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Proper cell fate determination relies on precise spatial and temporal genome-wide cooperation between regulatory elements (REs) and their targeted genes. However, the lengths of REs defined using different methods vary, which indicates that there is sequence redundancy and that the context of the genome may be unintelligible. We developed a method called MAE-seq (Massive Active Enhancers by Sequencing) to experimentally identify functional REs at a 25-bp scale. In this study, MAE-seq was used to identify 626879, 541617 and 554826 25-bp enhancers in mouse embryonic stem cells (mESCs), C2C12 and HEK 293T, respectively. Using â¼1.6 trillion 25 bp DNA fragments and screening 12 billion cells, we identified 626879 as active enhancers in mESCs as an example. Comparative analysis revealed that most of the histone modification datasets were annotated by MAE-Seq loci. Furthermore, 33.85% (212195) of the identified enhancers were identified as de novo ones with no epigenetic modification. Intriguingly, distinct chromatin states dictate the requirement for dissimilar cofactors in governing novel and known enhancers. Validation results show that these 25-bp sequences could act as a functional unit, which shows identical or similar expression patterns as the previously defined larger elements, Enhanced resolution facilitated the identification of numerous cell-specific enhancers and their accurate annotation as super enhancers. Moreover, we characterized novel elements capable of augmenting gene activity. By integrating with high-resolution Hi-C data, over 55.64% of novel elements may have a distal association with different targeted genes. For example, we found that the Cdh1 gene interacts with one novel and two known REs in mESCs. The biological effects of these interactions were investigated using CRISPR-Cas9, revealing their role in coordinating Cdh1 gene expression and mESC proliferation. Our study presents an experimental approach to refine the REs at 25-bp resolution, advancing the precision of genome annotation and unveiling the underlying genome context. This novel approach not only advances our understanding of gene regulation but also opens avenues for comprehensive exploration of the genomic landscape.
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Genoma , Sequências Reguladoras de Ácido Nucleico , Animais , Camundongos , Sequências Reguladoras de Ácido Nucleico/genética , Cromatina , Genômica/métodos , Regulação da Expressão Gênica , Elementos Facilitadores GenéticosRESUMO
Intact glycopeptide identification has long been known as a key and challenging barrier to the comprehensive and accurate understanding the role of glycosylation in an organism. Intact glycopeptide analysis is a blossoming field that has received increasing attention in recent years. MS-based strategies and relative software tools are major drivers that have greatly facilitated the analysis of intact glycopeptides, particularly intact N-glycopeptides. This article provides a systematic review of the intact glycopeptide-identification process using MS data generated in shotgun proteomic experiments, which typically focus on N-glycopeptide analysis. Particular attention is paid to the software tools that have been recently developed in the last decade for the interpretation and quality control of glycopeptide spectra acquired using different MS strategies. The review also provides information about the characteristics and applications of these software tools, discusses their advantages and disadvantages, and concludes with a discussion of outstanding tools.
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Glicopeptídeos/análise , Software , Animais , Humanos , Espectrometria de Massas , ProteômicaRESUMO
Chromosome conformation-capture technologies are widely used in 3D genomics; however, experimentally, such methods have high-noise limitations and, therefore, require significant bioinformatics efforts to extract reliable distal interactions. Miscellaneous undesired linear DNAs, present during proximity-ligation, represent a main noise source, which needs to be minimized or eliminated. In this study, different exonuclease combinations were tested to remove linear DNA fragments from a circularized DNA preparation. This method efficiently removed linear DNAs, raised the proportion of annulation and increased the valid-pairs ratio from â¼40% to â¼80% for enhanced interaction detection in standard Hi-C. This strategy is applicable for development of various 3D genomics technologies, or optimization of Hi-C sequencing efficiency.
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Cromossomos de Mamíferos , Exodesoxirribonucleases , Genômica/métodos , Animais , Linhagem Celular , Células Cultivadas , Cromatina , CamundongosRESUMO
The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid (BA)-functionalized mesoporous graphene-silica composite (denoted as GO@mSiO2-GLYMO-APB) for isolating intact glycopeptides from complex biological samples. The merits of this composite, including high surface area and synergistic effect from size exclusion functionality of mesoporous material, hydrophilic interaction of silica, and the reversible covalent binding with BA, enable the effective and specific enrichment of both intact N- and O-glycopeptides. The results from the enrichment performance of the strategy evaluated by standard glycoproteins and the application to global N- and O-glycosylation analyses in human serum indicate the robustness and potential of the strategy for intact glycopeptide analysis.
