Rapid and accurate demultiplexing of direct RNA nanopore sequencing datasets with SeqTagger.

Nanopore direct RNA sequencing (DRS) enables direct measurement of RNA molecules, including their native RNA modifications, without prior conversion to cDNA. However, commercial methods for molecular barcoding of multiple DRS samples are lacking, and community-driven efforts, such as DeePlexiCon, are not compatible with newer RNA chemistry flowcells and the latest-generation GPU cards. To overcome these limitations, we introduce SeqTagger, a rapid and robust method that can demultiplex direct RNA sequencing datasets with 99% precision and 95% recall.

Charting the epitranscriptomic landscape across RNA biotypes using native RNA nanopore sequencing.

RNA modifications are conserved chemical features found in all domains of life and across diverse RNA biotypes, shaping gene expression profiles and enabling rapid responses to environmental changes. Their broad chemical diversity and dynamic nature pose significant challenges for studying them comprehensively. These limitations can now be addressed through direct RNA nanopore sequencing (DRS), which allows simultaneous identification of diverse RNA modification types at single-molecule and single-nucleotide resolution.

Native RNA nanopore sequencing reveals antibiotic-induced loss of rRNA modifications in the A- and P-sites.

The biological relevance and dynamics of mRNA modifications have been extensively studied; however, whether rRNA modifications are dynamically regulated, and under which conditions, remains unclear. Here, we systematically characterize bacterial rRNA modifications upon exposure to diverse antibiotics using native RNA nanopore sequencing. To identify significant rRNA modification changes, we develop NanoConsensus, a novel pipeline that is robust across RNA modification types, stoichiometries and coverage, with very low false positive rates, outperforming all individual algorithms tested.

GLP-1 and GIP agonism has no direct actions in human hepatocytes or hepatic stellate cells.

The use of incretin agonists for managing metabolic dysfunction-associated steatohepatitis (MASH) is currently experiencing considerable interest. However, whether these compounds have a direct action on MASH is still under debate. This study aims to investigate whether GLP-1R/GIPR agonists act directly in hepatocytes and hepatic stellate cells (HSCs).

Dynamic rRNA modifications as a source of ribosome heterogeneity.

Ribosomal RNAs (rRNA) are the most abundant RNA molecules in almost all cell types. The general consensus in the field is that rRNA modifications are largely species-specific, with most previous works and databases solely stratifying modifications by the species of origin, without taking other levels of complexity into account. However, new evidence has emerged suggesting dynamic rRNA modifications may have additional layers of complexity and might play an important role in development and disease.

Natural-Looking Ears after Otoplasty with a Modified Anterior Scoring Technique.

The correction of prominent ears is a rewarding operation. When choosing a surgical technique, we prefer a cartilage-sparing technique to a cartilage-splitting technique to achieve natural-looking long-term results. In this article, we present our preferred technique of an anterior scoring technique combined with concha-mastoid sutures step by step.

Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus.

The genomes of positive-sense (+) single-stranded RNA (ssRNA) viruses are believed to be subjected to a wide range of RNA modifications. In this study, we focused on the chikungunya virus (CHIKV) as a model (+) ssRNA virus to study the landscape of viral RNA modification in infected human cells. Among the 32 distinct RNA modifications analysed by mass spectrometry, inosine was found enriched in the genomic CHIKV RNA.

Inhibition of hepatic p63 ameliorates steatohepatitis with fibrosis in mice.

Objective: p63 is a transcription factor involved in multiple biological functions. In the liver, the TAp63 isoform induces lipid accumulation in hepatocytes. However, the role of liver TAp63 in the progression of metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis is unknown.

Mitochondrial antiviral signaling protein enhances MASLD progression through the ERK/TNFα/NFκβ pathway.

Background And Aims: Mitochondrial antiviral signaling protein (MAVS) is a critical regulator that activates the host's innate immunity against RNA viruses, and its signaling pathway has been linked to the secretion of proinflammatory cytokines. However, the actions of MAVS on inflammatory pathways during the development of metabolic dysfunction-associated steatotic liver disease (MASLD) have been little studied.

Approach And Results: Liver proteomic analysis of mice with genetically manipulated hepatic p63, a transcription factor that induces liver steatosis, revealed MAVS as a target downstream of p63.

