesis about genome structure. Similarly to other levels of biological organization, a hierarchical compositional structure is prevalent in the genome. Once the compositional structure of a genome is identified, various measures can be derived to quantify the heterogeneity of such structure. The distribution of segment G+C content has recently been proposed as a new genome signature that proves to be useful for comparing complete genomes. Another meaningful measure is the sequence compositional complexity (SCC), which has been used for genome structure comparisons. Lastly, we review the recent genome comparisons in species of the ancient phylum Cyanobacteria, conducted by phylogenetic regression of SCC against time, which have revealed positive trends towards higher genome complexity. These findings provide the first evidence for a driven progressive evolution of genome compositional structure.

4.
Cell Rep Methods ; 3(5): 100480, 2023 May 22.
Article in English | MEDLINE | ID: mdl-37323569

ABSTRACT

IsomiRs, sequence variants of mature microRNAs, are usually detected and quantified using high-throughput sequencing. Many examples of their biological relevance have been reported, but sequencing artifacts identified as artificial variants might bias biological inference and therefore need to be ideally avoided. We conducted a comprehensive evaluation of 10 different small RNA sequencing protocols, exploring both a theoretically isomiR-free pool of synthetic miRNAs and HEK293T cells. We calculated that, with the exception of two protocols, less than 5% of miRNA reads can be attributed to library preparation artifacts. Randomized-end adapter protocols showed superior accuracy, with 40% of true biological isomiRs. Nevertheless, we demonstrate concordance across protocols for selected miRNAs in non-templated uridyl additions. Notably, NTA-U calling and isomiR target prediction can be inaccurate when using protocols with poor single-nucleotide resolution. Our results highlight the relevance of protocol choice for biological isomiRs detection and annotation, which has key potential implications for biomedical applications.


Subject(s)
MicroRNAs , Humans , MicroRNAs/genetics , HEK293 Cells , Base Sequence , Sequence Analysis, RNA
5.
Nucleic Acids Res ; 51(W1): W372-W378, 2023 07 05.
Article in English | MEDLINE | ID: mdl-37216599

ABSTRACT

RNA-sequencing has become one of the most used high-throughput approaches to gain knowledge about the expression of all different RNA subpopulations. However, technical artifacts, either introduced during library preparation and/or data analysis, can influence the detected RNA expression levels. A critical step, especially in large and low input datasets or studies, is data normalization, which aims at eliminating the variability in data that is not related to biology. Many normalization methods have been developed, each of them relying on different assumptions, making the selection of the appropriate normalization strategy key to preserve biological information. To address this, we developed NormSeq, a free web-server tool to systematically assess the performance of normalization methods in a given dataset. A key feature of NormSeq is the implementation of information gain to guide the selection of the best normalization method, which is crucial to eliminate or at least reduce non-biological variability. Altogether, NormSeq provides an easy-to-use platform to explore different aspects of gene expression data with a special focus on data normalization to help researchers, even without bioinformatics expertise, to obtain reliable biological inference from their data. NormSeq is freely available at: https://arn.ugr.es/normSeq.


Subject(s)
Gene Expression Profiling , RNA-Seq , Software , Gene Expression Profiling/methods , Gene Library , RNA/genetics , Sequence Analysis, RNA/methods
6.
Biomolecules ; 13(3)2023 02 26.
Article in English | MEDLINE | ID: mdl-36979375

ABSTRACT

Wheat is one of the most important food sources on Earth. MicroRNAs (miRNAs) play important roles in wheat productivity. To identify wheat miRNAs as well as their expression profiles under drought condition, we constructed and sequenced small RNA (sRNA) libraries from the leaves and roots of three wheat cultivars (Kukri, RAC875 and Excalibur) under water and drought conditions. A total of 636 known miRNAs and 294 novel miRNAs were identified, of which 34 miRNAs were tissue- or cultivar-specific. Among these, 314 were significantly regulated under drought conditions. miRNAs that were drought-regulated in all cultivars displayed notably higher expression than those that responded in a cultivar-specific manner. Cultivar-specific drought response miRNAs were mainly detected in roots and showed significantly different drought regulations between cultivars. By using wheat degradome library, 6619 target genes were identified. Many target genes were strongly enriched for protein domains, such as MEKHLA, that play roles in drought response. Targeting analysis showed that drought-downregulated miRNAs targeted more genes than drought-upregulated miRNAs. Furthermore, such genes had more important functions. Additionally, the genes targeted by drought-downregulated miRNAs had multiple interactions with each other, while the genes targeted by drought-upregulated miRNAs had no interactions. Our data provide valuable information on wheat miRNA expression profiles and potential functions in different tissues, cultivars and drought conditions.


