Transcriptional Activity Research Articles

Duck hepatitis A virus 1-encoded 2B protein disturbs ion and organelle homeostasis to promote NF-κB/NLRP3-mediated inflammatory response

Previous studies by our group and others have highlighted the critical role of hyperinflammation in the pathogenicity of duck hepatitis A virus 1 (DHAV-1), an avian picornavirus that has caused significant devastation in the duck industry worldwide for decades. However, the precise mechanisms by which DHAV-1 infection regulates the inflammatory responses, particularly the production of IL-1β, remain poorly understood. In this study, we demonstrate that DHAV-1 infection triggers NF-κB- and NLRP3 inflammasome-mediated IL-1β production. Mechanistically, DHAV-1 infection, particularly its replication and translation, disrupts cellular homeostasis of Ca2+, K+, ROS and cathepsin, which act cooperatively as assembly signals for NLRP3 inflammasome activation. By screening DHAV-1-encoded proteins, we identified that the viroporin 2B dominates NF-κB as well as NLRP3 inflammasome activation. Mutation analysis revealed that I43 within the 2B protein is the key amino acid for Ca2+ mobilization and subsequent activation of NF-κB transcriptional activity and NLRP3 inflammasome. Moreover, DHAV-1 infection and the 2B protein activate the MAVS- and MyD88-NF-κB pathways by relay, providing the necessary priming signals for NLRP3 inflammasome activation. In summary, our findings elucidate a mechanism through which DHAV-1 triggers inflammatory responses via NF-κB/NLRP3 inflammasome activation, offering new perspectives on DHAV-1 pathogenesis and informing the development of targeted anti-DHAV-1 treatments.

HOXC10 promotes hypertrophic scar fibroblast fibrosis through the regulation of STMN2 and the TGF-β/Smad signaling pathway.

The pathophysiology of hypertrophic scar (HS) shares similarities with cancer. HOXC10, a gene significantly involved in cancer development, exhibits higher expression levels in HS than in normal skin (NS), suggesting its potential role in HS regulation. And the precise functions and mechanisms by which HOXC10 influences HS require further clarification. Gene and protein expressions were analyzed using raeal-time quantitative polymerase chain reaction (RT-qPCR) and western blot techniques. Cell proliferation and migration were evaluated using EdU proliferation assays, CCK-8 assays, scratch assays, and Transwell assays. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were conducted to investigate the interactions between HOXC10 and STMN2. HOXC10 and STMN2 expression levels were significantly higher in HS tissues compared with NS tissues. Silencing HOXC10 led to decreased activation, proliferation, migration, and fibrosis in hypertrophic scar fibroblasts (HSFs). Our findings also indicate that HOXC10 directly targets STMN2. The promotional effects of HOXC10 knockdown on HSF activation, proliferation, migration, and fibrosis were reversed by STMN2 overexpression. We further demonstrated that HOXC10 regulates HSF activity through the TGF-β/Smad signaling pathway. HOXC10 induces the activation and fibrosis of HSFs by promoting the transcriptional activation of STMN2 and engaging the TGF-β/Smad signaling pathway. This study suggests that HOXC10 could be a promising target for developing treatments for HS.

Magnesium Lithospermate B Inhibits Colorectal Cancer Cell Progression Through JAK2-STAT3 Signaling.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide. The discovery of new effective therapeutic drugs is always a priority. Magnesium lithospermate B (MLB), a native polyphenol acid, is the major component of the aqueous extracts from Danshen, a traditional Chinese medicine derived from the dry root and rhizome of Salvia miltiorrhiza. MLB has been reported to have antioxidant, anti-inflammatory, and ion channel-regulating activities in several diseases, including cardiovascular, renal, and neuronal diseases. However, the effect of MLB on cancer progression has not been reported. In this study, a series of cellular and molecular experiments were conducted on two CRC cell lines (HCT116 and SW480) to investigate the effects of. The results demonstrated that MLB exerted inhibitory effects on cell proliferation, migration, and invasion. The administration of 50 mg/kg MLB inhibited tumor growth in HCT116 cells in xenografted models. Importantly, we found that MLB treatment inhibited the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) signaling pathway, and activation of JAK2-STAT3 signaling with interleukin 6 or overexpression STAT3 significantly suppressed the inhibitory effect of MLB. These findings provide evidence that MLB could inhibit CRC cell progression in vitro and might serve as a potential therapeutic drug for the treatment of CRC.

Global coupling of R-loopdynamics with RNA polymerase II modulates gene expression and early development of Drosophila.

