Histone Levels Research Articles

Linker histones H1 in human disease

Linker histones (H1) are basic proteins that are part of the nucleosome structure in the cell nucleus and are involved in the packaging of genetic material and the regulation of gene expression. As research progressed, it was discovered that linker histones constitute the largest group of histones in terms of variants found in humans. Even though the H1 variants differ slightly in the primary structure, they can perform different functions, undergo multiple post-translational modifications and differ in cellular localization. In addition to the nucleus, histones H1 can occur in the cytoplasm, on the cell surface and in the intercellular space. In these places, they play a supporting role for the immune system and act as signaling molecules. Changes in the levels of histones and their post-translational modifications have been associated with many human diseases and it is postulated that some of them may serve as biomarkers or therapeutic targets.

A novel dual-epigenetic inhibitor enhances recombinant monoclonal antibody expression in CHO cells

Epigenetic regulation plays a central role in the regulation of a number of cellular processes such as proliferation, differentiation, cell cycle, and apoptosis. In particular, small molecule epigenetic modulators are key elements that can effectively influence gene expression by precisely regulating the epigenetic state of cells. To identify useful small-molecule regulators that enhance the expression of recombinant proteins in Chinese hamster ovary (CHO) cells, we examined a novel dual-HDAC/LSD1 inhibitor I-4 as a supplement for recombinant CHO cells. Treatment with 2 μM I-4 was most effective in increasing monoclonal antibody production. Despite cell cycle arrest at the G1/G0 phase, which inhibits cell growth, the addition of the inhibitor at 2 µM to monoclonal antibody-expressing CHO cell cultures resulted in a 1.94-fold increase in the maximal monoclonal antibody titer and a 2.43-fold increase in specific monoclonal antibody production. In addition, I-4 significantly increased the messenger RNA levels of the monoclonal antibody and histone H3 acetylation and methylation levels. We also investigated the effect on HDAC-related isoforms and found that interference with the HDAC5 gene increased the monoclonal antibody titer by 1.64-fold. The results of this work provide an effective method of using epigenetic regulatory strategies to enhance the expression of recombinant proteins in CHO cells.Key points• HDAC/LSD1 dual-target small molecule inhibitor can increase the expression level of recombinant monoclonal antibodies in CHO cells.• By affecting the acetylation and methylation levels of histones in CHO cells and downregulating HDAC5, the production of recombinant monoclonal antibodies increased.• It provides an effective pathway for applying epigenetic regulation strategies to enhance the expression of recombinant proteins.

The Drosophila maternal-effect gene abnormal oocyte (ao) does not repress histone gene expression.

The abnormal oocyte (ao) gene of Drosophila melanogaster is a maternal-effect lethal gene previously identified as encoding a transcriptional regulator of core histones. However, background genetic mutations in existing ao mutant strains could compromise their utility in manipulating histone levels. To distinguish the true ao phenotype from background effects, we created two new ao reagents: a CRISPR/Cas9-mediated knockout of the ao allele for genetic and molecular analyses and an epitope-tagged ao allele for cytological experiments. Using these reagents, we confirm previous findings that ao exhibits maternal-effect lethality, which can be rescued by either a decrease in the histone gene copy number or by Y chromosome heterochromatin. We also confirm that the Ao protein localizes to the histone locus bodies in ovaries. Our data also suggest that ao genetically interacts with the histone genes and heterochromatin, as previously suggested. However, contrary to prior findings, we find that ao does not repress core histone transcript levels. Thus, the molecular basis for ao-associated maternal-effect lethality remains unknown.

Parvovirus B19 Infection Is Associated with the Formation of Neutrophil Extracellular Traps and Thrombosis: A Possible Linkage of the VP1 Unique Region.