Subject(s)
MicroRNAs , Water , Water/metabolism , Triticum/metabolism , Droughts , MicroRNAs/genetics , MicroRNAs/metabolism , Gene Library , Gene Expression Regulation, Plant , Stress, Physiological/genetics
7.
Genes Dev ; 37(5-6): 243-257, 2023 03 01.
Article in English | MEDLINE | ID: mdl-36810209

ABSTRACT

Transfer RNAs (tRNAs) are small adaptor RNAs essential for mRNA translation. Alterations in the cellular tRNA population can directly affect mRNA decoding rates and translational efficiency during cancer development and progression. To evaluate changes in the composition of the tRNA pool, multiple sequencing approaches have been developed to overcome reverse transcription blocks caused by the stable structures of these molecules and their numerous base modifications. However, it remains unclear whether current sequencing protocols faithfully capture tRNAs existing in cells or tissues. This is specifically challenging for clinical tissue samples that often present variable RNA qualities. For this reason, we developed ALL-tRNAseq, which combines the highly processive MarathonRT and RNA demethylation for the robust assessment of tRNA expression, together with a randomized adapter ligation strategy prior to reverse transcription to assess tRNA fragmentation levels in both cell lines and tissues. Incorporation of tRNA fragments not only informed on sample integrity but also significantly improved tRNA profiling of tissue samples. Our data showed that our profiling strategy effectively improves classification of oncogenic signatures in glioblastoma and diffuse large B-cell lymphoma tissues, particularly for samples presenting higher levels of RNA fragmentation, further highlighting the utility of ALL-tRNAseq for translational research.


Subject(s)
Protein Biosynthesis , RNA, Transfer , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA, Messenger/metabolism , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, RNA/methods
8.
Methods Mol Biol ; 2630: 179-213, 2023.
Article in English | MEDLINE | ID: mdl-36689184

ABSTRACT

The current versions of the microRNA databases MiRgeneDB, miRBase, and PmiREN contain annotations for a total of 358 different species. Public repositories, however, host small RNA sequencing data for over 800 species. This discrepancy implies that microRNA research is also very active in species that neither have an available high-quality genome assembly nor annotations for microRNAs or other types of noncoding genes. These cases are particularly challenging to analyze because reference sequences need to be collected from different sources and processed and formatted appropriately so that the dedicated small RNA analysis tools can make use of them. In this protocol we describe how small RNA sequencing data can be easily analyzed by means of a dockerized version of the well-established sRNAtoolbox/sRNAbench small RNA tools. We outline the analysis of two publicly available datasets to demonstrate basic aspects like the preparation of the local database, expression profiling, or differential expression analysis as well as more advanced features such as quantification of exogenous RNA content and data analysis in non-model species.


Subject(s)
MicroRNAs , Software , MicroRNAs/genetics , Sequence Analysis, RNA , Databases, Nucleic Acid , Base Sequence , High-Throughput Nucleotide Sequencing/methods
9.
Funct Integr Genomics ; 23(1): 15, 2022 Dec 23.
Article in English | MEDLINE | ID: mdl-36562829

ABSTRACT

Wheat is one of the most important food sources on Earth. MicroRNAs (miRNA) play important roles in wheat productivity. To identify wheat miRNAs, we constructed and sequenced sRNA libraries from leaves and roots of two wheat cultivars (RAC875 and Kukri) with many different traits. Given that available miRNA wheat complement in the plant-specific database PmiREN ( https://pmiren.com ) does not include root tissues and root-associated miRNAs might thus be missing, we performed first the prediction of novel miRNAs using the sRNAbench tool. We found a total of 150 putatively novel miRNA genes with expression of both arms from 289 unique mature sequences and nearly 30% of all miRNA reads in roots corresponded to novel miRNAs. In contrast, this figure in leaves dropped to under 3%, confirming the undersampling of roots in the complement of known miRNAs. By using 120 publicly available wheat datasets, 598 Zea mays small RNA libraries, 64 plant species genomes, wheat degradome library, and functional enrichment analysis, a subset of novel miRNAs were confirmed as bona-fide miRNAs. Of the total 605 miRNAs identified in this study inclusive of 316 known miRNAs, 528 miRNAs were shared by both cultivars, 429 miRNAs were shared by both root tissues and 329 miRNAs were shared by both leaf tissues. In addition, 32 miRNAs were specific to Kukri while 45 miRNAs were specific to RAC875. These miRNAs had diverse functions, such as regulation of gene transcription, protein translation, energy metabolism, and cell cycle progression. Our data provide a genome-wide miRNA expression profile in these two wheat cultivars and help functional studies of wheat genomics.


