SwePub - search: WFRF:(Chen Xingqi)

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1.	Bolin, Sara, 1988-, et al. (author) Dormant SOX9-positive cells behind MYC-driven medulloblastoma recurrence Journal article (peer-reviewed)abstract Tumor recurrence is a slow biological process involving therapy resistance, immune escape, and metastasis and is the leading cause of death in medulloblastoma, the most frequent malignant pediatric brain tumor. By studying paired primary-recurrent patient samples and patient-derived xenografts we identified a significant accumulation of SOX9-positive cells in relapses and metastases. They exist as rare, quiescent cells in Group 3 and Group 4 patients that constitute two-thirds of medulloblastoma. To follow relapse at the single-cell level we developed an inducible dual Tet model of MYC-driven MB, where MYC can be directed from treatment-sensitive bulk cells to resistant, dormant SOX9-positive cells by doxycycline. SOX9 promoted immune es-cape, DNA repair suppression and was essential for recurrence. Tumor cell dormancy was non-hierarchical, migratory, and depended on MYC suppression by SOX9 to promote relapse. By using computational modeling and treatment we further showed how doxorubicin and MGMT inhibitors are specifically targeting relapsing cells.
2.	Borgenvik, Anna, 1987-, et al. (author) Dormant SOX9-Positive Cells Facilitate MYC-Driven Recurrence of Medulloblastoma 2022 In: Cancer Research. - : AMER ASSOC CANCER RESEARCH. - 0008-5472 .- 1538-7445. ; 82:24, s. 4586-4603 Journal article (peer-reviewed)abstract Relapse is the leading cause of death in patients with medulloblas-toma, the most common malignant pediatric brain tumor. A better understanding of the mechanisms underlying recurrence could lead to more effective therapies for targeting tumor relapses. Here, we observed that SOX9, a transcription factor and stem cell/glial fate marker, is limited to rare, quiescent cells in high-risk medulloblastoma with MYC amplification. In paired primary-recurrent patient samples, SOX9-positive cells accumulated in medulloblastoma relapses. SOX9 expression anti-correlated with MYC expression in murine and human medulloblastoma cells. However, SOX9-positive cells were plastic and could give rise to a MYC high state. To follow relapse at the single-cell level, an inducible dual Tet model of medulloblastoma was developed, in which MYC expression was redirected in vivo from treatment-sensitive bulk cells to dormant SOX9-positive cells using doxycycline treatment. SOX9 was essential for relapse initiation and depended on suppression of MYC activity to promote therapy resistance, epithelial-mesenchymal transition, and immune escape. p53 and DNA repair pathways were downregulated in recurrent tumors, whereas MGMT was upregulated. Recurrent tumor cells were found to be sensitive to treatment with an MGMT inhibitor and doxorubicin. These findings suggest that recurrence-specific targeting coupled with DNA repair inhibition comprises a potential therapeutic strategy in patients affected by medulloblastoma relapse.Significance: SOX9 facilitates therapy escape and recurrence in medulloblastoma via temporal inhibition of MYC/MYCN genes, revealing a strategy to specifically target SOX9-positive cells to prevent tumor relapse.
3.	Cavalli, Marco, et al. (author) The Thioesterase ACOT1 as a Regulator of Lipid Metabolism in Type 2 Diabetes Detected in a Multi-Omics Study of Human Liver 2021 In: Omics. - : Mary Ann Liebert. - 1536-2310 .- 1557-8100. ; 25:10, s. 652-659 Journal article (peer-reviewed)abstract Type 2 diabetes (T2D) is characterized by pathophysiological alterations in lipid metabolism. One strategy to understand the molecular mechanisms behind these abnormalities is to identify cis-regulatory elements (CREs) located in chromatin-accessible regions of the genome that regulate key genes. In this study we integrated assay for transposase-accessible chromatin followed by sequencing (ATAC-seq) data, widely used to decode chromatin accessibility, with multi-omics data and publicly available CRE databases to identify candidate CREs associated with T2D for further experimental validations. We performed high-sensitive ATAC-seq in nine human liver samples from normal and T2D donors, and identified a set of differentially accessible regions (DARs). We identified seven DARs including a candidate enhancer for the ACOT1 gene that regulates the balance of acyl-CoA and free fatty acids (FFAs) in the cytoplasm. The relevance of ACOT1 regulation in T2D was supported by the analysis of transcriptomics and proteomics data in liver tissue. Long-chain acyl-CoA thioesterases (ACOTs) are a group of enzymes that hydrolyze acyl-CoA esters to FFAs and coenzyme A. ACOTs have been associated with regulation of triglyceride levels, fatty acid oxidation, mitochondrial function, and insulin signaling, linking their regulation to the pathogenesis of T2D. Our strategy integrating chromatin accessibility with DNA binding and other types of omics provides novel insights on the role of genetic regulation in T2D and is extendable to other complex multifactorial diseases.