Neutrophil extracellular traps (NETs) formation, namely NETosis, is implicated in antiphospholipid syndrome (APS)-related thrombosis in various autoimmune disorders such as systemic lupus erythematosus (SLE) and APS. Human parvovirus B19 (B19V) infection is closely associated with SLE and APS and causes various clinical manifestations such as blood disorders, joint pain, fever, pregnancy complications, and thrombosis. Additionally, B19V may trigger the production of autoantibodies, including those against nuclear and phospholipid components. Thus, exploring the connection between B19V, NETosis, and thrombosis is highly relevant. An in vitro NETosis model using differentiated HL-60 neutrophil-like cells (dHL-60) was employed to investigate the effect of B19V-VP1u IgG on NETs formation. A venous stenosis mouse model was used to test how B19V-VP1u IgG-mediated NETs affect thrombosis in vivo. The NETosis was observed in the dHL-60 cells treated with rabbit anti-B19V-VP1u IgG and was inhibited in the presence of either 8-Br-cAMP or CGS216800 but not GSK484. Significantly elevated reactive oxygen species (ROS), myeloperoxidase (MPO), and citrullinated histone (Cit-H3) levels were detected in the dHL60 treated with phorbol myristate acetate (PMA), human aPLs IgG and rabbit anti-B19V-VP1u IgG, respectively. Accordingly, a significantly larger thrombus was observed in a venous stenosis-induced thrombosis mouse model treated with PMA, human aPLs IgG, rabbit anti-B19V-VP1u IgG, and human anti-B19V-VP1u IgG, respectively, along with significantly increased amounts of Cit-H3-, MPO- and CRAMP-positive infiltrated neutrophils in the thrombin sections. This research highlights that anti-B19V-VP1u antibodies may enhance the formation of NETosis and thrombosis and implies that managing and treating B19V infection could lower the risk of thrombosis.

Centromere inactivation during aging can be rescued in human cells.

Aging involves a range of genetic, epigenetic, and physiological alterations. A key characteristic of aged cells is the loss of global heterochromatin, accompanied by a reduction in canonical histone levels. In this study, we track the fate of centromeres during aging in human cells. Our findings reveal that the centromeric histone H3 variant CENP-A is downregulated in aged cells, in a p53-dependent manner. We observe repression of centromeric noncoding transcription through an epigenetic mechanism via recruitment of a lysine-specific demethylase 1 (LSD1/KDM1A) to centromeres. This suppression results in defective de novo CENP-A loading at aging centromeres. By dual inhibition of p53 and LSD1/KDM1A in aged cells, we mitigate the reduction in centromeric proteins and centromeric transcripts, leading to mitotic rejuvenation of these cells. These results offer insights into a novel mechanism for centromeric inactivation during aging and provide potential strategies to reactivate centromeres.

Research Progress on the Mechanism of Histone Deacetylases in Ferroptosis of Glioma.

Glioma is the most prevalent primary malignant tumor of the central nervous system. While traditional treatment modalities such as surgical resection, radiotherapy, and chemotherapy have made significant advancements in glioma treatment, the prognosis for glioma patients remains often unsatisfactory. Ferroptosis, a novel form of programmed cell death, plays a crucial role in glioma and is considered to be the most functionally rich programmed cell death process. Histone deacetylases have emerged as a key focus in regulating ferroptosis in glioma. By inhibiting the activity of histone deacetylases, histone deacetylase inhibitors elevate acetylation levels of both histones and non-histone proteins, thereby influencing various cellular processes. Numerous studies have demonstrated that histone deacetylases are implicated in the development of glioma and hold promise for its treatment. This article provides an overview of research progress on the mechanism by which histone deacetylases contribute to ferroptosis in glioma.