R-loops are involved in many biological processes in cells, yet the regulatory principles for R-loops in vivo and their impact on development remain to be explored. Here, we modified the CUT&Tag strategy to profile R-loops in Drosophila at multiple developmental stages. While high GC content promotes R-loop formation in mammalian cells, it is not required in Drosophila. In contrast, RNAPII abundance appears to be a universal inducing factor for R-loop formation, including active promoters and enhancers, and H3K27me3 decorated repressive regions and intergenic repeat sequences. Importantly, such a regulatory relationship is dynamically maintained throughout development, and development-related transcription factors may regulate RNAPII activation and R-loop dynamics. By ablating Spt6, we further showed the global R-loop induction coupled with RNAPII pausing. Importantly, depending on the gene length, genes underwent up- or down-regulation, both of which were largely reversed by rnh1 overexpression, suggesting that R-loops play a significant role in the divergent regulation of transcription by Spt6 ablation. DNA damage, defects in survival, and cuticle development were similarly alleviated by rnh1 overexpression. Altogether, our findings indicate that dynamic R-loop regulation is dictated by RNAPII pausing and transcription activity, and plays a feedback role in gene regulation, genome stability maintenance, and Drosophila development.

TRPV1 alleviates APOE4-dependent microglial antigen presentation and T cell infiltration in Alzheimer's disease

BackgroundPersistent innate and adaptive immune responses in the brain contribute to the progression of Alzheimer’s disease (AD). APOE4, the most important genetic risk factor for sporadic AD, encodes apolipoprotein E4, which by itself is a potent modulator of immune response. However, little is known about the immune hub that governs the crosstalk between the nervous and the adaptive immune systems. Transient receptor potential vanilloid type 1 (TRPV1) channel is a ligand-gated, nonselective cation channel with Ca2+ permeability, which has been proposed as a neuroprotective target in AD.MethodsUsing Ca2+-sensitive dyes, dynamic changes of Ca2+ in microglia were measured, including exogenous Ca2+ uptake and endoplasmic reticulum Ca2+ release. The mRFP-GFP-tagged LC3 plasmid was expressed in microglia to characterize the role of TRPV1 in the autophagic flux. Transcriptomic analyses and flow cytometry were performed to investigate the effects of APOE4 on brain microglia and T cells from APOE-targeted replacement mice with microglia-specific TRPV1 gene deficiency.ResultsBoth APOE4 microglia derived from induced pluripotent stem cells of AD patients and APOE4-related tauopathy mouse model showed significantly increased cholesterol biosynthesis and accumulation compared to their APOE3 counterparts. Further, cholesterol dysregulation was associated with persistent activation of microglia and elevation of major histocompatibility complex II-dependent antigen presentation in microglia, subsequently accompanied by T cell infiltration. In addition, TRPV1-mediated transient Ca2+ influx mitigated cholesterol biosynthesis in microglia by suppressing the transcriptional activation of sterol regulatory element-binding protein 2, promoted autophagic activity and reduced lysosomal cholesterol accumulation, which were sufficient to resolve excessive immune response and neurodegeneration in APOE4-related tauopathy mouse model. Moreover, microglia-specific deficiency of TRPV1 gene accelerated glial inflammation, T cell response and associated neurodegeneration in an APOE4-related tauopathy mouse model.ConclusionsThe findings provide new perspectives for the treatment of APOE4-dependent neurodegeneration including AD.

A kind of protein affect the expression of bnk

The maternal gene products control the initial stages of embryo development in all animals. The role of controlling embryogenesis is taken up by the zygotic genome at some point after fertilization. This paper seeks to shed light on research on the effect of the zinc-finger protein Zelda on the expression of bnk in fruit flies. The zinc-finger protein Zelda (Zld) is a transcriptional activator of the early zygotic genome and plays an important role in early embryonic development. (Liang,2008) This paper mainly demonstrates the influence of Zld on the bnk, which encodes a kind of protein that controls the actinic organization and the time of cellular basal closure. In the absence of Zelda, the expression of bnk has subtle changes. This work highlights the importance of the zinc-finger protein Zelda in the expression of the bnk in fruit flies and also details the methodology followed in the research on the bnk expression in fruit flies, the results, and the conclusions drawn from the research work, showing the importance of Zld to bnk, especially in early embryonic development stages. The significance of this research is to open potential avenues for research on identifying genes whose expression has been influenced to some extent by Zelda. Also, understanding the effect of Zelda on bnk expression can aid in the manipulation of the two variables to establish a functional interaction.

