Seed transcriptome annotation reveals enhanced expression of genes related to ros homeostasis and ethylene metabolism at alternating temperatures in wild cardoon

The association among environmental cues, ethylene response, ABA signaling, and reactive oxygen species (ROS) homeostasis in the process of seed dormancy release is nowadays well-established in many species. Alternating temperatures are recognized as one of the main environmental signals determining dormancy release, but their underlying mechanisms are scarcely known. Dry after-ripened wild cardoon achenes germinated poorly at a constant temperature of 20, 15, or 10◦C, whereas germination was stimulated by 80% at alternating temperatures of 20/10◦C. Using an RNA-Seq approach, we identified 23,640 and annotated 14,078 gene transcripts expressed in dry achenes and achenes exposed to constant or alternating temperatures. Transcriptional patterns identified in dry condition included seed reserve and response to dehydration stress genes (i.e., HSPs, peroxidases, and LEAs). At a constant temperature, we observed an upregulation of ABA biosynthesis genes (i.e., NCED9), ABA-responsive genes (i.e., ABI5 and TAP), as well as other genes previously related to physiological dormancy and inhibition of germination. However, the alternating temperatures were associated with the upregulation of ethylene metabolism (i.e., ACO1, 4, and ACS10) and signaling (i.e., EXPs) genes and ROS homeostasis regulators genes (i.e., RBOH and CAT). Accordingly, the ethylene production was twice as high at alternating than at constant temperatures. The presence in the germination medium of ethylene or ROS synthesis and signaling inhibitors reduced significantly, but not completely, germination at 20/10◦C. Conversely, the presence of methyl viologen and salicylhydroxamic acid (SHAM), a peroxidase inhibitor, partially increased germination at constant temperature. Taken together, the present study provides the first insights into the gene expression patterns and physiological response associated with dormancy release at alternating temperatures in wild cardoon (Cynara cardunculus var. sylvestris).Fil: Huarte, Hector Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Lomas de Zamora; ArgentinaFil: Puglia, Giuseppe. D.. Consiglio Nazionale delle Ricerche; ItaliaFil: Prjibelski, Andrey D.. Academia de Ciencias de Rusia.; RusiaFil: Raccuia, Salvatore A.. Consiglio Nazionale delle Ricerche; Itali

MetaGT : A pipeline for de novo assembly of metatranscriptomes with the aid of metagenomic data

While metagenome sequencing may provide insights on the genome sequences and composition of microbial communities, metatranscriptome analysis can be useful for studying the functional activity of a microbiome. RNA-Seq data provides the possibility to determine active genes in the community and how their expression levels depend on external conditions. Although the field of metatranscriptomics is relatively young, the number of projects related to metatranscriptome analysis increases every year and the scope of its applications expands. However, there are several problems that complicate metatranscriptome analysis: complexity of microbial communities, wide dynamic range of transcriptome expression and importantly, the lack of high-quality computational methods for assembling meta-RNA sequencing data. These factors deteriorate the contiguity and completeness of metatranscriptome assemblies, therefore affecting further downstream analysis. Here we present MetaGT, a pipeline for de novo assembly of metatranscriptomes, which is based on the idea of combining both metatranscriptomic and metagenomic data sequenced from the same sample. MetaGT assembles metatranscriptomic contigs and fills in missing regions based on their alignments to metagenome assembly. This approach allows to overcome described complexities and obtain complete RNA sequences, and additionally estimate their abundances. Using various publicly available real and simulated datasets, we demonstrate that MetaGT yields significant improvement in coverage and completeness of metatranscriptome assemblies compared to existing methods that do not exploit metagenomic data. The pipeline is implemented in NextFlow and is freely available fromhttps://github.com/ablab/metaGT.Peer reviewe

The molecular characterisation of Escherichia coli K1 isolated from neonatal nasogastric feeding tubes

