RESUMEN
The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. 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. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.
Asunto(s)
Perfilación de la Expresión Génica , RNA-Seq , Humanos , Animales , Ratones , RNA-Seq/métodos , Perfilación de la Expresión Génica/métodos , Transcriptoma , Análisis de Secuencia de ARN/métodos , Anotación de Secuencia Molecular/métodosRESUMEN
Pseudomonas aeruginosa is a major cause of nosocomial infections and the leading cause of chronic lung infections in cystic fibrosis and chronic obstructive pulmonary disease patients. Antibiotic treatment remains challenging because P. aeruginosa is resistant to high concentrations of antibiotics and has a remarkable ability to acquire mutations conferring resistance to multiple groups of antimicrobial agents. Here we report that when P. aeruginosa is plated on ciprofloxacin (cipro) plates, the majority of cipro-resistant (ciproR) colonies observed at and after 48 h of incubation carry mutations in genes related to the Stringent Response (SR). Mutations in one of the major SR components, spoT, were present in approximately 40% of the ciproR isolates. Compared to the wild-type strain, most of these isolates had decreased growth rate, longer lag phase and altered intracellular ppGpp content. Also, 75% of all sequenced mutations were insertions and deletions, with short deletions being the most frequently occurring mutation type. We present evidence that most of the observed mutations are induced on the selective plates in a subpopulation of cells that are not instantly killed by cipro. Our results suggests that the SR may be an important contributor to antibiotic resistance acquisition in P. aeruginosa.
Asunto(s)
Ciprofloxacina , Infecciones por Pseudomonas , Humanos , Ciprofloxacina/farmacología , Pseudomonas aeruginosa/genética , Infecciones por Pseudomonas/tratamiento farmacológico , Antibacterianos/farmacología , Placas ÓseasRESUMEN
The cancer hazard associated with lifetime exposure to radiofrequency radiation (RFR) was examined in Sprague Dawley (SD) rats at the Ramazzini Institute (RI), Italy. There were increased incidences of gliomas and cardiac schwannomas. The translational relevance of these rare rat tumors for human disease is poorly understood. We examined the genetic alterations in RFR-derived rat tumors through molecular characterization of important cancer genes relevant for human gliomagenesis. A targeted next-generation sequencing (NGS) panel was designed for rats based on the top 23 orthologous human glioma-related genes. Single-nucleotide variants (SNVs) and small insertion and deletions (indels) were characterized in the rat gliomas and cardiac schwannomas. Translational relevance of these genetic alterations in rat tumors to human disease was determined through comparison with the Catalogue of Somatic Mutations in Cancer (COSMIC) database. These data suggest that rat gliomas resulting from life-time exposure to RFR histologically resemble low grade human gliomas but surprisingly no mutations were detected in rat gliomas that had homology to the human IDH1 p.R132 or IDH2 p.R172 suggesting that rat gliomas are primarily wild-type for IDH hotspot mutations implicated in human gliomas. The rat gliomas appear to share some genetic alterations with IDH1 wildtype human gliomas and rat cardiac schwannomas also harbor mutations in some of the queried cancer genes. These data demonstrate that targeted NGS panels based on tumor specific orthologous human cancer driver genes are an important tool to examine the translational relevance of rodent tumors resulting from chronic/life-time rodent bioassays.
Asunto(s)
Neoplasias Encefálicas , Glioma , Neurilemoma , Exposición a la Radiación , Humanos , Ratas , Animales , Ratas Sprague-Dawley , Glioma/genética , Glioma/patología , Mutación , Neurilemoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Isocitrato Deshidrogenasa/genética , Neoplasias Encefálicas/patologíaRESUMEN
Maintenance of the mitochondrial inner membrane potential (ΔΨM) is critical for many aspects of mitochondrial function, including mitochondrial protein import and ion homeostasis. While ΔΨM loss and its consequences are well studied, little is known about the effects of increased ΔΨM. In this study, we used cells deleted of ATPIF1, a natural inhibitor of the hydrolytic activity of the ATP synthase, as a genetic model of mitochondrial hyperpolarization. Our data show that chronic ΔΨM increase leads to nuclear DNA hypermethylation, regulating transcription of mitochondria, carbohydrate and lipid metabolism genes. Surprisingly, remodeling of phospholipids, but not metabolites or redox changes, mechanistically links the ΔΨM to the epigenome. These changes were also observed upon chemical exposures and reversed by decreasing the ΔΨM, highlighting them as hallmark adaptations to chronic mitochondrial hyperpolarization. Our results reveal the ΔΨM as the upstream signal conveying the mitochondrial status to the epigenome to regulate cellular biology, providing a new framework for how mitochondria can influence health outcomes in the absence of canonical dysfunction.