Mutations of PDS5 genes enhance TAD-like domain formation Arabidopsis thaliana

In eukaryotes, topologically associating domains (TADs) organize the genome into functional compartments. While TAD-like structures are common in mammals and many plants, they are challenging to detect in Arabidopsis thaliana. Here, we demonstrate that Arabidopsis PDS5 proteins play a negative role in TAD-like domain formation. Through Hi-C analysis, we show that mutations in PDS5 genes lead to the widespread emergence of enhanced TAD-like domains throughout the Arabidopsis genome, excluding pericentromeric regions. These domains exhibit increased chromatin insulation and enhanced chromatin interactions, without significant changes in gene expression or histone modifications. Our results suggest that PDS5 proteins are key regulators of genome architecture, influencing 3D chromatin organization independently of transcriptional activity. This study provides insights into the unique chromatin structure of Arabidopsis and the broader mechanisms governing plant genome folding.

LncRNA evf-2 Exacerbates Podocyte Injury in Diabetic Nephropathy by Inducing Cell Cycle Re-entry and Inflammation Through Distinct Mechanisms Triggered by hnRNPU.

Albuminuria is a hallmark of diabetic nephropathy (DN). Podocyte injury significantly contributes to proteinuria in DN. Our study found that lncRNA EVF-2 is upregulated in podocytes of DN patients, correlating with cell cycle re-entry and inflammation. Specific knockout or knockdown of lncRNA evf-2 in diabetic mice or cultured podocytes alleviated podocyte injury associated with these processes. RNA sequencing of evf-2-overexpressing podocytes unveiled a predominant enrichment of upregulated mRNAs in cell cycle and inflammation pathways, with alternative splicing in cell cycle-related mRNAs Ccnb1 and Tacc3. Chromatin isolation by RNA purification-mass spectrometry (ChIRP-MS) analysis highlighted the involvement of ribonucleoprotein complex and mRNA processing-related proteins, with hnRNPU as the main binding partner of evf-2 in spliceosomes. Knockdown of hnRNPU partially restored the upregulation of mRNAs induced by evf-2 overexpression, altering splice variants of Ccnb1 and Tacc3. This study is the first to reveal the splice variants of cell cycle-related genes in DN and elucidate the interaction between lncRNA evf-2 and hnRNPU. This interaction culminates in the upregulation of cell cycle-related genes and inflammatory factors through diverse pathways, potentially involving transcriptional activation, RNA stability modulation, alternative splicing or translational regulation. This highlights potential novel pathways for DN treatment.

Downregulation of the rice HRS1 HOMOLOG3 transcriptional repressor gene due to N deficiency directly co-activates ammonium and phosphate transporter genes.

Rice HRS1 HOMOLOG3 (OsHHO3) acts as a transcriptional repressor of AMMONIUM TRANSPORTER1 (OsAMT1) genes in rice; thus, reduced OsHHO3 expression in nitrogen (N)-deficient environments promotes ammonium uptake. In this study, we show that OsHHO3 also functions as a repressor of a specific subset of phosphate (Pi) transporter (PT) genes involved in the uptake and root-to-shoot translocation of Pi, including OsPT2, OsPT4, and OsPHO1;1. Consistently, disruption of OsHHO3 increased Pi uptake and Pi contents in shoots and roots, while overexpression of OsHHO3 generated the opposite effects. Furthermore, phosphorus (P) deficiency slightly decreased OsHHO3 expression, upregulating a specific subset of PT genes. However, N deficiency was more effective than P deficiency in suppressing OsHHO3 expression in roots, and unlike N deficiency-dependent activation of PT genes under the control of OsHHO3, the P deficiency-dependent activation of OsAMT1 genes was minimal. Interestingly, the simultaneous deficiency of both N and P promoted the OsHHO3-regulated expression of PT genes more significantly than the deficiency of either N or P, but diminished the expression of genes regulated by OsPHR2, a master regulator of Pi starvation-responsive transcriptional activation. Phenotypic analysis revealed that the inactivation and overexpression of OsHHO3 improved and reduced plant growth, respectively, under N-deficient and P-deficient conditions. These results suggest that OsHHO3 regulates a specific subset of PT genes independently of OsPHR2-mediated regulation and plays a critical role in the adaptation to diverse N and P environments.

Systematic Evaluation and Application of IDR Domain-Mediated Transcriptional Activation of NUP98 in Saccharomyces cerevisiae.