Subject(s)
MicroRNAs , Triticum , Triticum/genetics , Triticum/metabolism , Genomics , Base Sequence , MicroRNAs/genetics , MicroRNAs/metabolism , Genome, Plant , Gene Expression Regulation, Plant , RNA, Plant/genetics , RNA, Plant/metabolism , Gene Expression Profiling
10.
Int J Mol Sci ; 23(23)2022 Nov 26.
Article in English | MEDLINE | ID: mdl-36499112

ABSTRACT

The tropical common bean (Phaseolus vulgaris L.) is an obligatory short-day plant that requires relaxation of the photoperiod to induce flowering. Similar to other crops, photoperiod-induced floral initiation depends on the differentiation and maintenance of meristems. In this study, the global changes in transcript expression profiles were analyzed in two meristematic tissues corresponding to the vegetative and inflorescence meristems of two genotypes with different sensitivities to photoperiods. A total of 3396 differentially expressed genes (DEGs) were identified, and 1271 and 1533 were found to be up-regulated and down-regulated, respectively, whereas 592 genes showed discordant expression patterns between both genotypes. Arabidopsis homologues of DEGs were identified, and most of them were not previously involved in Arabidopsis floral transition, suggesting an evolutionary divergence of the transcriptional regulatory networks of the flowering process of both species. However, some genes belonging to the photoperiod and flower development pathways with evolutionarily conserved transcriptional profiles have been found. In addition, the flower meristem identity genes APETALA1 and LEAFY, as well as CONSTANS-LIKE 5, were identified as markers to distinguish between the vegetative and reproductive stages. Our data also indicated that the down-regulation of the photoperiodic genes seems to be directly associated with promoting floral transition under inductive short-day lengths. These findings provide valuable insight into the molecular factors that underlie meristematic development and contribute to understanding the photoperiod adaptation in the common bean.


Subject(s)
Arabidopsis , Phaseolus , Arabidopsis/genetics , Phaseolus/genetics , Phaseolus/metabolism , Gene Expression Regulation, Plant , Genes, Plant , Transcriptome , Meristem , Flowers/metabolism , Inflorescence/genetics , Inflorescence/metabolism , Plant Proteins/genetics
11.
Front Cell Infect Microbiol ; 12: 919786, 2022.
Article in English | MEDLINE | ID: mdl-35992165

ABSTRACT

Ticks are blood-feeding arthropods that use the components of their salivary glands to counter the host's hemostatic, inflammatory, and immune responses. The tick midgut also plays a crucial role in hematophagy. It is responsible for managing blood meals (storage and digestion) and protecting against host immunity and pathogen infections. Previous transcriptomic studies revealed the complexity of tick sialomes (salivary gland transcriptomes) and mialomes (midgut transcriptomes) which encode for protease inhibitors, lipocalins (histamine-binding proteins), disintegrins, enzymes, and several other tick-specific proteins. Several studies have demonstrated that mammalian hosts acquire tick resistance against repeated tick bites. Consequently, there is an urgent need to uncover how tick sialomes and mialomes respond to resistant hosts, as they may serve to develop novel tick control strategies and applications. Here, we mimicked natural repeated tick bites in a laboratory setting and analyzed gene expression dynamics in the salivary glands and midguts of adult female ticks. Rabbits were subjected to a primary (feeding on a naive host) and a secondary infestation of the same host (we re-exposed the hosts but to other ticks). We used single salivary glands and midguts dissected from individual siblings adult pathogen-free female Ixodes ricinus to reduce genetic variability between individual ticks. The comprehensive analysis of 88 obtained RNA-seq data sets allows us to provide high-quality annotated sialomes and mialomes from individual ticks. Comparisons between fed/unfed, timepoints, and exposures yielded as many as 3000 putative differentially expressed genes (DEG). Interestingly, when classifying the exposure DEGs by means of a clustering approach we observed that the majority of these genes show increased expression at early feeding time-points in the mid-gut of re-exposed ticks. The existence of clearly defined groups of genes with highly similar responses to re-exposure suggests the existence of molecular swiches. In silico functional analysis shows that these early feeding reexposure response genes form a dense interaction network at protein level being related to virtually all aspects of gene expression regulation and glycosylation. The processed data is available through an easy-to-use database-associated webpage (https://arn.ugr.es/IxoriDB/) that can serve as a valuable resource for tick research.