Histone lactylation-ROS loop contributes to light exposure-exacerbated neutrophil recruitment in zebrafish

Light serves as a crucial external zeitgeber for maintaining and restoring physiological homeostasis in most organisms. Disrupting of light rhythms often leads to abnormal immune function, characterized by excessive inflammatory responses. However, the underlying regulatory mechanisms behind this phenomenon remain unclear. To address this concern, we use in vivo imaging to establish inflammation models in zebrafish, allowing us to investigate the effects and underlying mechanisms of light disruption on neutrophil recruitment. Our findings reveal that under sustained light conditions (LL), neutrophil recruitment in response to caudal fin injury and otic vesicle inflammation is significantly increased. This is accompanied by elevated levels of histone (H3K18) lactylation and reactive oxygen species (ROS) content. Through ChIP-sequencing and ChIP‒qPCR analysis, we discover that H3K18 lactylation regulates the transcriptional activation of the duox gene, leading to ROS production. In turn, ROS further promote H3K18 lactylation, forming a positive feedback loop. This loop, driven by H3K18 lactylation-ROS, ultimately results in the over recruitment of neutrophils to inflammatory sites in LL conditions. Collectively, our study provides evidence of a mutual loop between histone lactylation and ROS, exacerbating neutrophil recruitment in light disorder conditions, emphasizing the significance of maintaining a proper light-dark cycle to optimize immune function.

The chromatin accessibility and transcriptomic landscape of the aging mice cochlea and the identification of potential functional super-enhancers in age-related hearing loss

BackgroundPresbycusis, also referred to as age-related hearing loss (ARHL), is a condition that results from the cumulative effects of aging on an individual's auditory capabilities. Given the limited understanding of epigenetic mechanisms in ARHL, our research focuses on alterations in chromatin-accessible regions.MethodsWe employed assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in conjunction with unique identifier (UID) mRNA-seq between young and aging cochleae, and conducted integrated analysis as well as motif/TF-gene prediction. Additionally, the essential role of super-enhancers (SEs) in the development of ARHL was identified by comparative analysis to previous research. Meanwhile, an ARHL mouse model and an aging mimic hair cell (HC) model were established with a comprehensive identification of senescence phenotypes to access the role of SEs in ARHL progression.ResultsThe control cochlear tissue exhibited greater chromatin accessibility than cochlear tissue affected by ARHL. Furthermore, the levels of histone 3 lysine 27 acetylation were significantly depressed in both aging cochlea and aging mimic HEI-OC1 cells, highlighting the essential role of SEs in the development of ARHL. The potential senescence-associated super-enhancers (SASEs) of ARHL were identified, most of which exhibited decreased chromatin accessibility. The majority of genes related to the SASEs showed obvious decreases in mRNA expression level in aging HCs and was noticeably altered following treatment with JQ1 (a commonly used SE inhibitor).ConclusionThe chromatin accessibility in control cochlear tissue was higher than that in cochlear tissue affected by ARHL. Potential SEs involved in ARHL were identified, which might provide a basis for future therapeutics targeting SASEs related to ARHL.

P-054 Paternal age and cryopreservation impact sperm chromatin status as indicated by changes in protamine and histone levels