Implementing dynamic control over gene transcription to decouple cell growth is essential for regulating protein expression in microbial cells. However, the availability of efficient regulatory elements in Saccharomyces cerevisiae remains limited. In this study, we present a novel β-estradiol-inducible gene expression system, termed DEN. This system combines a DNA-binding domain with an estradiol-binding domain and an intrinsically disordered region (IDR) from NUP98. Comparative analysis shows that the DEN system outperforms IDRs from other proteins, achieving an approximately 60-fold increase in EGFP expression upon β-estradiol induction. Moreover, our system is tightly controlled; nontoxic gene expression makes it a powerful tool for rapid and precise modulation of target gene expression. This system holds great potential for unlocking new functionalities from existing proteins in future research.

Deciphering STAT3’s negative regulation of LHPP in ESCC progression through single-cell transcriptomics analysis

BackgroundEsophageal Squamous Cell Carcinoma (ESCC) remains a predominant health concern in the world, characterized by high prevalence and mortality rates. Advances in single-cell transcriptomics have revolutionized cancer research by enabling a precise dissection of cellular and molecular diversity within tumors.ObjectiveThis study aims to elucidate the cellular dynamics and molecular mechanisms in ESCC, focusing on the transcriptional influence of STAT3 (Signal Transducer and Activator of Transcription 3) and its interaction with LHPP, thereby uncovering potential therapeutic targets.MethodsSingle-cell RNA sequencing was employed to analyze 44,206 cells from tumor and adjacent normal tissues of ESCC patients, identifying distinct cell types and their transcriptional shifts. We conducted differential gene expression analysis to assess changes within the tumor microenvironment (TME). Validation of key regulatory interactions was performed using qPCR in a cohort of 21 ESCC patients and further substantiated through experimental assays in ESCC cell lines.ResultsThe study revealed critical alterations in cell composition and gene expression across identified cell populations, with a notable shift towards pro-tumorigenic states. A significant regulatory influence of STAT3 on LHPP was discovered, establishing a novel aspect of ESCC pathogenesis. Elevated levels of STAT3 and suppressed LHPP expression were validated in clinical samples. Functional assays confirmed that STAT3 directly represses LHPP at the promoter level, and disruption of this interaction by promoter mutations diminished STAT3's repressive effect.ConclusionThis investigation underscores the central role of STAT3 as a regulator in ESCC, directly impacting LHPP expression and suggesting a regulatory loop crucial for tumor behavior. The insights gained from our comprehensive cellular and molecular analysis offer a deeper understanding of the dynamics within the ESCC microenvironment. These findings pave the way for targeted therapeutic interventions focusing on the STAT3-LHPP axis, providing a strategic approach to improve ESCC management and prognosis.

LncRNA BRE-AS1 regulates the JAK2/STAT3-mediated inflammatory activation via the miR-30b-5p/SOC3 axis in THP-1 cells

Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators in numerous biological processes, including macrophage-mediated inflammatory responses, which play a critical role in the progress of diverse diseases. This study focuses on the regulatory function of lncRNA brain and reproductive organ-expressed protein (BRE) antisense RNA 1 (BRE-AS1) in modulating the inflammatory activation of monocytes/macrophages. Employing the THP-1 cell line as a model, we demonstrate that lipopolysaccharide (LPS) treatment significantly upregulates BRE-AS1 expression. Notably, specific knockdown of BRE-AS1 via siRNA transfection enhances LPS-induced expression of interleukin (IL)-6 and IL-1β, while not affecting tumor necrosis factor (TNF)-α levels. This selective augmentation of pro-inflammatory cytokine production coincides with increased phosphorylation of Janus kinase (JAK)2 and signal transducer and activator of transcription (STAT)3. Furthermore, BRE-AS1 suppression results in the downregulation of suppressor of cytokine signaling (SOCS)3, an established inhibitor of the JAK2/STAT3 pathway. Bioinformatics analysis identified binding sites for miR-30b-5p on both BRE-AS1 and SOCS3 mRNA. Intervention with a miR-30b-5p inhibitor and a synthetic RNA fragment that represents the miR-30b-5p binding site on BRE-AS1 attenuates the pro-inflammatory effects of BRE-AS1 knockdown. Conversely, a miR-30b-5p mimic replicated the BRE-AS1 attenuation outcomes. Our findings elucidate the role of lncRNA BRE-AS1 in modulating inflammatory activation in THP-1 cells via the miR-30b-5p/SOCS3/JAK2/STAT3 signaling pathway, proposing that manipulation of macrophage BRE-AS1 activity may offer a novel therapeutic avenue in diseases characterized by macrophage-driven pathogenesis.