Subject(s)
Ixodes , Tick Bites , Animals , Arthropod Proteins/genetics , Arthropod Proteins/metabolism , Female , Ixodes/genetics , Mammals/genetics , Rabbits , Salivary Glands/metabolism , Transcriptome , Vertebrates
12.
Viruses ; 14(8)2022 07 28.
Article in English | MEDLINE | ID: mdl-36016282

ABSTRACT

Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus.


Subject(s)
Herpes Simplex , Herpesvirus 1, Human , MicroRNAs , Herpes Simplex/genetics , Herpesvirus 1, Human/physiology , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Up-Regulation , Virus Replication
13.
Nucleic Acids Res ; 50(W1): W710-W717, 2022 07 05.
Article in English | MEDLINE | ID: mdl-35556129

ABSTRACT

The NCBI Sequence Read Archive currently hosts microRNA sequencing data for over 800 different species, evidencing the existence of a broad taxonomic distribution in the field of small RNA research. Simultaneously, the number of samples per miRNA-seq study continues to increase resulting in a vast amount of data that requires accurate, fast and user-friendly analysis methods. Since the previous release of sRNAtoolbox in 2019, 55 000 sRNAbench jobs have been submitted which has motivated many improvements in its usability and the scope of the underlying annotation database. With this update, users can upload an unlimited number of samples or import them from Google Drive, Dropbox or URLs. Micro- and small RNA profiling can now be carried out using high-confidence Metazoan and plant specific databases, MirGeneDB and PmiREN respectively, together with genome assemblies and libraries from 441 Ensembl species. The new results page includes straightforward sample annotation to allow downstream differential expression analysis with sRNAde. Unassigned reads can also be explored by means of a new tool that performs mapping to microbial references, which can reveal contamination events or biologically meaningful findings as we describe in the example. sRNAtoolbox is available at: https://arn.ugr.es/srnatoolbox/.


Subject(s)
MicroRNAs , RNA, Small Untranslated , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Molecular Sequence Annotation , Sequence Analysis, RNA , Databases, Factual
14.
Hortic Res ; 2022 Jan 18.
Article in English | MEDLINE | ID: mdl-35039829

ABSTRACT

Trichomes are specialised epidermal cells developed in the aerial surface of almost every terrestrial plant. These structures form physical barriers, which combined with their capability of synthesis of complex molecules, prevent plagues from spreading and confer trichomes a key role in the defence against herbivores. In this work, the tomato gene HAIRPLUS (HAP) that controls glandular trichome density in tomato plants was characterised. HAP belongs to a group of proteins involved in histone tail modifications although some also bind methylated DNA. HAP loss of function promotes epigenomic modifications in the tomato genome reflected in numerous differentially methylated cytosines and causes transcriptomic changes in hap mutant plants. Taken together, these findings demonstrate that HAP links epigenome remodelling with multicellular glandular trichome development and reveal that HAP is a valuable genomic tool for pest resistance in tomato breeding.

15.
Sci Rep ; 10(1): 19073, 2020 11 04.
Article in English | MEDLINE | ID: mdl-33149190

ABSTRACT

Progressive evolution, or the tendency towards increasing complexity, is a controversial issue in biology, which resolution entails a proper measurement of complexity. Genomes are the best entities to address this challenge, as they encode the historical information of a species' biotic and environmental interactions. As a case study, we have measured genome sequence complexity in the ancient phylum Cyanobacteria. To arrive at an appropriate measure of genome sequence complexity, we have chosen metrics that do not decipher biological functionality but that show strong phylogenetic signal. Using a ridge regression of those metrics against root-to-tip distance, we detected positive trends towards higher complexity in three of them. Lastly, we applied three standard tests to detect if progressive evolution is passive or driven-the minimum, ancestor-descendant, and sub-clade tests. These results provide evidence for driven progressive evolution at the genome-level in the phylum Cyanobacteria.


