RESUMEN
The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is the most widely used method for measuring chromatin accessibility. Researchers have included multi-sample replication in ATAC-seq experimental designs. In epigenomic analysis, researchers should measure subtle changes in the peak by considering the read depth of individual samples. It is important to determine whether the peaks of each replication have an integrative meaning for the region of interest observed during multi-sample integration. We developed multi-epigenome sample integration approach for precise peak calling (MESIA), which integrates replication with high representativeness and reproducibility in multi-sample replication and determines the optimal peak. After identifying the reproducibility between all replications, our method integrated multiple samples determined as representative replicates. MESIA detected 6.06 times more peaks, and the value of the peaks was 1.32 times higher than the previously used method. MESIA is a shell-script-based open-source code that provides researchers involved in the epigenome with comprehensive insights.
Asunto(s)
Epigenoma , Secuenciación de Nucleótidos de Alto Rendimiento , Reproducibilidad de los Resultados , Cromatina/genética , Secuenciación de Inmunoprecipitación de Cromatina , Análisis de Secuencia de ADN/métodosRESUMEN
In the nucleus, distinct, discrete spots or regions called "foci" have been identified, each harboring a specific molecular function. Accurate and efficient quantification of these foci is essential for understanding cellular dynamics and signaling pathways. In this study, we present an innovative automated image analysis method designed to precisely quantify subcellular foci within the cell nucleus. Manual foci counting methods can be tedious and time-consuming. To address these challenges, we developed an open-source software that automatically counts the number of foci from the indicated image files. We compared the foci counting efficiency, velocity, accuracy, and convenience of Foci-Xpress with those of other conventional methods in foci-induced models. We can adjust the brightness of foci to establish a threshold. The Foci-Xpress method was significantly faster than other conventional methods. Its accuracy was similar to that of conventional methods. The most significant strength of Foci-Xpress is automation, which eliminates the need for analyzing equipment while counting. This enhanced throughput facilitates comprehensive statistical analyses and supports robust conclusions from experiments. Furthermore, automation completely rules out biases caused by researchers, such as manual errors or daily variations. Thus, Foci-Xpress is a convincing, convenient, and easily accessible focus-counting tool for cell biologists.
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Procesamiento de Imagen Asistido por Computador , Programas Informáticos , Procesamiento de Imagen Asistido por Computador/métodos , AutomatizaciónRESUMEN
Peptidylarginine deiminase (PADI) 2 catalyzes the post-translational conversion of peptidyl-arginine to peptidyl-citrulline in a process called citrullination. However, the precise functions of PADI2 in bone formation and homeostasis remain unknown. In this study, our objective was to elucidate the function and regulatory mechanisms of PADI2 in bone formation employing global and osteoblast-specific Padi2 knockout mice. Our findings demonstrate that Padi2 deficiency leads to the loss of bone mass and results in a cleidocranial dysplasia (CCD) phenotype with delayed calvarial ossification and clavicular hypoplasia, due to impaired osteoblast differentiation. Mechanistically, Padi2 depletion significantly reduces RUNX2 levels, as PADI2-dependent stabilization of RUNX2 protected it from ubiquitin-proteasomal degradation. Furthermore, we discovered that PADI2 binds to RUNX2 and citrullinates it, and identified ten PADI2-induced citrullination sites on RUNX2 through high-resolution LC-MS/MS analysis. Among these ten citrullination sites, the R381 mutation in mouse RUNX2 isoform 1 considerably reduces RUNX2 levels, underscoring the critical role of citrullination at this residue in maintaining RUNX2 protein stability. In conclusion, these results indicate that PADI2 plays a distinct role in bone formation and osteoblast differentiation by safeguarding RUNX2 against proteasomal degradation. In addition, we demonstrate that the loss-of-function of PADI2 is associated with CCD, thereby providing a new target for the treatment of bone diseases.