RESUMEN
Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25-2.5 µM cisplatin (CisPt) or 12.5-100 µM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 µM CisPt from 3 to 96 h expressing 2000-5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity.
Asunto(s)
Células Epiteliales , Túbulos Renales Proximales , Humanos , Ratones , Animales , RNA-Seq , Línea Celular , Túbulos Renales Proximales/metabolismo , Cisplatino/metabolismoRESUMEN
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.
RESUMEN
The selection and firing of DNA replication origins play key roles in ensuring that eukaryotes accurately replicate their genomes. This process is not well documented in plants due in large measure to difficulties in working with plant systems. We developed a new functional assay to label and map very early replicating loci that must, by definition, include at least a subset of replication origins. Arabidopsis (Arabidopsis thaliana) cells were briefly labeled with 5-ethynyl-2'-deoxy-uridine, and nuclei were subjected to two-parameter flow sorting. We identified more than 5500 loci as initiation regions (IRs), the first regions to replicate in very early S phase. These were classified as strong or weak IRs based on the strength of their replication signals. Strong initiation regions were evenly spaced along chromosomal arms and depleted in centromeres, while weak initiation regions were enriched in centromeric regions. IRs are AT-rich sequences flanked by more GC-rich regions and located predominantly in intergenic regions. Nuclease sensitivity assays indicated that IRs are associated with accessible chromatin. Based on these observations, initiation of plant DNA replication shows some similarity to, but is also distinct from, initiation in other well-studied eukaryotic systems.
Asunto(s)
Arabidopsis/metabolismo , Cromatina/metabolismo , ADN de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN de Plantas/fisiología , Origen de Réplica/genética , Origen de Réplica/fisiologíaRESUMEN
Formaldehyde (FA) is a simple biological aldehyde that is produced inside cells by several processes such as demethylation of DNA and proteins, amino acid metabolism, lipid peroxidation and one carbon metabolism (1-C). Although accumulation of excess FA in cells is known to be cytotoxic, it is unknown if an increase in FA level might be associated with mitochondrial dysfunction. We choose to use primary human fibroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might affect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and different cellular parameters were studied. Elevation in intracellular FA level was achieved and was found to be cytotoxic by virtue of both apoptosis and necrosis and was accompanied by both G2/M arrest and reduction in the time spent in S phase. A gene expression assessment by microarray analysis revealed FA affected FSK cells by altering expression of many genes including genes involved in mitochondrial function and electron transport. We were surprised to observe increased DNA double-strand breaks (DSBs) in mitochondria after exposure to FA, as revealed by accumulation of γH2A.X and 53BP1 at mitochondrial DNA foci. This was associated with mitochondrial structural rearrangements, loss of mitochondrial membrane potential and activation of mitophagy. Collectively, these results indicate that an increase in the cellular level of FA can trigger mitochondrial DNA double-strand breaks and dysfunction.
Asunto(s)
Daño del ADN/genética , Fibroblastos/metabolismo , Formaldehído/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Apoptosis/genética , Ciclo Celular/genética , Línea Celular , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , ADN Mitocondrial/genética , Humanos , Potencial de la Membrana Mitocondrial/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Eukaryotes use a temporally regulated process, known as the replication timing program, to ensure that their genomes are fully and accurately duplicated during S phase. Replication timing programs are predictive of genomic features and activity and are considered to be functional readouts of chromatin organization. Although replication timing programs have been described for yeast and animal systems, much less is known about the temporal regulation of plant DNA replication or its relationship to genome sequence and chromatin structure. We used the thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to describe, at high-resolution, the genome-wide replication timing program for Arabidopsis (Arabidopsis thaliana) Col-0 suspension cells. We identified genomic regions that replicate predominantly during early, mid, and late S phase, and correlated these regions with genomic features and with data for chromatin state, accessibility, and long-distance interaction. Arabidopsis chromosome arms tend to replicate early while pericentromeric regions replicate late. Early and mid-replicating regions are gene-rich and predominantly euchromatic, while late regions are rich in transposable elements and primarily heterochromatic. However, the distribution of chromatin states across the different times is complex, with each replication time corresponding to a mixture of states. Early and mid-replicating sequences interact with each other and not with late sequences, but early regions are more accessible than mid regions. The replication timing program in Arabidopsis reflects a bipartite genomic organization with early/mid-replicating regions and late regions forming separate, noninteracting compartments. The temporal order of DNA replication within the early/mid compartment may be modulated largely by chromatin accessibility.
Asunto(s)
Arabidopsis/genética , Cromatina/genética , Cromosomas de las Plantas , Momento de Replicación del ADN , Cromatina/metabolismo , Elementos Transponibles de ADN , Citometría de Flujo , Genoma de Planta , Estudio de Asociación del Genoma Completo , Fase S/genética , Análisis de Secuencia de ADN/métodosRESUMEN
5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems.