ASTRO: Automated Spatial Whole-Transcriptome RNA-Expression Workflow

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 27, 2025

Abstract Motivation Despite significant advances in spatial transcriptomics, the analysis of formalin-fixed paraffin-embedded (FFPE) tissues, which constitute most clinically available samples, remains challenging. Additionally, capturing both coding and noncoding RNAs a context poses challenges. We recently introduced Patho-DBiT, technology designed to address these unmet needs. However, marked differences between Patho-DBiT existing transcriptomics protocols necessitate specialized computational tools for comprehensive whole-transcriptome FFPE samples. Results Here, we present ASTRO, an automated pipeline developed process data. In addition supporting standard datasets, ASTRO is optimized analyses enabling detection various RNA species, including non-coding such as miRNAs. To compensate reduced quality incorporates filtering step optimizes barcode calling, increasing mapping rate. These optimizations allow spatially quantify species entire transcriptome achieve robust performance Availability Codes are at GitHub ( https://github.com/gersteinlab/ASTRO ).

Language: Английский

---

Stress-Related LncRNAs and Their Roles in Diabetes and Diabetic Complications

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(5), P. 2194 - 2194

Published: Feb. 28, 2025

Diabetes mellitus (DM) is a chronic metabolic disorder and one of the most significant global health burdens worldwide. Key pathophysiological mechanisms underlying its onset associated complications include hyperglycemia-related stresses, such as oxidative stress endoplasmic reticulum (ER stress). Long non-coding RNAs (lncRNAs), defined RNA transcripts longer than 200 nucleotides lacking protein-coding capacity, play crucial roles in various biological processes have emerged regulators pathogenesis diabetes. This review provides comprehensive overview lncRNA biogenesis functional roles, emphasizing recent findings that link stress-related lncRNAs to diabetic pathology complications. Also, we discuss how influence diabetes by modulating pathways involved cell death, proliferation, inflammation, fibrosis, which contribute pancreatic β dysfunction, insulin resistance, nephropathy, retinopathy. By analyzing current research, aim enhance understanding involvement while identifying potential therapeutic targets guiding future research directions elucidate complex this pervasive condition.

Language: Английский

---

Evaluation of siRNA dependent knockdowns of drug metabolizing enzymes in multi-well array culture of primary human hepatocyte spheroids for estimation of fraction metabolized

Drug Metabolism and Disposition, Journal Year: 2025, Volume and Issue: 53(4), P. 100062 - 100062

Published: March 10, 2025

The determination of the relative contribution different drug-metabolizing enzymes to metabolism slowly metabolized compounds is a challenging task. quantification low compound turnover in standard vitro systems, such as liver microsomes or hepatocyte suspension cultures, can be difficult. Thus, use long-term models, HepatoPac (BioIVT) spheroids, has been suggested. Inhibitors cytochrome P450 (P450) enzymes, most important group represent current evaluate route drug metabolism. However, inhibition systems spheroid models may technically due limited stability some commonly used inhibitors. Small interfering RNA (siRNA)-dependent knockdown cultures primary human hepatocytes represents novel alternative established methods. In study, we report successful attenuation CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoforms via siRNA on gene expression, well functional level, for at least 7 days. analysis revealed that knockdowns had only minor effects overall transcriptome. They also showed acceptable selectivity towards except CYP2C19. Applicability system fraction clearance substances was examined using 6 by P450s. By introducing siRNA-dependent phenotypically relevant hope provide elucidate pathways vitro. SIGNIFICANCE STATEMENT: RNA-mediated were shown effective representing reaction phenotyping. This method potential improve assessment pharmacokinetic variability victim drug-drug interaction risks enzyme polymorphism inhibition/induction with more confidence, particularly candidates. Furthermore, small cell viability transcriptome observed which implies this useful deconvoluting toxicity caused metabolites.