Asunto(s)
Citrulinación , Displasia Cleidocraneal , Animales , Ratones , Osteogénesis , Cromatografía Liquida , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Espectrometría de Masas en Tándem , Ratones NoqueadosRESUMEN
Although the normal physiological level of oxidative stress is beneficial for maintaining bone homeostasis, imbalance between reactive oxygen species (ROS) production and antioxidant defense can cause various bone diseases. The purpose of this study was to determine whether nicotinamide (NAM), an NAD+ precursor, can support the maintenance of bone homeostasis by regulating osteoblasts. Here, we found that NAM enhances osteoblast differentiation and mitochondrial metabolism. NAM increases the expression of antioxidant enzymes, which is due to increased FOXO3A transcriptional activity via SIRT3 activation. NAM has not only a preventive effect against weak and chronic oxidative stress but also a therapeutic effect against strong and acute exposure to H2O2 in osteoblast differentiation. Collectively, the results indicate that NAM increases mitochondrial biogenesis and antioxidant enzyme expression through activation of the SIRT3-FOXO3A axis, which consequently enhances osteoblast differentiation. These results suggest that NAM could be a potential preventive or therapeutic agent for bone diseases caused by ROS.
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Enfermedades Óseas , Sirtuina 3 , Humanos , Antioxidantes/farmacología , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Niacinamida/farmacología , Sirtuina 3/genética , Sirtuina 3/metabolismo , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo , Osteoblastos/metabolismoRESUMEN
Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.
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Senescencia Celular/efectos de los fármacos , Quimiocinas CC/metabolismo , Peróxido de Hidrógeno/farmacología , FN-kappa B/metabolismo , Arginina Deiminasa Proteína-Tipo 2/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL5/antagonistas & inhibidores , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Quimiocina CCL7/antagonistas & inhibidores , Quimiocina CCL7/genética , Quimiocina CCL7/metabolismo , Quimiocinas CC/antagonistas & inhibidores , Quimiocinas CC/genética , Daño del ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Ratones , Osteoblastos/citología , Osteoblastos/metabolismo , Arginina Deiminasa Proteína-Tipo 2/antagonistas & inhibidores , Arginina Deiminasa Proteína-Tipo 2/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The complete mitochondrial (mt) genome of Trachemys scripta elegans in Korea was sequenced and characterized. The mt genome is constituted of 37 genes (13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes) and a noncoding control region. Phylogenetic analysis based on the complete mt genome showed that T. s. elegans Korea has closer relationship with T. scripta Canada than T. s. elegans China. This is the first complete mt genome from T. s. elegans in Korea, which provides information for biogeographical studies and management plan for invasive species.
RESUMEN
The complete mitochondrial (mt) genome of Trachemys scripta ssp. in Korea was sequenced and characterized. The mt genome is constituted of 37 genes (13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes) and a control region. Phylogenetic analysis based on the complete mt genome showed that the unidentified turtle had the mt genome closely related to that of T. s. elegans, though it had distinct morphology compared to T. s. elegans. This study can provide information for biogeographical studies and management plan for invasive species.
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The complete mitochondrial (mt) genome of Trachemys scripta troostii was sequenced and was characterized, which comprised 37 genes (13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs) and a non-coding control region. Phylogenetic analysis based on the full mt genome indicated that T. s. troostii was more closely related to T. scripta from Canada than to T. s. elegans from China or T. s. scripta fom China. This is the first complete mt genome from T. s. troostii, which provides data for further study of phylogeny in Emydidae.
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The complete mitochondrial (mt) genome of Hynobius unisacculus was sequenced and characterized. The circular mt genome constituted of 37 genes (13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs) and a non-coding region (NCR). Phylogenetic analysis based on the full mt genome sequences confirmed that H. unisacculus was closely related to Hynobius leechii rather than other Hynobius species. This is the first completed mt genome from H. unisacculus, which provides data for further study of phylogeny in Hynobiidaes.
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The complete mitochondrial (mt) genome of Lithobates catesbeianus was sequenced and characterized. The circular mt genome was constituted of of 37 genes (13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs) and a non-coding region (NCR). Phylogenetic analysis based on the full mt genome sequences confirmed that among the genus Lithobates, L. catesbeianus Korea is included in a monophyletic group with L. catesbeianus China, but not with either L. catesbeianus Japan or L. catesbeianus Canada. This is the first completed mt genome from L. catesbeianus Korea, which provide data for further study of phylogeny in Lithobates spp. that have been introduced into a number of different countries originally from North America.
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We sequenced the complete mitochondrial (mt) genome of Gavia arctica. The circular mt genome is 17,065 bp long, consisting of 37 genes (13 proteins, 22 transfer RNAs, and two ribosomal RNAs) and a control region. Phylogenetic analysis based on the full mt genome sequences confirmed that the genus Gavia is a monophyletic group, containing the G. stellata, G. arctica, and G. pacifica. These data can provide insights into the phylogenetic relationships for inferring the pattern and degree of mt genome evolution among the loon species.