Porcine circovirus type 2 infection promotes the SUMOylation of nucleophosmin-1 to facilitate the viral circular single-stranded DNA replication.

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.

Profiling chromatin accessibility in formalin-fixed paraffin-embedded samples.

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.

Decoding Connectivity Map-based drug repurposing for oncotherapy.

The rising global burden of cancer has driven considerable efforts into the research and development of effective anti-cancer agents. Fortunately, with impressive advances in transcriptome profiling technology, the Connectivity Map (CMap) database has emerged as a promising and powerful drug repurposing approach. It provides an important platform for systematically discovering of the associations among genes, small-molecule compounds and diseases, and elucidating the mechanism of action of drug, contributing toward efficient anti-cancer pharmacotherapy. Moreover, CMap-based computational drug repurposing is gaining attention because of its potential to overcome the bottleneck constraints faced by traditional drug discovery in terms of cost, time and risk. Herein, we provide a comprehensive review of the applications of drug repurposing for anti-cancer drug discovery and summarize approaches for computational drug repurposing. We focus on the principle of the CMap database and novel CMap-based software/algorithms as well as their progress achieved for drug repurposing in the field of oncotherapy. This article is expected to illuminate the emerging potential of CMap in discovering effective anti-cancer drugs, thereby promoting efficient healthcare for cancer patients.

Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy.

Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-ß (PDGFRB) as a potential therapeutic target.

Circular ecDNA promotes accessible chromatin and high oncogene expression.

Oncogenes are commonly amplified on particles of extrachromosomal DNA (ecDNA) in cancer1,2, 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.

Sensing Self and Foreign Circular RNAs by Intron Identity.

Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail with largely unknown functions. Here we show that transfection of purified in vitro generated circRNA into mammalian cells led to potent induction of innate immunity genes and confers protection against viral infection. The nucleic acid sensor RIG-I is necessary to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of a 5' triphosphate, double-stranded RNA structure, or the primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA and highlight introns-the predominant output of mammalian transcription-as arbiters of self-nonself identity.

Super-enhancers conserved within placental mammals maintain stem cell pluripotency.

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 evolution 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 placental 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 pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals.

3D ATAC-PALM: super-resolution imaging of the accessible genome.

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.

Deciphering the functional roles of transporter proteins in subcellular metal transportation of plants.

MAIN CONCLUSION: The role of transporters in subcellular metal transport is of great significance for plants in coping with heavy metal stress and maintaining their proper growth and development. Heavy metal toxicity is a serious long-term threat to plant growth and agricultural production, becoming a global environmental concern. Excessive heavy metal accumulation not only damages the biochemical and physiological functions of plants but also causes chronic health hazard to human beings through the food chain. To deal with heavy metal stress, plants have evolved a series of elaborate mechanisms, especially a variety of spatially distributed transporters, to strictly regulate heavy metal uptake and distribution. Deciphering the subcellular role of transporter proteins in controlling metal absorption, transport and separation is of great significance for understanding how plants cope with heavy metal stress and improving their adaptability to environmental changes. Hence, we herein introduce the detrimental effects of excessive common essential and non-essential heavy metals on plant growth, and describe the structural and functional characteristics of transporter family members, with a particular emphasis on their roles in maintaining heavy metal homeostasis in various organelles. Besides, we discuss the potential of controlling transporter gene expression by transgenic approaches in response to heavy metal stress. This review will be valuable to researchers and breeders for enhancing plant tolerance to heavy metal contamination.

PARP1- and CTCF-Mediated Interactions between Active and Repressed Chromatin at the Lamina Promote Oscillating Transcription.

Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.

FACT-seq: profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers.

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 ∼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.

Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to define a pluripotency gene regulatory network.

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.

ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing.

Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.

Pentoxifylline intervention and drug action in scalping forehead flap for large temporal cutaneous defects.

Pentoxifylline is widely used in the treatment of cerebrovascular disease, at present, there are a variety of drugs in the clinical expansion of the role of the skin flap, by improving or promoting blood circulation, to ensure the survival of the flap. We designed scalping forehead flap to reconstruct large temporal cutaneous defects. This flap is reliable and can give us excellent postoperative result. This design has several advantages and is worth to be promoted. The scalping forehead flap we designed is reliable. The posterior auricular artery and it branches provided enough blood supply to the scalp and forehead tissue of the flap. The frontal muscle also make the flap have a high survival rate. Large temporal cutaneous defects can be reconstructed by this novel scalping forehead flap we described. This technique is useful especially for patients who suffered malignant tumor and can not choose free flap.

Clonorchis sinensis infection in a non­endemic area: A case report.

Non-endemic Clonorchis sinensis infection is challenging to diagnose. The present study reports the case of a 40-year-old female patient with severe epigastric pain, initially suspected to be a liver lesion, who was admitted to The Affiliated Hospital of Zunyi Medical University (Zunyi, China). A combination of diagnostic procedures, including computed tomography and magnetic resonance imaging, revealed an abnormality in the left hepatic lobe. Postoperative evaluation and the epidemiologic history of the patient (consumption of raw fish slices) revealed characteristics of Clonorchis sinensis infection, including chronic bile duct inflammation and eosinophilic liver infiltration. The present case highlights the unexpected emergence of this disease outside of recognized endemic areas and advocates clinical vigilance. Even in non-endemic areas, individuals should be reminded not to eat raw fish and shrimp.