Language: Английский

---

Defining expansions and perturbations to the RNA polymerase III transcriptome and epitranscriptome by modified direct RNA nanopore sequencing

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: March 12, 2025

ABSTRACT RNA polymerase III (Pol III) transcribes cytosolic transfer RNAs (tRNAs) and other non-coding (ncRNAs) essential to cellular function. However, many aspects of Pol transcription processing, including modifications, remain poorly understood, mainly due a lack available sensitive systematic methods for their analysis. Here, we present DRAP3R (Direct Read Analysis Polymerase transcribed RNAs), modified nanopore direct sequencing approach analysis framework that enables the specific capture nascent RNAs. Applying distinct cell types, identify previously unconfirmed tRNA genes novel RNAs, thus expanding known transcriptome. Critically, also discrimination between co- post-transcriptional modifications such as pseudouridine (Ψ) N 6 -methyladenosine (m A) at single-nucleotide resolution across all examined transcript types reveals differential Ψ installation patterns isodecoders ncRNAs. Finally, applying epithelial cells infected with Herpes Simplex Virus Type 1 an extensive remodelling both transcriptome epitranscriptome. Our findings establish powerful tool systematically studying in diverse contexts.

Language: Английский

---

Gap-App: A Sex-Distinct AI-Based Predictor for Pancreatic Ductal Adenocarcinoma Survival as A Web Application Open to Patients and Physicians

Cancer Letters, Journal Year: 2025, Volume and Issue: unknown, P. 217689 - 217689

Published: April 1, 2025

Language: Английский

---

RIBOSS detects novel translational events by combining long- and short-read transcriptome and translatome profiling

Briefings in Bioinformatics, Journal Year: 2025, Volume and Issue: 26(2)

Published: March 1, 2025

Ribosome profiling is a high-throughput sequencing technique that captures the positions of translating ribosomes on RNAs. Recent advancements in ribosome include achieving highly phased footprints for plant translatomes and more recently bacterial translatomes. This substantially increases specificity detecting open reading frames (ORFs) can be translated, such as small ORFs located upstream downstream annotated ORFs. However, most genomes (e.g. genomes) lack annotations transcription start termination sites. hinders systematic discovery novel 'untranslated' regions data. Here, we develop new computational pipeline called RIBOSS to discover noncanonical assess their translational potential against The Python modules are versatile, use them analyse both prokaryotic eukaryotic We present resulting list with high Homo sapiens, Arabidopsis thaliana, Salmonella enterica. further illustrate utility when studying organisms incomplete transcriptome annotations. leverage long-read short-read data reference-guided assembly events assembled S. In sum, first integrated ORF detection assessment incorporates long- technologies investigate translation. freely available at https://github.com/lcscs12345/riboss.

Language: Английский

---

Facilitating genome annotation using ANNEXA and long-read RNA sequencing

Published: April 20, 2025

Abstract With the advent of complete genome assemblies, annotation has become essential for functional interpretation genomic data. Long-read RNA sequencing (LR-RNAseq) technologies have significantly improved transcriptome by enabling full-length transcript reconstruction both coding and non-coding RNAs. However, challenges such as fragmentation incomplete isoform representation persist, highlighting need robust quality control (QC) strategies. This study presents an updated version ANNEXA, a pipeline designed to enhance using LR-RNAseq data while also providing QC reconstructed genes transcripts. ANNEXA integrates two tools, StringTie2 Bambu, applying stringent filtering criteria improve accuracy. It incorporates deep learning models evaluate transcription start sites (TSSs) employs tool FEELnc systematic long RNAs (lncR-NAs). Additionally, offers intuitive visualizations comparative analyses repertoires. Benchmarking against multiple reference annotations revealed distinct patterns sensitivity precision known novel transcripts mRNAs lncRNAs. To demonstrate its utility, was applied in oncology involving human eight canine cancer cell lines. The successfully identified across species, expanding catalog protein-coding lncRNA species. Implemented Nextflow scalability reproducibility, is available open-source tool: https://github.com/IGDRion/ANNEXA .