Background: The most common cause of Gram-negative bacterial neonatal meningitis is E. coli K1. It has a mortality rate of 10–15%, and neurological sequelae in 30– 50% of cases. Infections can be attributable to nosocomial sources, however the pre-colonisation of enteral feeding tubes has not been considered as a specific risk factor. Methods: Thirty E. coli strains, which had been isolated in an earlier study, from the residual lumen liquid and biofilms of neonatal nasogastric feeding tubes were genotyped using pulsed-field gel electrophoresis, and 7-loci multilocus sequence typing. Potential pathogenicity and biofilm associated traits were determined using specific PCR probes, genome analysis, and in vitro tissue culture assays. Results: The E. coli strains clustered into five pulsotypes, which were genotyped as sequence types (ST) 95, 73, 127, 394 and 2076 (Achman scheme). The extra-intestinal pathogenic E. coli (ExPEC) phylogenetic group B2 ST95 serotype O1:K1:NM strains had been isolated over a 2 week period from 11 neonates who were on different feeding regimes. The E. coli K1 ST95 strains encoded for various virulence traits associated with neonatal meningitis and extracellular matrix formation. These strains attached and invaded intestinal, and both human and rat brain cell lines, and persisted for 48 h in U937 macrophages. E. coli STs 73, 394 and 2076 also persisted in macrophages and invaded Caco-2 and human brain cells, but only ST394 invaded rat brain cells. E. coli ST127 was notable as it did not invade any cell lines. Conclusions: Routes by which E. coli K1 can be disseminated within a neonatal intensive care unit are uncertain, however the colonisation of neonatal enteral feeding tubes may be one reservoir source which could constitute a serious health risk to neonates following ingestion

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis

Understanding complex dynamics of behavioral, neurochemical and transcriptomic changes induced by prolonged chronic unpredictable stress in zebrafish

Stress-related neuropsychiatric disorders are widespread, debilitating and often treatment-resistant illnesses that represent an urgent unmet biomedical problem. Animal models of these disorders are widely used to study stress pathogenesis. A more recent and historically less utilized model organism, the zebrafish (Danio rerio), is a valuable tool in stress neuroscience research. Utilizing the 5-week chronic unpredictable stress (CUS) model, here we examined brain transcriptomic profiles and complex dynamic behavioral stress responses, as well as neurochemical alterations in adult zebrafish and their correction by chronic antidepressant, fluoxetine, treatment. Overall, CUS induced complex neurochemical and behavioral alterations in zebrafish, including stable anxiety-like behaviors and serotonin metabolism deficits. Chronic fluoxetine (0.1 mg/L for 11 days) rescued most of the observed behavioral and neurochemical responses. Finally, whole-genome brain transcriptomic analyses revealed altered expression of various CNS genes (partially rescued by chronic fluoxetine), including inflammation-, ubiquitin- and arrestin-related genes. Collectively, this supports zebrafish as a valuable translational tool to study stress-related pathogenesis, whose anxiety and serotonergic deficits parallel rodent and clinical studies, and genomic analyses implicate neuroinflammation, structural neuronal remodeling and arrestin/ubiquitin pathways in both stress pathogenesis and its potential therapy. © 2020, The Author(s).The research was supported by the Russian Science Foundation (RSF) Grant 19‐15‐00053. KAD is supported by the President of Russia Graduate Fellowship, the Special Rector’s Productivity Fellowship for SPSU PhD Students, and the Russian Foundation for Basic Research (RFBR) grant 18‐34‐00996. ADP was supported by St. Petersburg University (project ID 51555422). The research team was supported by St. Petersburg State University state budgetary funds (project ID 51130521). AVK is the Chair of the International Zebrafish Neuroscience Research Consortium (ZNRC) and President of the International Stress and Behavior Society (ISBS, www.stress-and-behavior.com) that coordinated this collaborative multi-laboratory project. The consortium provided a collaborative idea exchange platform for this study. It is not considered as an affiliation, and did not fund the study. AVK is supported by the Southwest University Zebrafish Platform Construction Fund. TGA’s research is supported by the budgetary funding for basic research from the Scientific Research Institute of Physiology and Basic Medicine (AAAA-A16-116021010228-0, Novosibirsk, Russia). This study utilized equipment of the Core Facilities Centre “Centre for Molecular and Cell Technologies” of St. Petersburg State University. The funders had no role in the design, analyses, and interpretation of the submitted study, or decision to publish

Accurate isoform discovery with IsoQuant using long reads

Publisher Copyright: © 2023, The Author(s).Annotating newly sequenced genomes and determining alternative isoforms from long-read RNA data are complex and incompletely solved problems. Here we present IsoQuant—a computational tool using intron graphs that accurately reconstructs transcripts both with and without reference genome annotation. For novel transcript discovery, IsoQuant reduces the false-positive rate fivefold and 2.5-fold for Oxford Nanopore reference-based or reference-free mode, respectively. IsoQuant also improves performance for Pacific Biosciences data.Peer reviewe

A spatially resolved brain region- and cell type-specific isoform atlas of the postnatal mouse brain

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 (www.isoformAtlas.com). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity.Pattern Recognition and Bioinformatic