Interactions between methyl octabromo ether flame retardant and expanded polystyrene microplastics in the photoaging process.

Expanded polystyrene (EPS) plastic is widely used because of its low density and lightweight properties, enabling it to float on water and increase its exposure to sunlight. In this study, we simulated the photoaging process of flame retardant-added EPS (FR-EPS) and common original EPS (OR-EPS) microplastic (MP) particles with and without methyl octabromoether flame retardant (MOBE) in the laboratory to explore the effect of MOBE on the photodegradation of EPS. Results showed that MOBE accelerated size reduction and surface hole formation on the particles, hastening the shedding and replacement of particle surfaces. FR-EPS particles exhibited a weight loss exceeding that of OR-EPS, reaching 40.85 ± 3.72% after 36 days of irradiation. Moreover, rapid physical peeling of the FR-EPS surface was accompanied by continuous chemical oxidation and fluctuations of the carbonyl index and O/C ratio. A diffusion model based on Fick's second law fitted well for the concentration of MOBE remaining in FR-EPS particles. MOBE's sensitivity to direct photochemical reactions inhibited the early-stage photoaging of EPS MP particles by competing for photons. However, MOBE as chromophores could absorb photons and produce •OH to promote the aging of EPS. Moreover, the capacity of EPS to absorb light energy also accelerated MOBE degradation. These findings suggested that the photoaging behavior of commercial EPS products containing flame retardants in the environment is quite different from that of pure EPS, indicating that additive-plastic interactions significantly alter MP fate and environmental risks.

Genome-wide DNA methylation-analysis of blastic plasmacytoid dendritic cell neoplasm identifies distinct molecular features.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) constitutes a rare and aggressive malignancy originating from plasmacytoid dendritic cells (pDCs) with a primarily cutaneous tropism followed by dissemination to the bone marrow and other organs. We conducted a genome-wide analysis of the tumor methylome in an extended cohort of 45 BPDCN patients supplemented by WES and RNA-seq as well as ATAC-seq on selected cases. We determined the BPDCN DNA methylation profile and observed a dramatic loss of DNA methylation during malignant transformation from early and mature DCs towards BPDCN. DNA methylation profiles further differentiate between BPDCN, AML, CMML, and T-ALL exhibiting the most striking global demethylation, mitotic stress, and merely localized DNA hypermethylation in BPDCN resulting in pronounced inactivation of tumor suppressor genes by comparison. DNA methylation-based analysis of the tumor microenvironment by MethylCIBERSORT yielded two, prognostically relevant clusters (IC1 and IC2) with specific cellular composition and mutational spectra. Further, the transcriptional subgroups of BPDCN (C1 and C2) differ by DNA methylation signatures in interleukin/inflammatory signaling genes but also by higher transcription factor activity of JAK-STAT and NFkB signaling in C2 in contrast to an EZH2 dependence in C1-BPDCN. Our integrative characterization of BPDCN offers novel molecular insights and potential diagnostic applications.

ATAC-See: A Tn5 Transposase-Mediated Assay for Detection of Chromatin Accessibility with Imaging.

Assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that enables direct imaging of the accessible genome in situ and deep sequencing to reveal the identity of the imaged elements. Here we image spatial organization of the accessible genome in HT1080 cells with this method.

Inverse association between type 2 diabetes and hepatocellular carcinoma in East Asian populations.

Aims: To investigate the potential association between type 2 diabetes (T2D) and hepatocellular carcinoma (HCC) in East Asian populations using Mendelian randomization (MR) analyses. Methods: Bidirectional Mendelian randomization (MR) studies were conducted using summary statistics from genome-wide association studies (GWAS) related to T2D and HCC. The potential effects of confounders such as chronic hepatitis B, chronic hepatitis C, body mass index, and alcohol intake frequency were corrected using a multivariate MR study. Various MR methods, including the inverse variance weighted (IVW) approach, were used to estimate the associations between T2D and HCC. Sensitivity analysis and assessment of heterogeneity were performed to ensure the robustness of the results. Results: In the forward MR study, the IVW approach of MR analysis suggested an inverse association between T2D and HCC, with a risk odds ratio of 0.8628 (95% confidence interval [CI], 0.7888-0.9438). Furthermore, even after adjusting for BMI, chronic hepatitis B, and alcohol intake frequency, this study still supports the inverse association between T2D and HCC. Additional MR methods provided further support for this relationship. Sensitivity analysis and assessment of heterogeneity confirmed the robustness of the results. The reverse MR analysis did not show a clear impact of genetic liability to HCC on reduced risk of T2D(OR=0.9788; 95% CI, 0.9061-1.0574). Conclusion: This study provides evidence of an inverse association between T2D and HCC in East Asian populations using MR analysis. Further studies are warranted to validate these findings.

Heparanase Modulates Chromatin Accessibility.

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.