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Grafite , Ácidos Borônicos , Glicopeptídeos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dióxido de SilícioRESUMO
Precise and automated analysis of site-specific O-glycosylation on single proteins is crucial for comprehensive characterization of some important glycoproteins, such as tumor biomarkers and recombinant drug proteins. Mass spectrometry has been proven to be a powerful technique for protein sequencing and N-glycosylation analysis. However, challenges remain in developing computational tools for intact O-glycopeptide analysis, which has greatly hindered the development of mass-spectrometry-based O-glycosylation analysis. Herein, an integrated strategy together with a dedicated automated computational tool termed AOGP was developed for intact O-glycopeptide analysis on single proteins. AOGP utilized de novo sequencing for O-glycans and a database search strategy for peptide backbones. The false discovery rate (FDR) of the identification results was controlled and validated by a mixed Gaussian distribution estimation method. AOGP exhibited superior performance in identifying intact O-glycopeptides of the human erythropoietin with a total of 188 O-glycopeptide spectra reported under 1% FDR. AOGP is developed in Python, is fully open-sourced, and is equipped with a user-friendly interface. Such an easy-operating and robust tool would greatly facilitate O-glycosylation analysis on single proteins in tumor biomarker and recombinant drug protein development.
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Algoritmos , Assialoglicoproteínas/análise , Automação , Eritropoetina/análise , Fetuínas/análise , Glicopeptídeos/análise , Animais , Bovinos , Glicosilação , Humanos , Espectrometria de Massas em TandemRESUMO
Introduction: Glycomics, which aims to define the glycome of a biological system to better assess the biological attributes of the glycans, has attracted increasing interest. However, the complexity and diversity of glycans present challenging barriers to glycome definition. Technological advances are major drivers in glycomics.Areas covered: This review summarizes the main methods and emphasizes the most recent advances in mass spectrometry-based methods regarding glycomics following the general workflow in glycomic analysis.Expert opinion: Recent mass spectrometry-based technological advances have significantly lowered the barriers in glycomics. The field of glycomics is moving toward both generic and precise analysis.
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Glicômica/métodos , Espectrometria de Massas/métodos , Animais , Humanos , Polissacarídeos/químicaRESUMO
Efficient detection of aberrant glycoproteins in serum is particularly important for biomarker discovery. However, direct quantitation of glycoproteins in serum remains technically challenging because of the extraordinary complexity of the serum proteome. In the current work, we proposed a straightforward and highly efficient strategy by using the nonglycopeptides releasing from the specifically enriched glycoproteins for targeted glycoprotein quantification. With this so-called nonglycopeptide-based mass spectrometry (NGP-MS) strategy, a powerful and nondiscriminatory pipeline for hepatocellular carcinoma (HCC) glycoprotein biomarker discovery, verification, and validation has been developed. First, a data set of 234 NGPs was strictly established for multiple-reaction monitoring (MRM) quantification in serum. Second, the NGPs enriched from 20 HCC serum mixtures and 20 normal serum mixtures were labeled with mTRAQ reagents (Δ0 and Δ8, respectively) to find the differentially expressed glycoproteins in HCC. A total of 97 glycoprotein candidates were preliminarily screened and submitted for absolute quantitation with NGP-based stable-isotope-labeled (SID)-MRM in the individual samples of 38 HCC serum and 24 normal controls. Finally, 21 glycoproteins were absolutely quantified with high quality. The diagnostic sensitivity results showed that three glycoproteins, ß-2-glycoprotein 1 (APOH), α-1-acid glycoprotein 2 (ORM2), and complement C3 (C3), could be used for the discrimination between HCC patients and healthy people. A novel glycoprotein biomarker panel [APOH, ORM2, C3, and α-fetoprotein (AFP)] has proven to outperform AFP, the known HCC serum biomarker, alone, in this study. We believe that this strategy and the panel of glycoproteins might hold great clinical value for HCC detection in the future.
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Carcinoma Hepatocelular/sangue , Glicoproteínas/sangue , Neoplasias Hepáticas/sangue , Espectrometria de Massas/métodos , Biomarcadores/sangue , Humanos , alfa-Fetoproteínas/metabolismoRESUMO
Hi-C is a commonly used technology in 3D genomics which can depict global chromatin interactions across eukaryotic genome. Integrating with different datasets, it can also be applied to studying various biological questions, such as nuclear organization, gene transcription regulation, spatiotemporal development, genome assembly, and cancer genomics. During the last decade, the development and application of Hi-C have dramatically changed the view of genome architecture, chromatin conformation, and gene interaction. So far, Hi-C-related studies remain vivacious and controversial; thus, a unified standard of library construction and bioinformatics analysis are urgently needed. In this review, we have summarized its history, development, methodologies, advances, applications, shortages, and future perspectives. We discuss a few limitations of the current Hi-C technologies and future directions for improvement and highlight how Hi-C can bridge 3D structure to gene function. This review will be helpful for scientists who want to engage in the 3D genomics field; it also shows some future tracks.