4.	Chen, Xingqi, et al. (author) Chromatin in situ proximity (ChrISP) : Single-cell analysis of chromatin proximities at a high resolution 2014 In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 56:3, s. 117-124 Journal article (peer-reviewed)abstract Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of similar to 170 angstrom in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA.with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.
5.	Chen, Xingqi (author) Dynamics of higher order chromatin structures 2014 Doctoral thesis (other academic/artistic)abstract During the last few decades, the intensive focus on microscopy observations and genome sequencing analyses has proved that the genomic DNA is packaged in the non-random manner in the nucleus of interphase cells. Accumulated evidence have thus documented that the chromatin organization in 3D plays key roles in central biological processes, such as transcription, replication and DNA repair. In the interphase nucleus, each chromosome is expanded and organized in a manner depending on structural hallmarks of the nucleus. Thus, repressed domains localize to the nuclear periphery to form lamina associated domains (LADs) or large organized chromatin K9 modifications (LOCKs). In addition, prevalent chromatin interactions can be formed from same chromosome (in cis) or different chromosome (in trans). It is still not clear how such dynamic interactions between chromatin fibers control the expressivity of the genome and to what extent these depend on epigenetic chromatin states. The study in this thesis had focused on the dynamics of higher order chromatin structures, particularly on the relationship between the dynamics of chromatin structure and chromatin states. Since the resolution of current single cell techniques in the chromatin organization research, such as DNA FISH and immuno-staining, are limited by the resolution of the microscopy, we invented a new in situ single cell technique termed ChrISP (paper I). Using this technique we could detect chromatin proximities with a resolution less than 17nm even though the analysis was implemented using the low resolution confocal microscope. In paper II, the scope of the ChrISP technique was extended to include an analysis of chromatin states within a single chromosome in a single cell to document that compacted chromatin at the nuclear periphery depends on the H3K9me2 mark that impinges on the nuclear periphery in finger-like structures. Moreover, upon the removal of these marks the rest of the chromosome showed signs of compaction, potentially related to chromosome condensation. These results are consistent with the interpretation that the H3K9me2 mark regulates pleiotropic features of higher order chromatin structure. In paper III, we had used the view point of a single locus to explore the dynamics of chromatin interactions in developmental window using the circular chromatin conformation capture (4C) technique. The resulting inter-chromosomal network connected, surprisingly, both active and repressive chromatin domains involving LADs. Moreover, this network depended on the circadian recruitment of active chromatin hubs to the repressed chromatin structures at the nuclear periphery mediated by the physical proximities between CTCF and PARP1. This circadian pattern was required to attenuate transcription of the active chromatin hubs in a rhythmic manner. In summary, a new high-resolution technique termed ChrISP was invented in this thesis to enable quantitative analyses of dynamic of higher order chromatin structures. This technique could, moreover, be used to visualize specific chromatin marks, notably H3K9me2, within a specific chromosome in relation to structural hallmarks of the nucleus within a single cell. The compact chromatin structure thus identified was discovered to transiently harbor active chromatin hubs, which was recruited to the nuclear periphery in oscillating manner. We show that this feature likely underlies the attenuation of genes under circadian control. These findings open new perspectives to understand the function of dynamics of higher order chromatin structure.
6.	Chen, Xingqi, et al. (author) Structural phase transition in monolayer gold(I) telluride : From a room-temperature topological insulator to an auxetic semiconductor 2021 In: Physical Review B. - : American Physical Society. - 2469-9950 .- 2469-9969. ; 103:7 Journal article (peer-reviewed)abstract Structural phase transitions between semiconductors and topological insulators have rich applications in nanoelectronics but are rarely found in two-dimensional (2D) materials. In this work, by combining ab initio computations and evolutionary structure search, we investigate two stable 2D forms of gold(I) telluride (Au2Te) with square symmetry, noted as s(I)- and s(II)-Au2Te. s(II)-Au2Te is the global minimum structure and is a room-temperature topological insulator. s(I)-Au2Te is a direct-gap semiconductor with high carrier mobilities and unusual in-plane negative Poisson's ratio. Both s(I) and s(II) phases have ultralow Young's modulus, implying high flexibility. By applying a small tensile strain, s(II)-Au2Te can be transformed into s(I)-Au2Te. Hence, a structural phase transition from a room-temperature topological insulator to an auxetic semiconductor is found in the 2D forms of Au2Te, which enables potential applications in phase-change electronic devices. Moreover, we elucidate the mechanism of the phase transition with the help of phonon spectra and group theory analysis.