HECT domain containing-3 (HECTD3) E3 ubiquitin ligase attenuates cardiac ischemia and hypertrophy in mice

Abstract HECT domain containing 3 (HECTD3) is one of the E3 ubiquitin ligases, the cardiac function of which is not completely understood yet. We aimed here to understand the role of HECTD3 in cardiomyocytes using loss- and gain-of-function approaches in in vitro and in vivo models. Interestingly, we found that HECTD3 was significantly downregulated in cardiac tissue obtained from human patients suffering from cardiac hypertrophy and ischemia. We thus employed loss- and gain-of-function approaches in neonatal rat cardiomyocytes (NRVCMs) to get further insights into potential cardiac function of HECTD3. Overexpression of HECTD3 in NRVCMs attenuated cardiomyocyte hypertrophy and lipopolysaccharide (LPS) or interferon-γ (IFN- γ) induced cellular inflammation; its knockdown on the other hand aggravated these effects. Mechanistically, using Yeast two-hybrid screen, mass-spectrometry, and transcriptomics analyses, we identified Small Ubiquitin like Modifier 2 (SUMO2) Signal Transducer and Activator of Transcription-1 (STAT1) as novel substrates for HECTD3, regulation of which resulted into attenuation of hypertrophy and inflammation, respectively. To translate these in vitro results in vivo, we employed Adeno-associated virus-9 (AAV-9) mediated gene therapy approach to overexpress HECTD3 specifically in the heart of mouse models of cardiac ischemia due to permanent ligation of the left anterior descending artery (LAD), and cardiac hypertrophy due to transverse aortic constriction (TAC). Overexpression of HECTD3 in mice, though moderate but significantly improved cardiac function upon ischemic or hypertrophic stress compared to respective control groups. Importantly, in vitro findings were consistent in vivo, where AAV-9 mediated overexpression of HECTD3 substantially reduced cardiac hypertrophy and fibrosis, improved cardiac function as assessed by echocardiography, inhibited activation of STAT1-mediated downstream signaling, and attenuated infiltration of inflammatory cells to the heart after TAC or LAD ligation. This data indicates that HECTD3 is a novel modulator of cardiac ischemia, hypertrophy, and inflammation via regulation of SUMO2 and STAT1-signaling. In conclusion, we report here a novel cardioprotective mechanism involving the E3 ubiquitin ligase HECTD3, which exerts anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and its SUMOylating target STAT1. We believe that this "dual pathway" inhibition exhibits translational importance and could further be exploited for the prevention and/or therapy of heart failure due to cardiac ischemia or hypertrophy.

Transcriptome-wide association study discovers functional long non-coding RNAs in coronary artery disease

Abstract Genetically regulated long non-coding RNAs (lncRNAs) in coronary artery disease (CAD) have not been investigated. To discover CAD-relevant lncRNAs, we performed the transcriptome-wide association study (TWAS) using a subset genomic data of CARDIoGRAMplusC4D Consortium and transcriptomic data of nine CAD-relevant tissue types from Genotyped Tissue Expression (GTEx) and Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) projects. We identified 20 lncRNAs associated with CAD in a tissue-specific fashion. The eQTL-GWAS colocalization analysis associated 15 lncRNAs with cardiometabolic traits which could bridge the lncRNA to CAD risk. The co-expression analysis identified co-expressed protein-coding genes of 16 lncRNAs and suggested their involved biological pathways. Four antisense lncRNAs from the same locus, CDH13-AS2, CDH13-AS1, CEDORA, and CTD-3253I12.1, not only shared cardiometabolic traits but also exclusively co-expressed with the host gene, CDH13. Our biological experiments using dRfxCas13d-based RNA immunoprecipitation, CRISPR/Cas9 gene knockdown, and dCas9-VP64-based transcriptional activation in endothelial cells, elucidated CDH13-AS2 as a stabilizer of CDH13 mRNA and suggested therapeutic potential of CDH13-AS2 overexpression.