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Cromatina/metabolismo , Genoma , Genômica/métodos , Animais , Doença/genética , Evolução Molecular , Saúde , HumanosRESUMO
Nujiangexathone A (NJXA), a novel compound derived from Garcinia nujiangensis, has been demonstrated to inhibit the proliferation of several human cancer cell lines. This study is the first to demonstrate the apoptosis inductive activities of NJXA and the possible underlying mechanisms. Our results demonstrated that NJXA inhibited colony formation by HeLa and SiHa cells in a dose-dependent manner. An Annexin V-FITC/PI staining assay showed that NJXA strongly triggered apoptosis in a dose-dependent manner. Western blotting analyses showed that NJXA induced the caspase-dependent apoptosis of HeLa and SiHa cells by triggering a series of events, including changes in the levels of Bcl-2 family proteins, cytochrome c release, caspase-3 activation, and chromosome fragmentation. Furthermore, we demonstrated that NJXA induced cell apoptosis by activating the reactive oxygen species (ROS)-mediated JNK signaling pathway. Consistent with this finding, a ROS scavenger, N-acetyl-l-cysteine (NAC, 10 mM), hindered NJXA-induced apoptosis and attenuated the sensitivity of HeLa and SiHa cells to NJXA. In vivo results further confirmed that the tumor inhibitory effect of NJXA was partially through the induction of apoptosis. Taken together, our results demonstrated that NJXA induced the apoptosis of HeLa and SiHa cells through the ROS/JNK signaling pathway, indicating that NJXA could be important candidate for the clinical treatment of cervical cancer.
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Antineoplásicos Fitogênicos/administração & dosagem , Caspases/metabolismo , Garcinia/química , Extratos Vegetais/administração & dosagem , Neoplasias do Colo do Útero/tratamento farmacológico , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Neoplasias do Colo do Útero/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. We report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-ß stimulated mouse embryonic fibroblasts (MEFs). The data clearly discriminate two IFN-ß stimulated MEFs. Moreover, our results indicate 81% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on Cap-Analysis Gene Expression (CAGE) (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.
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Fibroblastos , Estudo de Associação Genômica Ampla , Animais , Camundongos , Regiões Promotoras Genéticas , Genoma , Genômica , Mamíferos/genéticaRESUMO
The Microbiome Protocols eBook (MPB) serves as a crucial bridge, filling gaps in microbiome protocols for both wet experiments and data analysis. The first edition, launched in 2020, featured 152 meticulously curated protocols, garnering widespread acclaim. We now extend a sincere invitation to researchers to participate in the upcoming 2nd version of MPB, contributing their valuable protocols to advance microbiome research.
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Protein glycosylation is a widespread posttranslational modification that can impact the function of proteins. Dysregulated protein glycosylation has been linked to several diseases, including chronic respiratory diseases (CRDs). CRDs pose a significant public health threat globally, affecting the airways and other lung structures. Emerging researches suggest that glycosylation plays a significant role in regulating inflammation associated with CRDs. This review offers an overview of the abnormal glycoenzyme activity and corresponding glycosylation changes involved in various CRDs, including chronic obstructive pulmonary disease, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, pulmonary arterial hypertension, non-cystic fibrosis bronchiectasis, and lung cancer. Additionally, this review summarizes recent advances in glycomics and glycoproteomics-based protein glycosylation analysis of CRDs. The potential of glycoenzymes and glycoproteins for clinical use in the diagnosis and treatment of CRDs is also discussed.
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Transtornos Respiratórios , Humanos , Glicosilação , Glicoproteínas/metabolismo , Pulmão/metabolismo , InflamaçãoRESUMO
The morphogenesis of hair follicle structure is accompanied by the differentiation of skin tissue. Mammalian coats are produced by hair follicles. The formation of hair follicles requires signal transmission between the epidermis and dermis. However, knowledge of the transcriptional regulatory mechanism is still lacking. We used single-cell RNA sequencing to obtain 26,573 single cells from the scapular skin of yaks at hair follicle telogen and anagen stages. With the help of known reference marker genes, 11 main cell types were identified. In addition, we further analyzed the DP cell and dermal fibroblast lineages, drew a single-cell map of the DP cell and dermal fibroblast lineages, and elaborated the key genes, signals, and functions involved in cell fate decision making. The results of this study provide a very valuable resource for the analysis of the heterogeneity of DP cells and dermal fibroblasts in the skin and provide a powerful theoretical reference for further exploring the diversity of hair follicle cell types and hair follicle morphogenesis.