7.	Du, Qian, et al. (author) Porcine circovirus type 2 infection promotes the SUMOylation of nucleophosmin-1 to facilitate the viral circular single-stranded DNA replication 2024 In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 20:2 Journal article (peer-reviewed)abstract The mechanism of genome DNA replication in circular single-stranded DNA viruses is currently a mystery, except for the fact that it undergoes rolling-circle replication. Herein, we identified SUMOylated porcine nucleophosmin-1 (pNPM1), which is previously reported to be an interacting protein of the viral capsid protein, as a key regulator that promotes the genome DNA replication of porcine single-stranded DNA circovirus. Upon porcine circovirus type 2 (PCV2) infection, SUMO2/3 were recruited and conjugated with the K263 site of pNPM1's C-terminal domain to SUMOylate pNPM1, subsequently, the SUMOylated pNPM1 were translocated in nucleoli to promote the replication of PCV2 genome DNA. The mutation of the K263 site reduced the SUMOylation levels of pNPM1 and the nucleolar localization of pNPM1, resulting in a decrease in the level of PCV2 DNA replication. Meanwhile, the mutation of the K263 site prevented the interaction of pNPM1 with PCV2 DNA, but not the interaction of pNPM1 with PCV2 Cap. Mechanistically, PCV2 infection increased the expression levels of Ubc9, the only E2 enzyme involved in SUMOylation, through the Cap-mediated activation of ERK signaling. The upregulation of Ubc9 promoted the interaction between pNPM1 and TRIM24, a potential E3 ligase for SUMOylation, thereby facilitating the SUMOylation of pNPM1. The inhibition of ERK activation could significantly reduce the SUMOylation levels and the nucleolar localization of pNPM1, as well as the PCV2 DNA replication levels. These results provide new insights into the mechanism of circular single-stranded DNA virus replication and highlight NPM1 as a potential target for inhibiting PCV2 replication. Different types of DNA viruses employ different mechanisms to replicate their genome DNA. Porcine circovirus type 2 (PCV2) is the most representative circular single-stranded DNA virus that harms the pig industry all over the world. In this study, we found that the PCV2 Cap interacting protein pNPM1 also interacts with PCV2 DNA in a SUMOylated form to promote PCV2 DNA replication. The SUMOylation of pNPM1 at the conserved K263 site is critical for the interaction of pNPM1 with PCV2 DNA and the replication of PCV2 DNA. Furthermore, we found that PCV2 infection promotes the SUMO2/3 mediated SUMOylation of pNPM1, while does not significantly alter the expression level of pNPM1. PCV2 Cap is the major component that promotes pNPM1 SUMOylation by activating ERK/Ubc9/TRIM24 signalings. These results contribute to a better understanding of the replication mechanism of circular single-stranded DNA viruses, particularly PCV2.
8.	Husain, S., et al. (author) Large Dzyaloshinskii-Moriya interaction and atomic layer thickness dependence in a ferromagnet- WS2 heterostructure 2022 In: Physical Review B. - : American Physical Society. - 2469-9950 .- 2469-9969. ; 105:6 Journal article (peer-reviewed)abstract Two-dimensional transition metal dichalcogenides (TMDs) have immense potential for spintronics applications. Here, we report atomic layer thickness dependence in WS2/Co3FeB heterostructures. The layer dependence is predicted by density functional theory and demonstrated experimentally by the layer dependence of the Dzyaloshinskii-Moriya interaction (DMI). Notably, we have observed the DMI in WS2 to be larger than that for heavy metals such as W and Ta, which is important to stabilize chiral structures. Inversion symmetry is not preserved with an odd number of layers, while it exists with an even number of layers. This symmetry rule is reflected in the temperature dependence of the effective damping parameter of the heterostructure. That the damping parameter decreases (increases) in odd (even) layers can be resolved at low temperature. This suggests that the layer dependence has its origin at the WS2 interface, where the spin-valley coupling and spin-orbit coupling activate these features. Large DMI, pure spin current, and unique layer dependence in TMDs provide valuable information and fundamental understanding for designing TMD-based quantum information storage devices. © 2022 American Physical Society.