Language: Английский

---

Identifying non-coding variant effects at scale via machine learning models of cis-regulatory reporter assays

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: April 18, 2025

Abstract The inability to interpret the functional impact of non-coding variants has been a major impediment in promise precision medicine. While high-throughput experimental approaches such as Massively Parallel Reporter Assays (MPRAs) have made progress identifying causal and their underlying molecular mechanisms, these tools cannot exhaustively measure variant effects genome-wide. Here we present MPAC, an ensemble machine-learning models trained on MPRA data that provides accurate scalable prediction cis-regulatory variants. Using MPAC predict allelic for 575M single nucleotide (SNVs) across diverse applications, including complex trait genetics, clinical tumor sequencing, evolutionary analyses, saturation mutagenesis. We find predictions match performance empirical MPRAs trait-associated alleles. demonstrate utility by applying it ClinVar, pathogenic variation with higher accuracy than other sequence-to-function models. also nominate 1,892 candidate cancer drivers predicting somatic SNVs COSMIC database. Next, evaluate population-level genetic all 514M gnomAD, quantifying relationship between regulatory function constraint. Finally, generate prospective maps using in-silico mutagenesis 18,658 human promoters, observing widespread selection against predicted disrupt promoter activity. Collectively, this study establishes value comprehensive, publicly available resource interpretation.

Language: Английский

---

PPARα Genetic Deletion Reveals Global Transcriptional Changes in the Brain and Exacerbates Cerebral Infarction in a Mouse Model of Stroke

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(9), P. 4082 - 4082

Published: April 25, 2025

Ischemic stroke is a leading cause of death and disability worldwide. Currently, there an unmet clinical need for pharmacological treatments that can improve ischemic outcomes. In this study, we investigated the role brain peroxisome proliferator-activated receptor alpha (PPARα) in pathophysiology. We used well-established model cerebral ischemia PPARα transgenic mice conducted RNA sequencing (RNA-seq) mouse brains harvested 48 h post-middle artery occlusion (MCAO). knockout (KO) increased infarct size following stroke, indicating protective ischemia. Our RNA-seq analysis showed KO altered expression genes with known roles also identified many other differentially expressed (DEGs) upon loss correlated our model. Gene set enrichment (GSEA) Ontology (GO) revealed upregulation gene signatures positive regulation leukocyte proliferation, apoptotic processes, acute-phase response, cellular component disassembly KO. addition, pathway data TNFα signaling, IL6/STAT3 epithelial–mesenchymal transition (EMT) were brains. study highlights as attractive drug target due to its transcriptional inflammation-, apoptosis-, EMT-related tissue

Language: Английский

---

The 2025 Nucleic Acids Research database issue and the online molecular biology database collection

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 53(D1), P. D1 - D9

Published: Dec. 10, 2024

The 2025 Nucleic Acids Research database issue contains 185 papers spanning biology and related areas. Seventy three new databases are covered, while resources previously described in the account for 101 update articles. Databases most recently published elsewhere a further 11 papers. acid include EXPRESSO multi-omics of 3D genome structure (this issue's chosen Breakthrough Resource Article) NAIRDB Fourier transform infrared data. New protein predictions human isoforms at ASpdb viral proteins BFVD. UniProt, Pfam InterPro have all provided updates: metabolism signalling covered by descriptions STRING, KEGG CAZy, updated microbe-oriented Enterobase, VFDB PHI-base. Biomedical research is supported, among others, ClinVar, PubChem DrugMAP. Genomics-related Ensembl, UCSC Genome Browser dbSNP. plant cover Solanaceae (SolR) Asteraceae (AMIR) families an from NCBI Taxonomy also features. Database Issue freely available on website (https://academic.oup.com/nar). At NAR online Molecular Biology Collection (http://www.oxfordjournals.org/nar/database/c/), 932 entries been reviewed last year, 74 added 226 discontinued URLs eliminated bringing current total to 2236 databases.

Language: Английский

---