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In view of the good adsorption properties of graphene and carbon foam, they were combined to achieve the optimal matching of microstructures. Taking mesophase pitch as a raw material, pitch-based carbon foam was prepared by the self-foaming method. Graphene gel was prepared as the second phase to composite with the carbon foam matrix; graphene-modified, pitch-based carbon foam composites were finally obtained. Graphene gel was dispersed in the rich pore structure of carbon foam to improve its agglomeration and the porosity, and the active sites of the composite were further increased; the adsorption properties and mechanical properties of the composites were also significantly improved. The microstructure and morphology of the composites were studied by SEM, XRD and Raman spectroscopy; the compressive property and porosity were also tested. Methylene blue (MB) solution was used to simulate a dye solution for the adsorption test, and the influence of the composite properties and MB solution on the adsorption property was studied. Results showed that the compressive strength of the composite was 13.5 MPa, increased by 53.41%, and the porosity was 58.14%, increased by 24.15%, when compared to raw carbon foam. When the mass of the adsorbent was 150 mg, the initial concentration of the MB solution was 5 mg/L, and the pH value of the MB solution was 11; the graphene-modified carbon foam composites showed the best adsorption effect, with an adsorption rate of 96.3% and an adsorption capacity of 144.45 mg/g. Compared with the raw carbon foam, the adsorption rate and adsorption capacity of the composites were increased by 158.18% and 93.50%, respectively.
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Genetic information is loaded on chromatin, which involves DNA sequence arrangement and the epigenetic landscape. The epigenetic information including DNA methylation, nucleosome positioning, histone modification, 3D chromatin conformation, and so on, has a crucial impact on gene transcriptional regulation. Out of them, nucleosomes, as basal chromatin structural units, play an important central role in epigenetic code. With the discovery of nucleosomes, various nucleosome-level technologies have been developed and applied, pushing epigenetics to a new climax. As the underlying methodology, next-generation sequencing technology has emerged and allowed scientists to understand the epigenetic landscape at a genome-wide level. Combining with NGS, nucleosome-omics (or nucleosomics) provides a fresh perspective on the epigenetic code and 3D genome landscape. Here, we summarized and discussed research progress in technology development and application of nucleosome-omics. We foresee the future directions of epigenetic development at the nucleosome level.
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Cromatina , Nucleossomos , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nucleossomos/genéticaRESUMO
Over the past decade, with the development of high-throughput single-cell sequencing technology, single-cell omics has been emerged as a powerful tool to understand the molecular basis of cellular mechanisms and refine our knowledge of diverse cell states. They can reveal the heterogeneity at different genetic layers and elucidate their associations by multiple omics analysis, providing a more comprehensive genetic map of biological regulatory networks. In the post-GWAS era, the molecular biological mechanisms influencing human diseases will be further elucidated by single-cell omics. This review mainly summarizes the development and trend of single-cell omics. This involves single-cell omics technologies, single-cell multi-omics technologies, multiple omics data integration methods, applications in various human organs and diseases, classic laboratory cell lines, and animal disease models. The review will reveal some perspectives for elucidating human diseases and constructing animal models.
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The prevalence of intracranial aneurysm (IA) is increasing, and the consequences of its rupture are severe. This study aimed to reveal specific, sensitive, and non-invasive biomarkers for diagnosis and classification of ruptured and unruptured IA, to benefit the development of novel treatment strategies and therapeutics altering the course of the disease. We first assembled an extensive candidate biomarker bank of IA, comprising up to 717 proteins, based on altered proteins discovered in the current tissue and serum proteomic analysis, as well as from previous studies. Mass spectrometry assays for hundreds of biomarkers were efficiently designed using our proposed deep learning-based method, termed DeepPRM. A total of 113 potential markers were further quantitated in serum cohort I (n = 212) & II (n = 32). Combined with a machine-learning-based pipeline, we built two sets of biomarker combinations (P6 & P8) to accurately distinguish IA from healthy controls (accuracy: 87.50%) or classify IA rupture patients (accuracy: 91.67%) upon evaluation in the external validation set (n = 32). This extensive circulating biomarker development study provides valuable knowledge about IA biomarkers.