Abstract Study question Do paternal age and cryopreservation alter sperm chromatin status in normospermic men? Summary answer Paternal age and cryopreservation appear to impact sperm chromatin condensation by altering protamine and histone DNA levels. What is known already Semen parameters do not accurately reflect male fertility and thus are not adequate to assess the impact of patient profile and semen manipulation/conservation on sperm functional quality. DNA protamination leads to sperm chromatin condensation, which is associated with sperm maturity but also appears to function as a defence mechanism against oxidative attack. In the face of contrasting/insufficient previous data, the impact of advanced paternal age (APA) and cryopreservation on sperm chromatin condensation and histone/protamine balance remains unclear. Study design, size, duration Twenty-two normospermic men, 11 of which with advanced paternal age (APA; ≥42) and 11 pre-APA (≤35), provided semen samples by masturbation (1-5 days abstinence). Fresh and cryopreserved/thawed samples from all patients were assessed for chromatin histone staining, while samples from a subgroup of 10 patients (5 pre-APA and 5 APA) were assessed for protamine presence. The effects of age and cryopreservation on the percentage of stained cells were assessed with the Fisher’s exact test. Participants/materials, setting, methods Semen samples were provided by male partners of couples treated at our fertility centre. Sample aliquots were cryopreserved and analyzed after thawing (CryoSperm & Sperm Preparation Medium, Origio). Histone presence was assessed through aniline blue staining and protamine presence was indirectly assessed through staining with the protamine competitor Chromomycin A3 (CMA3); stained cells were visualized by bright field and fluorescence microscopy, respectively. Two hundred sperm cells were evaluated in each sample by the same embryologist. Main results and the role of chance Mean paternal age was 31.5±3.9 and 44.4±2.1 years for pre-APA and APA patient-groups, respectively. Major parameters observed in fresh semen samples were: 2.3±0.7 vs. 3.6±1.6mL (volume); 39±28 vs. 38±12 x10ˆ6/mL (concentration); 7±12 vs. 6±5% (rapid progressive motility); 41±12 vs. 38±13% (slow progressive motility); 12±5 vs. 11±4% (non-progressive motility); 40±17 vs. 45±16% (immotile) and 5±1 vs. 6±2% (normal morphology), for pre-APA and APA, respectively. The percentage of aniline blue (histone marker) positive cells was less than half in fresh samples from APA as compared to pre-APA patients (10.2% and 23.4%, respectively, p < 0.0001). Consistently, the percentage of CMA3 (protamine absence marker) positive cells was less than half in APA as compared to pre-APA patients (6.4% and 16.4%, respectively, p < 0.0001). In both age groups, the percentages of aniline blue and CMA3 positive cells were higher in cryopreserved/thawed samples as compared to their fresh counterparts (aniline blue positive: 35.2% vs. 23.4% and 22.0% vs. 10.2%; CMA3 positive: 27.2% vs. 16.4% and 18.8% vs. 6.4%, in cryopreserved vs. fresh in pre-APA and APA, respectively, p < 0.0001). Limitations, reasons for caution Our study is limited by the potential interference of confounding factors not equally distributed in age groups. The conclusions from this study must be confirmed in other patient populations with different race/genetics and habits. Wider implications of the findings Our findings suggest that APA increases, while cryopreservation decreases, sperm chromatin condensation, as indicated by changes in DNA histone and protamine abundance. Our study thus sheds light on the impact of paternal age and cryopreservation on sperm quality, providing new valuable parameters for reproductive medicine practice and research. Trial registration number na

Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3

Epithelial cells are the first point of contact for bacteria entering the respiratory tract. Streptococcus pneumoniae is an obligate human pathobiont of the nasal mucosa, carried asymptomatically but also the cause of severe pneumoniae. The role of the epithelium in maintaining homeostatic interactions or mounting an inflammatory response to invasive S. pneumoniae is currently poorly understood. However, studies have shown that chromatin modifications, at the histone level, induced by bacterial pathogens interfere with the host transcriptional program and promote infection. Here, we uncover a histone modification induced by S. pneumoniae infection maintained for at least 9 days upon clearance of bacteria with antibiotics. Di-methylation of histone H3 on lysine 4 (H3K4me2) is induced in an active manner by bacterial attachment to host cells. We show that infection establishes a unique epigenetic program affecting the transcriptional response of epithelial cells, rendering them more permissive upon secondary infection. Our results establish H3K4me2 as a unique modification induced by infection, distinct from H3K4me3 or me1, which localizes to enhancer regions genome-wide. Therefore, this study reveals evidence that bacterial infection leaves a memory in epithelial cells after bacterial clearance, in an epigenomic mark, thereby altering cellular responses to subsequent infections and promoting infection.