9.	Li, Dongqing, et al. (author) Single-Cell Analysis Reveals Major Histocompatibility Complex II-Expressing Keratinocytes in Pressure Ulcers with Worse Healing Outcomes 2022 In: Journal of Investigative Dermatology. - : Elsevier. - 0022-202X .- 1523-1747. ; 142:3, s. 705-716 Journal article (peer-reviewed)abstract Pressure ulcer (PU) is a chronic wound often seen in patients with spinal cord injury and other bed-bound individuals, particularly in the elderly population. Despite its association with high mortality, the pathophysiology of PU remains poorly understood. In this study, we compared single-cell transcriptomic profiles of human epidermal cells from PU wound edges with those from uninjured skin and acute wounds in healthy donors. We identified significant shifts in the cell composition and gene expression patterns in PU. In particular, we found that major histocompatibility complex class II-expressing keratinocytes were enriched in patients with worse healing outcomes. Furthermore, we showed that the IFN-gamma in PU-derived wound fluid could induce major histocompatibility complex II expression in keratinocytes and that these wound fluid-treated keratinocytes inhibited autologous T-cell activation. In line with this observation, we found that T cells from PUs enriched with major histocompatibility complex II+ keratinocytes produced fewer inflammatory cytokines. Overall, our study provides a high-resolution molecular map of human PU compared with that of acute wounds and intact skin, providing insights into PU pathology and the future development of tailored wound therapy.
10.	Li, Honglian, et al. (author) Heparanase Modulates Chromatin Accessibility 2023 In: Cells. - : MDPI. - 2073-4409. ; 12:6 Journal article (peer-reviewed)abstract Heparanase is the sole endoglucuronidase that degrades heparan sulfate in the cell surface and extracellular matrix (ECM). Several studies have reported the localization of heparanase in the cell nucleus, but the functional role of the nuclear enzyme is still obscure. Subjecting mouse embryonic fibroblasts (MEFs) derived from heparanase knockout (Hpse-KO) mice and applying transposase-accessible chromatin with sequencing (ATAC-seq), we revealed that heparanase is involved in the regulation of chromatin accessibility. Integrating with genome-wide analysis of chromatin states revealed an overall low activity in the enhancer and promoter regions of Hpse-KO MEFs compared with wild-type (WT) MEFs. Western blot analysis of MEFs and tissues derived from Hpse-KO vs. WT mice confirmed reduced expression of H3K27ac (acetylated lysine at N-terminal position 27 of the histone H3 protein). Our results offer a mechanistic explanation for the well-documented attenuation of inflammatory responses and tumor growth in Hpse-KO mice.
11.	Lu, Xi, et al. (author) Cell-lineage controlled epigenetic regulation in glioblastoma stem cells determines functionally distinct subgroups and predicts patient survival 2022 In: Nature Communications. - : Springer Nature. - 2041-1723. ; 13 Journal article (peer-reviewed)abstract The epigenetic regulation of glioblastoma stem cell (GSC) function remains poorly understood. Here, the authors compare the chromatin accessibility landscape of GSC cultures from mice and patients and suggest that the epigenome of GSCs is cell lineage-regulated and could predict patient survival. There is ample support for developmental regulation of glioblastoma stem cells. To examine how cell lineage controls glioblastoma stem cell function, we present a cross-species epigenome analysis of mouse and human glioblastoma stem cells. We analyze and compare the chromatin-accessibility landscape of nine mouse glioblastoma stem cell cultures of three defined origins and 60 patient-derived glioblastoma stem cell cultures by assay for transposase-accessible chromatin using sequencing. This separates the mouse cultures according to cell of origin and identifies three human glioblastoma stem cell clusters that show overlapping characteristics with each of the mouse groups, and a distribution along an axis of proneural to mesenchymal phenotypes. The epigenetic-based human glioblastoma stem cell clusters display distinct functional properties and can separate patient survival. Cross-species analyses reveals conserved epigenetic regulation of mouse and human glioblastoma stem cells. We conclude that epigenetic control of glioblastoma stem cells primarily is dictated by developmental origin which impacts clinically relevant glioblastoma stem cell properties and patient survival.
12.	Lu, Xi, et al. (author) Identification of ATF3 as a novel protective signature of quiescent colorectal tumor cells 2023 In: Cell Death and Disease. - : Springer Nature. - 2041-4889. ; 14:10 Journal article (peer-reviewed)abstract Colorectal cancer (CRC) is the third most common cancer and the second leading cause of death in the world. In most cases, drug resistance and tumor recurrence are ultimately inevitable. One obstacle is the presence of chemotherapy-insensitive quiescent cancer cells (QCCs). Identification of unique features of QCCs may facilitate the development of new targeted therapeutic strategies to eliminate tumor cells and thereby delay tumor recurrence. Here, using single-cell RNA sequencing, we classified proliferating and quiescent cancer cell populations in the human colorectal cancer spheroid model and identified ATF3 as a novel signature of QCCs that could support cells living in a metabolically restricted microenvironment. RNA velocity further showed a shift from the QCC group to the PCC group indicating the regenerative capacity of the QCCs. Our further results of epigenetic analysis, STING analysis, and evaluation of TCGA COAD datasets build a conclusion that ATF3 can interact with DDIT4 and TRIB3 at the transcriptional level. In addition, decreasing the expression level of ATF3 could enhance the efficacy of 5-FU on CRC MCTS models. In conclusion, ATF3 was identified as a novel marker of QCCs, and combining conventional drugs targeting PCCs with an option to target QCCs by reducing ATF3 expression levels may be a promising strategy for more efficient removal of tumor cells.