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Aneurisma Roto , Aneurisma Intracraniano , Aneurisma Roto/diagnóstico , Aneurisma Roto/metabolismo , Biomarcadores , Humanos , Aneurisma Intracraniano/diagnóstico , Aneurisma Intracraniano/metabolismo , Proteômica , Medição de RiscoRESUMO
Large-scale intact glycopeptide identification has been advanced by software tools. However, tools for quantitative analysis remain lagging behind, which hinders exploring the differential site-specific glycosylation. Here, we report pGlycoQuant, a generic tool for both primary and tandem mass spectrometry-based intact glycopeptide quantitation. pGlycoQuant advances in glycopeptide matching through applying a deep learning model that reduces missing values by 19-89% compared with Byologic, MSFragger-Glyco, Skyline, and Proteome Discoverer, as well as a Match In Run algorithm for more glycopeptide coverage, greatly expanding the quantitative function of several widely used search engines, including pGlyco 2.0, pGlyco3, Byonic and MSFragger-Glyco. Further application of pGlycoQuant to the N-glycoproteomic study in three different metastatic HCC cell lines quantifies 6435 intact N-glycopeptides and, together with in vitro molecular biology experiments, illustrates site 979-core fucosylation of L1CAM as a potential regulator of HCC metastasis. We expected further applications of the freely available pGlycoQuant in glycoproteomic studies.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Biologia MolecularRESUMO
BACKGROUND: Pyroptosis, an inflammatory form of programmed cell death (PCD), is reported to play important roles in the treatment of tumors. In our previous studies, we found that neobractatin (NBT), a caged prenylxanthone isolated from edible fruits of Garcinia bracteata C. Y. Wu ex Y. H. Li, showed anticancer effects against different cancer cells. However, the effect of NBT on pyroptosis is not well understood. PURPOSE: This study aims to investigate whether and how GSDME-mediated pyroptosis contributes to NBT-induced antitumor effects in esophageal cancer (EC) cells. METHODS: Cell viability assay and colony formation assay were used to determine the anticancer effects of NBT in esophageal cancer cells. Lactate dehydrogenase (LDH) release assay and microscopy imaging were used to detect the main characteristic of pyroptosis. CRISPR-Cas9 knockout and siRNA knockdown were performed to verify the roles of GSDME and caspase-3 in NBT-induced pyroptosis. Flow cytometry was used to measure the reactive oxygen species (ROS) level and cell apoptosis. The changes of related protein level were detected by Western blot. Furthermore, animal experiments were used to verify the in vivo effect of NBT. RESULTS: The results showed that NBT reduced the viability of EC cells mainly through GSDME-mediated pyroptosis. Morphologically, NBT induced cell swelling and formed large bubbles emerging from plasma membrane in wild type EC cells. Furthermore, NBT induced the cleavage of GSDME by activating caspase-3 in EC cells. On the other hand, caspase-3 activated by NBT also induced apoptosis especially at high dosage. Knocking down GSDME switched NBT-induced cell death from mainly pyroptosis to apoptosis in vivo and in vitro. Mechanistic studies indicated that NBT led to accumulation of ROS, which then regulated the phosphorylation of both JNK and MEK/ERK. In the absence of ROS or caspase-3, NBT-induced pyroptosis and apoptosis were completely reversed. Moreover, NBT showed a significant antitumor effect in both the KYSE150 and GSDME knockout KYSE150-/- xenograft models by inducing pyroptosis and apoptosis, respectively. CONCLUSION: Our results indicated that natural compound NBT could induce GSDME-mediated pyroptosis and apoptosis in esophageal cancer cells, making it a potential therapeutic drug in clinical treatment.
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Neoplasias Esofágicas , Garcinia , Animais , Caspase 3/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Humanos , Piroptose , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismoRESUMO
We demonstrate the possibility of conducting synchronous, repeated, multi-game economic decision-making experiments with hundreds of subjects in-person or remotely with live streaming using entirely mobile platforms. Our experiment provides important proof-of-concept that such experiments are not only possible, but yield recognizable results as well as new insights, blurring the line between laboratory and field experiments. Specifically, our findings from 8 different experimental economics games and tasks replicate existing results from traditional laboratory experiments despite the fact that subjects play those games/task in a specific order and regardless of whether the experiment was conducted in person or remotely. We further leverage our large subject population to study the effect of large (N = 100) versus small (N = 10) group sizes on behavior in three of the scalable games that we study. While our results are largely consistent with existing findings for small groups, increases in group size are shown to matter for the robustness of those findings.