Spectral libraries from nucleobases and deoxyribonucleosides facilitate the identification of ribonucleosides by nano-flow liquid chromatography-tandem mass spectrometry.

Rationale: The study addresses the challenge of identifying RNA post-transcriptional modifications when commercial standards are not available to generate reference spectral libraries. It proposes employing homologous nucleobases and deoxyribonucleosides as alternative reference spectral libraries to aid in identifying modified ribonucleosides and distinguishing them from their positional isomers when the standards are unavailable.

Methods: Complete sets of ribonucleoside, deoxyribonucleoside and nucleobase standards were analyzed using high-performance nano-flow liquid chromatography coupled to an Orbitrap Eclipse Tribrid mass spectrometer.

N-methyladenosine modification is not a general trait of viral RNA genomes.

Despite the nuclear localization of the mA machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively mA-modified. However, these findings are mostly based on mA-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of mA in CHIKV and DENV RNAs.

Reclassification of and species as members of the genus Bae . 2007 and emended description of the genus .

The recently proposed genus Deshmukh and Oren 2023 is illegitimate because it includes the type species of the genera Bae . 2007 and Yoon and Oh 2012, contravening Rule 51b(1) of the International Code of Nomenclature of Prokaryotes. As Bae .

Comprehensive map of ribosomal 2'-O-methylation and C/D box snoRNAs in Drosophila melanogaster.

During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs).

The lncRNA Snhg11, a new candidate contributing to neurogenesis, plasticity, and memory deficits in Down syndrome.

Down syndrome (DS) stands as the prevalent genetic cause of intellectual disability, yet comprehensive understanding of its cellular and molecular underpinnings remains limited. In this study, we explore the cellular landscape of the hippocampus in a DS mouse model, the Ts65Dn, through single-nuclei transcriptional profiling. Our findings demonstrate that trisomy manifests as a highly specific modification of the transcriptome within distinct cell types.

p63 controls metabolic activation of hepatic stellate cells and fibrosis via an HER2-ACC1 pathway.

The p63 protein has pleiotropic functions and, in the liver, participates in the progression of nonalcoholic fatty liver disease (NAFLD). However, its functions in hepatic stellate cells (HSCs) have not yet been explored. TAp63 is induced in HSCs from animal models and patients with liver fibrosis and its levels positively correlate with NAFLD activity score and fibrosis stage.

Hypothalamic free fatty acid receptor-1 regulates whole-body energy balance.

Objective: Free fatty acid receptor-1 (FFAR1) is a medium- and long-chain fatty acid sensing G protein-coupled receptor that is highly expressed in the hypothalamus. Here, we investigated the central role of FFAR1 on energy balance.

Methods: Central FFAR1 agonism and virogenic knockdown were performed in mice.

The lncRNA Snhg11, a new candidate contributing to neurogenesis, plasticity and memory deficits in Down syndrome.

Down syndrome (DS) stands as the prevalent genetic cause of intellectual disability, yet comprehensive understanding of its cellular and molecular underpinnings remains limited. In this study, we explore the cellular landscape of the hippocampus in a DS mouse model through single-nuclei transcriptional profiling. Our findings demonstrate that trisomy manifests as a highly specific modification of the transcriptome within distinct cell types.

Hepatocyte-specific O-GlcNAc transferase downregulation ameliorates nonalcoholic steatohepatitis by improving mitochondrial function.

Objective: O-GlcNAcylation is a post-translational modification that directly couples the processes of nutrient sensing, metabolism, and signal transduction, affecting protein function and localization, since the O-linked N-acetylglucosamine moiety comes directly from the metabolism of glucose, lipids, and amino acids. The addition and removal of O-GlcNAc of target proteins are mediated by two highly conserved enzymes: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase (OGA), respectively. Deregulation of O-GlcNAcylation has been reported to be associated with various human diseases such as cancer, diabetes, and cardiovascular diseases.

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment.

Nanopore Direct RNA Sequencing Data Processing and Analysis Using MasterOfPores.

This chapter describes MasterOfPores v.2 (MoP2), an open-source suite of pipelines for processing and analyzing direct RNA Oxford Nanopore sequencing data. The MoP2 relies on the Nextflow DSL2 framework and Linux containers, thus enabling reproducible data analysis in transcriptomic and epitranscriptomic studies.