Machine learning methods for diagnostic disease prediction and treatment decisions in parvovirus B19 positive viral cardiomyopathy

Abstract Background Parvovirus B19 (B19V) is the most common type of virus found in endomyocardial biopsies (EMB) from patients with inflammatory heart disease. However, the detection of genomic B19V-DNA appears to have little clinical significance and is often considered an incidental finding. Recent analyses of a large cohort of B19V-positive patients revealed that the detection of RNA transcripts rather than DNA is of clinical importance, as B19V-RNA-positive patients have a significantly worse cardiovascular outcome. Here, we applied machine learning (ML) methods to assess the impact of B19V transcriptional activity on cardiac inflammation and patient clinical outcome. Methods and results A total of 305 patients with a known clinical course and a positive endomyocardial B19V-DNA test were included in this study. Follow-up measurements of the B19V-specific transcripts VP1/2 and NS1 revealed significantly worse clinical courses than in patients without B19V-RNA detection (P=0.019, hazard ratio (95% CI)=1.62 (1.10-2.57)). LVEF at follow-up was significantly lower in patients with detected B19V-RNA (P=0.007), and adverse events such as worsening LV function (P=0.003) or stable systolic dysfunction (P=0.013) were more frequent in this group. Importantly, analysis of inflammatory markers in EMB showed no difference in patients with and without RNA detection (P=0.432), suggesting that the deterioration in clinical outcome was related to B19V transcriptional activity rather than an increase in inflammation. The analysis of area under the receiver operating characteristic curve (AUROC) of B19V-RNA-negative patients revealed that the macrophage marker MAC-1 (AUROC=0.66, P=0.002) was the single most prognostically relevant parameter, while the T cell marker CD3 (AUROC=0.73, P<0.001) was identified as the most prognostically relevant marker in B19V-RNA-positive patients. The linear combination of several inflammatory markers increased the AUROC value to 0.67 in B19V-RNA-negative patients and to 0.74 in B19V-RNA-positive patients. To further increase prognostic accuracy, we trained ML models on features derived from EMB inflammatory markers. For the prediction of major adverse cardiac events within 60 months, the best ML-models achieved an AUROC of 0.87 in B19V-RNA-positive and of 0.86 in B19V-RNA-negative patients, while models that neglect the differentiation of patients with regard to the presence of B19V RNA transcripts only achieved an AUROC of up to 0.76. Conclusions These results show that B19V transcriptional activity is an independent risk marker for adverse cardiac events in patients with viral cardiomyopathy and thus confirm the clinical-therapeutic importance of myocardial biopsy. Furthermore, we present an ML-based algorithm that accurately indicates disease progression in B19V-positive cardiomyopathy patients and is thus helpful for appropriate therapy recommendation.

Asiatic acid inhibits HBV cccDNA transcription by promoting HBx degradation

BackgroundHepatitis B virus (HBV) infection is a persistent global public health problem, and curing for chronic hepatitis B (CHB) through the application of existing antiviral drugs is beset by numerous challenges. The viral protein HBx is a critical regulatory factor in the life cycle of HBV. Targeting HBx is a promising possibility for the development of novel therapeutic strategies.MethodsThe Nano-Glo® HiBiT Lysis Detection System was used to screen the herbal monomer compound library for compounds that inhibit HBx expression. Western blotting was used to examine proteins expression. Southern blotting or Northern blotting were used to detect HBV DNA or HBV RNA. ELISA was performed to detect the HBsAg level. The effect of asiatic acid on HBV in vivo was investigated by using recombinant cccDNA mouse model.ResultsAsiatic acid, an extract of Centella asiatica, significantly reduced the HBx level. Mechanistic studies demonstrated that asiatic acid may promote the degradation of HBx in an autophagy pathway-dependent manner. Subsequently, asiatic acid was found to reduce the amount of HBx bound to covalently closed circular DNA (cccDNA) microchromosomes, and repressive chromatin modifications then occurred, ultimately inhibiting cccDNA transcriptional activity. Moreover, in HBV-infected cells and a mouse model of persistent HBV infection, asiatic acid exhibited potent anti-HBV activity, as evidenced by decreased levels of HBV RNAs, HBV DNA and HBsAg.ConclusionsAsiatic acid was identified as a compound that targets HBx, revealing its potential for application as an anti-HBV agent.

Soil viral–host interactions regulate microplastic-dependent carbon storage

Microplastic is globally regarded as an important factor impacting biogeochemical cycles, yet our understanding of such influences is limited by the uncertainties of intricate microbial processes. By multiomics analysis, coupled with soil chemodiversity characterization and microbial carbon use efficiency (CUE), we investigated how microbial responses to microplastics impacted soil carbon cycling in a long-term field experiment. We showed that biodegradable microplastics promoted soil organic carbon accrual by an average of 2.47%, while nondegradable microplastics inhibited it by 17.4%, as a consequence of the virus-bacteria coadaptations to the microplastics disturbance. In the relevant functional pathways, nondegradable microplastics significantly (P < 0.05) enhanced the abundance and transcriptional activity related to complex carbohydrate metabolism, whereas biodegradable microplastics significantly (P < 0.05) promoted functions involved in amino acid metabolism and glycolysis. Accordingly, viral lysis enhanced in nondegradable microplastics treatments to introduce more complex organic compounds to soil dissolved organic matters, thus benefiting the oligotrophs with high carbon metabolic capabilities in exploitation competition. In contrast, biodegradable microplastics enriched viral auxiliary metabolic genes of carbon metabolism through "piggyback-the-winner" strategy, conferring to dominant copiotrophs, enhanced substrate utilization capabilities. These virus-host interactions were also demonstrated in the corresponding soil plastisphere, which would alter microbial resource allocation and metabolism via CUE, affecting carbon storage consequently. Overall, our results underscore the importance of viral-host interactions in understanding the microplastics-dependent carbon storage in the soil ecosystem.