Subject(s)
Cyanobacteria/genetics , Evolution, Molecular , Genome, Bacterial , Cyanobacteria/classification , Phylogeny
16.
Nucleic Acids Res ; 48(W1): W262-W267, 2020 07 02.
Article in English | MEDLINE | ID: mdl-32484556

ABSTRACT

Although miRNA-seq is extensively used in many different fields, its quality control is frequently restricted to a PhredScore-based filter. Other important quality related aspects like microRNA yield, the fraction of putative degradation products (such as rRNA fragments) or the percentage of adapter-dimers are hard to assess using absolute thresholds. Here we present mirnaQC, a webserver that relies on 34 quality parameters to assist in miRNA-seq quality control. To improve their interpretability, quality attributes are ranked using a reference distribution obtained from over 36 000 publicly available miRNA-seq datasets. Accepted input formats include FASTQ and SRA accessions. The results page contains several sections that deal with putative technical artefacts related to library preparation, sequencing, contamination or yield. Different visualisations, including PCA and heatmaps, are available to help users identify underlying issues. Finally, we show the usefulness of this approach by analysing two publicly available datasets and discussing the different quality issues that can be detected using mirnaQC.


Subject(s)
MicroRNAs/chemistry , Sequence Analysis, RNA/standards , Software , Artifacts , Female , Humans , MicroRNAs/metabolism , Quality Control , Uterine Cervical Neoplasms/genetics
17.
Plants (Basel) ; 9(4)2020 Apr 22.
Article in English | MEDLINE | ID: mdl-32331491

ABSTRACT

Pod maturation of common bean relies upon complex gene expression changes, which in turn are crucial for seed formation and dispersal. Hence, dissecting the transcriptional regulation of pod maturation would be of great significance for breeding programs. In this study, a comprehensive characterization of expression changes has been performed in two common bean cultivars (ancient and modern) by analyzing the transcriptomes of five developmental pod stages, from fruit setting to maturation. RNA-seq analysis allowed for the identification of key genes shared by both accessions, which in turn were homologous to known Arabidopsis maturation genes and furthermore showed a similar expression pattern along the maturation process. Gene- expression changes suggested a role in promoting an accelerated breakdown of photosynthetic and ribosomal machinery associated with chlorophyll degradation and early activation of alpha-linolenic acid metabolism. A further study of transcription factors and their DNA binding sites revealed three candidate genes whose functions may play a dominant role in regulating pod maturation. Altogether, this research identifies the first maturation gene set reported in common bean so far and contributes to a better understanding of the dynamic mechanisms of pod maturation, providing potentially useful information for genomic-assisted breeding of common bean yield and pod quality attributes.

18.
Nucleic Acids Res ; 47(W1): W530-W535, 2019 07 02.
Article in English | MEDLINE | ID: mdl-31114926

ABSTRACT

Since the original publication of sRNAtoolbox in 2015, small RNA research experienced notable advances in different directions. New protocols for small RNA sequencing have become available to address important issues such as adapter ligation bias, PCR amplification artefacts or to include internal controls such as spike-in sequences. New microRNA reference databases were developed with different foci, either prioritizing accuracy (low number of false positives) or completeness (low number of false negatives). Additionally, other small RNA molecules as well as microRNA sequence and length variants (isomiRs) have continued to gain importance. Finally, the number of microRNA sequencing studies deposited in GEO nearly triplicated from 2014 (280) to 2018 (764). These developments imply that fast and easy-to-use tools for expression profiling and subsequent downstream analysis of miRNA-seq data are essential to many researchers. Key features in this sRNAtoolbox release include addition of all major RNA library preparation protocols to sRNAbench and improvements in sRNAde, a tool that summarizes several aspects of small RNA sequencing studies including the detection of consensus differential expression. A special emphasis was put on the user-friendliness of the tools, for instance sRNAbench now supports parallel launching of several jobs to improve reproducibility and user time efficiency.


Subject(s)
MicroRNAs/chemistry , MicroRNAs/metabolism , Software , Gene Expression Profiling , Genetic Variation , Sequence Analysis, RNA
19.
Methods Mol Biol ; 1766: 31-47, 2018.
Article in English | MEDLINE | ID: mdl-29605846

ABSTRACT

The promoter region of around 70% of all genes in the human genome is overlapped by a CpG island (CGI). CGIs have known functions in the transcription initiation and outstanding compositional features like high G+C content and CpG ratios when compared to the bulk DNA. We have shown before that CGIs manifest as clusters of CpGs in mammalian genomes and can therefore be detected using clustering methods. These techniques have several advantages over sliding window approaches which apply compositional properties as thresholds. In this protocol we show how to determine local (CpG islands) and global (distance distribution) clustering properties of CG dinucleotides and how to generalize this analysis to any k-mer or combinations of it. In addition, we illustrate how to easily cross the output of a CpG island prediction algorithm with our methylation database to detect differentially methylated CGIs. The analysis is given in a step-by-step protocol and all necessary programs are implemented into a virtual machine or, alternatively, the software can be downloaded and easily installed.