13.	Lu, Xi (author) Integrative Modeling of Epigenetic Regulation in Human Disease 2023 Doctoral thesis (other academic/artistic)abstract Glioblastoma is the most common and aggressive primary brain tumor. Standard of treatment prove ineffective, leading to tumor recurrence and an average survival rate of 14 months. This treatment resistance is attributed to both inter-tumor and intra-tumor heterogeneity observed across diverse tumor samples and within individual cells. Dravet syndrome is a severe early-onset refractory epilepsy characterized by an unfavorable long-term outcome and medication resistance. In this thesis, patient-derived stem cell culture models were utilized to study both diseases. We employed sequencing technologies, including the Assay for Transposase-Accessible Chromatin with high-throughput sequencing and single-cell multi-omics technologies to delve into cell-lineage-controlled epigenetic regulation in glioblastoma, the mechanism behind the recurrence of glioblastoma and epigenetic dysfunction in Dravet syndrome patients during neural development. In Paper I, we validated the impacts of the developmental origin on human glioblastoma stem cell groups and revealed conserved epigenetic regulation between mouse and human glioblastoma stem cells through cross-species epigenome analysis. Human glioblastoma stem cell clusters exhibited distinct functional properties and have significant clinical outcomes. In Paper II, we discovered that the infiltrative region of the primary bulk tumor was more invasive and less self-renewing and tumorigenic compared to its paired bulk culture. Single-cell multi-omics sequencing showed an inclination towards astrocyte-like/mesenchymal-like cell states in edge cultures across all patients from transcriptomic aspect and chromatin-accessibility profiles highlighting edge cells associated with cell invasion, inflammation, and myeloid cells. In Paper III, we offered critical insights into the dysfunction of regulatory chromatin in Dravet syndrome patients using time-series ATAC sequencing. We observed that heathy individuals and Dravet syndrome patients were regulated by different transcription factors during development. Treatment with VPA effectively reshaped the chromatin landscape and rescued the observed dysfunctional development in some Dravet syndrome patients.
14.	Meijer, Mandy, et al. (author) Epigenomic priming of immune genes implicates oligodendroglia in multiple sclerosis susceptibility 2022 In: Neuron. - : Elsevier. - 0896-6273 .- 1097-4199. ; 110:7, s. 1193-1210 Journal article (peer-reviewed)abstract Multiple sclerosis (MS) is characterized by a targeted attack on oligodendroglia (OLG) and myelin by immune cells, which are thought to be the main drivers of MS susceptibility. We found that immune genes exhibit a primed chromatin state in single mouse and human OLG in a non-disease context, compatible with transitions to immune-competent states in MS. We identified BACH1 and STAT1 as transcription factors involved in immune gene regulation in oligodendrocyte precursor cells (OPCs). A subset of immune genes presents bivalency of H3K4me3/H3K27me3 in OPCs, with Polycomb inhibition leading to their increased activation upon interferon gamma (IFN-g) treatment. Some MS susceptibility single-nucleotide polymorphisms (SNPs) overlap with these regulatory regions in mouse and human OLG. Treatment of mouse OPCs with IFN-g leads to chromatin architecture remodeling at these loci and altered expression of interacting genes. Thus, the susceptibility for MS may involve OLG, which therefore constitutes novel targets for immunological based therapies for MS.
15.	Neves, Inês, et al. (author) Paired glioblastoma cell cultures of the fluorescent bulk tumor and non-fluorescent tumor margin display differential phenotypes and cell states across patients Other publication (other academic/artistic)abstract Glioblastoma is an aggressive and therapy-resistant primary brain tumor with a dismal prognosis. The inevitable recurrence is in almost all patients in contact with the resection cavity, suggesting the local peritumoral area as its origin. Glioblastoma cells of this region have seldom been studied and few authenticated models exist. We have explanted matched tissue samples from the bulk tumor and local tumor edge of 13 glioblastoma patients of which 7 were sustainable beyond passage 6. Each edge culture was more invasive and less self-renewing and tumorigenic compared to its paired bulk culture. Three pairs of edge and bulk cultures were profiled with a combined single nucleus (sn) RNA- and ATAC-sequencing. Transcriptome analysis displayed for all patients a shift towards AC-MES cell states in the edge cultures. Chromatin-accessibility profiles uncovered differential regulatory networks with edge cells being enriched for transcription factor (TF) motifs of invasion, neurons, and immune cells. We propose that edge cells have been epigenetically reprogrammed by their unique interactions with various cell types in the peritumoral region. The fact that glioblastoma edge cells display distinct epigenetic regulation compared to their bulk tumor cells has implications for therapy development that should be targeted to and tested on the relapse-causing glioblastoma edge cells.