Cardiomyocyte proliferation is promoted by intracellular Gas6-mediated Yap activity during neonatal heart regeneration

Abstract Purpose The adult mammalian heart has limited regenerative capacity after injury, mostly attributable to postnatal cardiomyocyte (CM) cell cycle arrest. The key to unlocking the regenerative potential of the adult hearts may lie within the developmental transitions occurring during neonatal life. Gas6, a secretory protein, stimulates the proliferation of various type cells through binding to TAM receptor family, such as endothelial progenitor cells, tumor cells, and hair follicle stem-cell. However, no study to date has studied the role of Gas6 during the proliferation of CMs. Herein, our study uncovers that Gas6 is transiently expressed during early postnatal heart and regenerative heart, and functions through intracellular pathway by regulating Yap activity to promote CM proliferation. Methods CM isolation was conducted to assess the change of Gas6 expression in both postnatal development and regenerated stages using the CM-specific Tdtomato mice. Cardiac deletion of Gas6 gene mice was used to evaluate the impaired heart regeneration. CM-specific Gas6 overexpression mice were generated using adeno-associated virus system to evaluate Gas6 pro-proliferate and protect effect after ischemic injury. Secreted signal peptide truncation (ΔSP-Gas6) and predicted functional region truncation (ΔCC-Gas6) were generated to identify the intracellular mechanism of Gas6 via Sav1/p-mst1/p-lats1/ p-yap axis. Results We found that Gas6 expression is enriched in CM4 and the expression profile correlates with the expression of the CM4 marker Acta2, as well as cell-cycle genes Mki67 and Aurora B, in individual CM. Gas6 was highly expressed in the neonatal heart and continued to increase during the neonatal heart regenerative process, but was nearly absent in the adult heart under physiological and ischemia conditions. CM-specific Gas6 deletion resulted in significantly reduced CM proliferation rate and increased CM size in P0 hearts, and congestive heart failure in P28 hearts. Conversely, sustained CM-specific overexpression of Gas6 in postnatal hearts increased CM division, elevated CM numbers, and reduced CM size; and reduced infarct scar area and enhanced cardiac function after ischemia injury. Pre-overexpression of Gas6 in adult CMs protected adult cardiac function against ischemia injury, indicated by the activation of mitosis, angiogenesis and reduced oxidative phosphorylation. In primary neonatal CMs, both Gas6 and ΔSP-Gas6promoted CM proliferation with sarcomere disassembly. Mechanistically, Gas6 interacted with Sav1to inhibit Mst1recruitment and Mst1/Lats1/Yap phosphorylation cascade, therefore, promoted Yap translocation into nucleus. Conclusions We demonstrate that Gas6 is an important intrinsic regulator of neonatal heart regeneration, which acts as a promoter of Yap transcriptional activity to enhance CM proliferation. Supplementing Gas6 in the adult heart represents a potential therapeutic approach for cardiac repair.

The orchestration of coding and non-coding genes at the CDH13 locus in coronary artery disease