Subject(s)
CpG Islands/genetics , DNA Methylation , Genome, Human/genetics , Animals , Base Composition , Base Sequence , DNA/chemistry , DNA/genetics , DNA/metabolism , Humans , Promoter Regions, Genetic/genetics , Software , Transcription Initiation, Genetic
20.
Cir. plást. ibero-latinoam ; 43(supl.1): s117-s128, sept. 2017. ilus, tab, graf
Article in Spanish | IBECS | ID: ibc-169065

ABSTRACT

Introducción y Objetivo. Las quemaduras en las manos suponen un volumen total muy importante en la atención sanitaria. Prácticamente la totalidad de la población ha sufrido o sufrirá a lo largo de su vida una quemadura en sus manos. Afortunadamente, la inmensa mayoría no requerirá una atención especializada por parte de un cirujano plástico. Aun así, el impacto de los que sí precisan dicha atención es importante y debemos tener siempre planificado cómo actuar en cada situación. La función final que se obtenga de una mano quemada será de vital importancia, tanto si es la única zona del cuerpo afectada, como si entra en el contexto del gran quemado, y más en estos casos puesto que tendrá una gran repercusión positiva en su calidad de vida si se logra un resultado óptimo y/o funcional. El objetivo de este artículo es revisar nuestra experiencia como Unidad de Quemados en el tratamiento de la mano quemada y mostrar una serie de ejemplos de abordaje de las secuelas de quemaduras en manos que permita al paciente reincorporarse de forma completa a su actividad habitual. Material y Método. Revisamos los pacientes atendidos en nuestra Unidad entre 2012 y 2014, la afectación en manos, su epidemiología, distribución por variables, y revisamos varios casos de secuelas, algunos típicos y otros menos frecuentes. Resultados. Atendimos a un total de 307 pacientes, de los cuales 68 sufrieron quemaduras en 1 mano y 113 en las 2, requiriendo finalmente cirugía 155 manos. Del total, 39 pacientes provenían del ámbito laboral, de los cuales sufrieron quemaduras en manos 20, siendo en estos casos más frecuente la afectación de ambas manos que en los pacientes que no sufrieron accidentes laborales (4 afectados en una sola mano y 20 en ambas manos). Conclusiones. Nuestros pilares básicos se centran en: un diagnóstico preciso y precoz de profundidad, extensión y repercusiones; un tratamiento quirúrgico temprano y adecuado; un manejo rehabilitador continuo; y un seguimiento detallado de las posibles secuelas para poder tratarlas con la mejor indicación y en el momento preciso (AU)


Background and Objective. Hand burns have a very important total volume in healthcare. Virtually all of the population has suffered or will suffer throughout the life a burn on hands. Fortunately, the vast majority do not require a specialized attention by a plastic surgeon. Still, the impact of those who need such specialist attention is important and we must always plan what to do in every situation. The final function that is obtained from a burned hand will be vital whether it is the only area affected as if within the context of a critical burn patient, and more so in these cases as it will have a positive impact on their quality of life if results achieved are optimal and/or functional. The aim of this study is to present our experience as Burns Unit in the treatment of burned hands, and to review some examples of management of burns sequels in hands in order to get a complete reintegration of patients to their usual activities. Methods. We reviewed patients at our Unit involvement in hands, epidemiology, distribution in variables, and reviewed several cases, some typical and others less frequent. Results. We attended a total of 307 patients, 68 of them with burns in 1 hand, and 113 in both hands, finally needing surgery 155 hands. From the total, 39 patients came from the laboral ambient, 20 with hand burns, being both hands burns more frequent in those cases than in those who didn't suffered laboral accidents (4 burns in only 1 hand and 20 in both hands). Conclusions. Our cornerstones are: an accurate and early diagnosis of depth, extent and impact; an early and appropriate surgical treatment; continuous rehabilitation management; and a close follow up of the possible sequelae to treat them with the best indication and at the right time (AU)


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Burns/surgery , Hand/surgery , Burns/epidemiology , Early Diagnosis , Quality of Life , Cicatrix, Hypertrophic/diagnosis , Cicatrix, Hypertrophic/rehabilitation , Retrospective Studies
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