16.	Probst, Hans Christian, et al. (author) Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues 2023 In: European Journal of Immunology. - : Wiley. - 0014-2980 .- 1521-4141. ; 53:11 Journal article (peer-reviewed)abstract This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.
17.	Schuster, Jens, Assistant Professor, 1972-, et al. (author) Epigenetic Insights into GABAergic development in Dravet Syndrome iPSC and Therapeutic Implications Other publication (other academic/artistic)abstract Dravet syndrome (DS) is a devastating early onset refractory epilepsy syndrome caused by variants in the SCN1A gene. A disturbed GABAergic interneuron function is implicated in the progression to DS but the underlying developmental and pathophysiological mechanisms remain elusive, in particularly at the chromatin level. In this study, we utilized induced pluripotent stem cells (iPSCs) derived from DS cases and healthy donors to model disease-associated epigenetic abnormalities of GABAergic development. Employing the ATAC-seq technique, we assessed chromatin accessibility at multiple time points (Day 0, Day 19, Day 35, and Day 65) of GABAergic differentiation. Additionally, we elucidated the effects of the commonly used anti-seizure drug valproic acid (VPA) on chromatin accessibility in GABAergic cells. The distinct dynamics in chromatin profile of DS iPSC predicted accelerated early GABAergic development, evident at D19, and diverged further from the pattern in control iPSC with continued differentiation, indicating a disrupted GABAergic maturation. Exposure to VPA at D65 reshaped the chromatin landscape at a variable extent in different iPSC-lines and rescued the observed dysfunctional development in some DS iPSC-GABA. This study provides the first comprehensive investigation on the chromatin landscape of GABAergic differentiation in DS-patient iPSC, offering valuable insights into the epigenetic dysregulations associated with interneuronal dysfunction in DS. Moreover, our detailed analysis of the chromatin changes induced by VPA in iPSC-GABA holds the potential to improve development of personalized and targeted anti-epileptic therapies.
18.	Wu, Sihan, et al. (author) Circular ecDNA promotes accessible chromatin and high oncogene expression 2019 In: Nature. - : NATURE PUBLISHING GROUP. - 0028-0836 .- 1476-4687. ; 575:7784, s. 699-703 Journal article (peer-reviewed)abstract Oncogenes are commonly amplified on particles of extrachromosomal DNA (ecDNA) in cancer(1,2), but our understanding of the structure of ecDNA and its effect on gene regulation is limited. Here, by integrating ultrastructural imaging, long-range optical mapping and computational analysis of whole-genome sequencing, we demonstrate the structure of circular ecDNA. Pan-cancer analyses reveal that oncogenes encoded on ecDNA are among the most highly expressed genes in the transcriptome of the tumours, linking increased copy number with high transcription levels. Quantitative assessment of the chromatin state reveals that although ecDNA is packaged into chromatin with intact domain structure, it lacks higher-order compaction that is typical of chromosomes and displays significantly enhanced chromatin accessibility. Furthermore, ecDNA is shown to have a significantly greater number of ultra-long-range interactions with active chromatin, which provides insight into how the structure of circular ecDNA affects oncogene function, and connects ecDNA biology with modern cancer genomics and epigenetics.
19.	Xie, Liangqi, et al. (author) 3D ATAC-PALM : super-resolution imaging of the accessible genome 2020 In: Nature Methods. - : NATURE PUBLISHING GROUP. - 1548-7091 .- 1548-7105. ; 17:4, s. 430-436 Journal article (peer-reviewed)abstract 3D ATAC-PALM integrates ATAC with super-resolution imaging for nanoscale views of the accessible genome. When combined with FISH, protein fluorescence and genetic perturbation, the method enables investigation of accessible chromatin in situ. To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA-fluorescence in situ hybridization (FISH), RNA-FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.