Abstract Background The CDH13 locus was associated with coronary artery disease (CAD) by genome-wide association studies (GWAS). However, the causal gene(s) and underlying disease mechanisms at this locus remain undetermined. Purpose We intended to pinpoint and functionally validate the causal coding and non-coding genes at the CDH13 locus and delineate the underlying mechanisms for CAD. Methods and results We conducted the transcriptome-wide association analysis (TWAS) using nine types of CAD-relevant tissues from Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) and Genotyped Tissue Expression (GTEx) projects and identified CDH13 (T-cadherin) and four long non-coding RNA (lncRNA) genes as candidate causal genes at The CDH13 locus. One of the antisense(AS) lncRNAs, CDH13-AS2, was predicted to bind on the 3’ untranslated region (UTR) of CDH13 using LncRRIsearch and RNAup servers. The binding interaction was validated by dCas13-medicated RNA immunoprecipitation. Weighted correlation network analysis (WGCNA) using transcriptomic data from CAD-relevant tissues of ~600 human subjects suggested a positive expression correlation between CDH13 and CDH13-AS2 in artery tissues. CRISPR/Cas9-based knockout (KO) of CDH13 or CDH13-AS2 in human umbilical vein endothelial cells (HUVECs) indicated synergistically atherogenic phenotypes, including reduced proliferation and migration, and increased apoptosis and monocyte adhesion. The data matched with the downregulation of CDH13 in artery samples of atherosclerosis patients. The cellular phenotypes were also in line with our in vivo data of Cdh13 and Apoe double-KO (Cdh13-/-/Apoe-/-) mice, which showed increased aortic lesion areas when fed on a high-fat diet compared to Apoe-/- mice. Furthermore, prediction using TargetScan, miRWalk, and scanMiRApp databases identified the potential binding of several EC-expression microRNAs (mir) on 3’UTR of CDH13. Several disease-associated variants were mapped to the predicted binding sites of a few microRNAs, including mir-let7, mir-30, and mir-125. The dual luciferase-based RNA interference (RNAi) assay confirmed the binding of mir-let7 on 3’UTR of CDH13. Transcriptional activation of CDH13-AS2 by the enzymatically inactivated Cas9 (dCas9) system diminished the binding of mir-let7 and increased CDH13 expression. Conclusions At the CDH13 locus, CDH13 and CDH13-AS2 are two causal genes, whose downregulation likely contributes to CAD. CDH13-AS2 bound and stabilized CDH13 partially by preventing mir-let7 mediated degradation of CDH13 mRNA, suggesting therapeutic potentials.

Burst-like transcription of MYH7, allelic and contractile imbalance already in early stage hiPSC-cardiomyocytes indicate these as pathogenic factors in Hypertrophic Cardiomyopathy

Abstract Background Hypertrophic Cardiomyopathy (HCM) is caused by heterozygous mutations in sarcomeric proteins, which can cause hyper- or hypocontractiliy of cardiomyocytes. Previously we also observed highly variable force generation (contractile imbalance) among individual cardiomyocytes of HCM-patients with myosin (MYH7) or myosin-binding-protein-C mutations (MYBPC3), ranging from normal to highly altered values. Contractile imbalance is presumably caused by stochastic, burst-like transcription of mutant and wildtype allele, which induces unequal ratios of mutant vs. wildtype mRNA among individual cardiomyocytes. Variable force generation most likely is the result of also unequal fractions of mutated protein from cell to cell. Aim With this study we aimed to examine whether in addition to the effects of the mutation on sarcomere function, also allelic and contractile imbalance already exist at the beginning of disease development or whether both result from disease-related alterations in symptomatic HCM-patients. Methods and Results We studied this in patient-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with MYH7-mutation R723G as model for an early disease stage. These early, fetal-like cardiomyocytes developed typical HCM-related features like hypertrophy and increased myofibrillar disarray compared to WT-hiPSC-CMs. Calcium sensitivity was reduced, twitch kinetics were slowed down. To test whether MYH7 in these early-stage hiPSC-CMs also is transcribed in bursts or continuously, we performed RNA-fluorescence in situ hybridization (RNA-FISH). Nuclei without, with one and with two or more active transcription sites were observed which indicates stochastic, burst-like and independent transcription of the two MYH7-alleles. Ratios of mRNA from both alleles were quantified in individual cardiomyocytes by allele-specific single cell RT-PCR. We found highly variable ratios of mutated vs. wildtype mRNA among individual cardiomyocytes, similar to HCM-patient’s cardiac tissue, indicating unequal expression of mutant and wildtype mRNA from cell-to-cell. Furthermore, functional measurements on myofibrils from R723G-hiPSC-CMs revealed substantial variability in force generation at physiological calcium, compared to much smaller heterogeneity among WT-hiPSC-CMs. In addition, variability of twitch kinetics of intact hiPSC-CMs was also substantially increased among R723G- compared to WT-hiPSC-CMs. Altogether, this suggests that functional heterogeneity results from burst-like transcription in heterozygous HCM cardiomyocytes. Conclusions Our results in HCM-patient derived hiPSC-cardiomyocytes with a fetal-like developmental stage suggest that allelic and contractile imbalance among individual cardiomyocytes together with the mutation are present at the beginning of the disease. Thus, they most likely are exacerbating factors that contribute development of HCM hallmarks.