20.	Xie, Liangqi, et al. (author) BRD2 compartmentalizes the accessible genome 2022 In: Nature Genetics. - : Springer Nature. - 1061-4036 .- 1546-1718. ; 54:4, s. 481-491 Journal article (peer-reviewed)abstract BRD2 facilitates mixing and compartmentalization of active chromatin upon cohesin depletion. BRD2's function is counteracted by cohesin and BRD4. Mammalian chromosomes are organized into megabase-sized compartments that are further subdivided into topologically associating domains (TADs). While the formation of TADs is dependent on cohesin, the mechanism behind compartmentalization remains enigmatic. Here, we show that the bromodomain and extraterminal (BET) family scaffold protein BRD2 promotes spatial mixing and compartmentalization of active chromatin after cohesin loss. This activity is independent of transcription but requires BRD2 to recognize acetylated targets through its double bromodomain and interact with binding partners with its low-complexity domain. Notably, genome compartmentalization mediated by BRD2 is antagonized on the one hand by cohesin and on the other hand by the BET homolog protein BRD4, both of which inhibit BRD2 binding to chromatin. Polymer simulation of our data supports a BRD2-cohesin interplay model of nuclear topology, in which genome compartmentalization results from a competition between loop extrusion and chromatin-state-specific affinity interactions.
21.	Yu, Yawei, et al. (author) Ferromagnetism with in-plane magnetization, Dirac spin-gapless semiconducting properties, and tunable topological states in two-dimensional rare-earth metal dinitrides 2022 In: Physical Review B. - : American Physical Society. - 2469-9950 .- 2469-9969. ; 105:2 Journal article (peer-reviewed)abstract Since the successful synthesis of bulk single crystals MoN2 and ReN2, which have a layered structure, transition-metal dinitrides have attracted considerable attention in recent years. Here, we focus on rare-earth metal (Rem) elements, and propose seven stable Rem dinitride monolayers with a 1T structure, namely, 1T-RemN2. We use first-principles calculations, and find that these monolayers have a ferromagnetic ground state with in-plane magnetization. Without spin-orbit coupling (SOC), the band structures are spin-polarized with Dirac points at the Fermi level. Remarkably, the 1T-LuN2 monolayer exhibits an isotropic magnetocrystalline anisotropy energy in the xy plane with in-plane magnetization, indicating easy tunability of the magnetization direction. When rotating the magnetization vector in the xy plane, we propose a model that accurately describes the variation of the SOC band gap and the two possible topological states (Weyl-like semimetal and Chern insulator states) whose properties are tunable. The Weyl-like semimetal state is a critical point between the two Chern insulator states with opposite sign of the Chern numbers (+/- 1). The nontrivial band gap (up to 60.3 meV) and the Weyl-like semimetal state are promising for applications in spintronic devices.
22.	Zhang, Hua, et al. (author) Profiling chromatin accessibility in formalin-fixed paraffin-embedded samples 2022 In: Genome Research. - : Cold Spring Harbor Laboratory Press (CSHL). - 1088-9051 .- 1549-5469. ; 32:1, s. 150-161 Journal article (peer-reviewed)abstract Archived formalin-fixed paraffin-embedded (FFPE) samples are the global standard format for preservation of the majority of biopsies in both basic research and translational cancer studies, and profiling chromatin accessibility in the archived FFPE tissues is fundamental to understanding gene regulation. Accurate mapping of chromatin accessibility from FFPE specimens is challenging because of the high degree of DNA damage. Here, we first showed that standard ATAC-seq can be applied to purified FFPE nuclei but yields lower library complexity and a smaller proportion of long DNA fragments. We then present FFPE-ATAC, the first highly sensitive method for decoding chromatin accessibility in FFPE tissues that combines Tn5-mediated transposition and T7 in vitro transcription. The FFPE-ATAC generates high-quality chromatin accessibility profiles with 500 nuclei from a single FFPE tissue section, enables the dissection of chromatin profiles from the regions of interest with the aid of hematoxylin and eosin (H&E) staining, and reveals disease-associated chromatin regulation from the human colorectal cancer FFPE tissue archived for >10 yr. In summary, the approach allows decoding of the chromatin states that regulate gene expression in archival FFPE tissues, thereby permitting investigators to better understand epigenetic regulation in cancer and precision medicine.
23.	Zhang, Juqing, et al. (author) Super-enhancers conserved within placental mammals maintain stem cell pluripotency 2022 In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 119:40 Journal article (peer-reviewed)abstract Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share an evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolu-tion in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that displayed remarkable conservation in pla-cental mammals and were sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR-Cas9 approach severely impaired stem cell pluripotency. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of plu-ripotency as well as species-specific modulation of the pluripotency-associated regula-tory networks in mammals.
24.	Zhang, Juqing, et al. (author) Super enhancers-Functional cores under the 3D genome 2021 In: Cell Proliferation. - : John Wiley & Sons. - 0960-7722 .- 1365-2184. ; 54:2 Research review (peer-reviewed)abstract Complex biochemical reactions take place in the nucleus all the time. Transcription machines must follow the rules. The chromatin state, especially the three-dimensional structure of the genome, plays an important role in gene regulation and expression. The super enhancers are important for defining cell identity in mammalian developmental processes and human diseases. It has been shown that the major components of transcriptional activation complexes are recruited by super enhancer to form phase-separated condensates. We summarize the current knowledge about super enhancer in the 3D genome. Furthermore, a new related transcriptional regulation model from super enhancer is outlined to explain its role in the mammalian cell progress.
25.	Zhao, Linxuan, et al. (author) A Highly Sensitive Method to Efficiently Profile the Histone Modifications of FFPE Samples 2022 In: Bio-protocol. - : Bio-Protocol. - 2331-8325. ; 12:10 Journal article (peer-reviewed)abstract The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10-20 tissue sections or whole tissue blocks, which prevents better resolved analyses. Nevertheless, it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissue of interest are limited. Here, we present FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), highly sensitive method to efficiently profile histone modifications in FFPE tissue by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7-pA-Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We showed a very small piece of FFPE tissue section containing similar to 4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. In archived FFPE human colorectal and human glioblastoma cancer tissue, H3K27ac FACT-seq revealed disease specific super enhancers. In summary, FACT-seq allows researchers to decode histone modifications like H3K27ac and H3K27me3 in archival FFPE tissues with high sensitivity, thus allowing us to understand epigenetic regulation.
26.	Zhao, Linxuan, et al. (author) FACT-seq : profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers 2021 In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 49:21 Journal article (peer-reviewed)abstract The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10-20 tissue sections or whole tissue blocks, which prevents better resolved analyses. But it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissues of interest are limited. Here, we present FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), the first highly sensitive method to efficiently profile histone modifications in FFPE tissues by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7-pA-Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We proved a very small piece of FFPE tissue section containing similar to 4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. H3K27ac FACT-seq revealed disease-specific super enhancers in the archived FFPE human colorectal and human glioblastoma cancer tissue. In summary, FACT-seq allows decoding the histone modifications in archival FFPE tissues with high sensitivity and help researchers to better understand epigenetic regulation in cancer and human disease.
27.	Zhao, Xue-Ke, et al. (author) Focal amplifications are associated with chromothripsis events and diverse prognoses in gastric cardia adenocarcinoma 2021 In: Nature Communications. - : Springer Nature. - 2041-1723. ; 12 Journal article (peer-reviewed)abstract The role of focal amplifications and extrachromosomal DNA (ecDNA) is unknown in gastric cardia adenocarcinoma (GCA). Here, we identify frequent focal amplifications and ecDNAs in Chinese GCA patient samples, and find focal amplifications in the GCA cohort are associated with the chromothripsis process and may be induced by accumulated DNA damage due to local dietary habits. We observe diverse correlations between the presence of oncogene focal amplifications and prognosis, where ERBB2 focal amplifications positively correlate with prognosis and EGFR focal amplifications negatively correlate with prognosis. Large-scale ERBB2 immunohistochemistry results from 1668 GCA patients show survival probability of ERBB2 positive patients is lower than that of ERBB2 negative patients when their surviving time is under 2 years, however, the tendency is opposite when their surviving time is longer than 2 years. Our observations indicate that the ERBB2 focal amplifications may represent a good prognostic marker in GCA patients.
28.	Zhu, Zhenshuo, et al. (author) Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to define a pluripotency gene regulatory network 2021 In: The FASEB Journal. - : John Wiley & Sons. - 0892-6638 .- 1530-6860. ; 35:6 Journal article (peer-reviewed)abstract The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs.
29.	Zou, Zhixiang, et al. (author) Design of Filter-Based Stabilizing Control for PLL-Synchronized Converters 2024 In: IEEE Transactions on Industrial Electronics. - : Institute of Electrical and Electronics Engineers (IEEE). - 0278-0046 .- 1557-9948. ; , s. 1-12 Journal article (peer-reviewed)abstract The phase-locked loop (PLL) has an important effect on the system stability of grid-connected converters in weak grids. In the literature, parameter tuning methods and stabilizing control strategies have been proposed to deal with this problem. However, most of them target at tuning parameters of PLL or mitigating the negative damping as well as the phase lag introduced by the PLL, while the interactions between the PLL and the current control and the cross coupling effects are still not fully explored in the design procedure of stabilizing control. To address this issue, this article extends the study of the interaction between the PLL and the current control using a multiple-input multiple-output (MIMO) model, and then proposes a design criterion of the stabilizing control using design-oriented analysis from the perspective of MIMO impedance model. A category of stabilizing control schemes that uses different types of biquad filters is proposed following the criterion, which not only improves system stability but also removes the coupling between the PLL and the current control according to the sensitivity analysis. Hardware-in-the-loop and experimental results are provided to verify the effectiveness of the proposed control schemes.

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