Restoring discarded porcine lungs by ex vivo removal of neutrophil extracellular traps

BackgroundBy causing inflammation and tissue damage, neutrophil extracellular traps (NETs) constitute an underlying mechanism of aspiration-induced lung injury, a major factor of the low utilization of donor lungs in lung transplantation (LTx). MethodTo determine whether NET removal during ex vivo lung perfusion (EVLP) can restore lung function and morphology in aspiration-damaged lungs, gastric aspiration lung injury was induced in 12 pigs. After confirmation of acute respiratory distress syndrome, the lungs were explanted and assigned to NET removal connected to EVLP (treated) (n=6) or EVLP only (non-treated) (n=6). Hemodynamic measurements were taken, and blood and tissue samples were collected to assess lung function, morphology, levels of cell-free DNA, extracellular histones, and nucleosomes as markers of NETs as well as cytokine levels. ResultsAfter EVLP and NET removal in porcine lungs, PaO2/FiO2 ratios increased significantly compared to those undergoing EVLP alone (p=0.0411). Treated lungs had lower cell-free DNA (p=0.0260) and lower levels of extracellular histones in EVLP perfusate than non-treated lungs (p=0.0260). According to histopathology, treated lungs showed less immune cell infiltration and less oedema compared with non-treated lungs which was reflected in decreased levels of pro-inflammatory cytokines in EVLP perfusate and BALF. ConclusionTo conclude, removing NETs during EVLP improved lung function and morphology in aspiration-damaged donor lungs. The ability to remove NETs during EVLP could represent a new therapeutic approach for LTx and potentially expand the donor pool for transplantation.

Immune markers of severe acute pancreatitis.

Acute pancreatitis is a common acute inflammatory disorder of the pancreas, and its incidence has been increasing worldwide. Approximately 10% of acute pancreatitis progresses to severe acute pancreatitis (SAP), which carries significant morbidity and mortality. Disordered immune response to pancreatic injury is regarded as a key event that mediates systemic injury in SAP. In this article, we review recent developments in immune biomarkers of SAP and future directions for research. Given the importance of the NLRP3-inflammasome pathway in mediating systemic inflammatory response syndrome and systemic injury, recent studies have investigated associations of SAP with systemic levels of activators of NLRP3, such as the damage associated molecular patterns (DAMPs) for the first time in human SAP. For example, circulating levels of histones, mitochondrial DNAs, and cell free DNAs have been associated with SAP. A panel of mechanistically relevant immune markers (e.g., panel of Angiopoeitin-2, hepatocyte growth factor, interleukin-8 (IL-8), resistin and sTNF-α R1) carried higher predictive accuracies than existing clinical scores and individual immune markers. Of the cytokines with established relevance to SAP pathogenesis, phase 2 trials of immunotherapies, including tumor necrosis factor (TNF)-alpha inhibition and stimulation of IL-10 production, are underway to determine if altering the immunologic response can reduce the severity of acute pancreatitis (AP). Circulating systemic levels of various DAMPs and a panel of immune markers that possibly reflect activities of different pathways that drive SAP appear promising as predictive biomarkers for SAP. But larger multicenter studies are needed for external validation. Studies investigating immune cellular pathways driving SAP using immunophenotyping techniques are scarce. Interdisciplinary efforts are also needed to bring some of the promising biomarkers to the bedside for validation and testing for clinical utility. Studies investigating the role of and characterization of altered gut-lymph and gut-microbiota in severe AP are needed.

DIPG-45. TARGETING HYPERACETYLATION IN PEDIATRIC DIFFUSE MIDLINE GLIOMAS USING ONCOLYTIC ADENOVIRUSES

Abstract BACKGROUND H3K27-altered pediatric diffuse midline glioma (DMG) is a highly aggressive type of brain tumor with a 5-year survival rate of less than 2%. Conventional treatments are restricted to palliative radiation therapy. Therefore, effective treatments are urgently needed. Results from a recent phase I clinical trial for Diffuse Intrinsic Pontine Glioma showed that intratumoral administration of Delta-24-RGD oncolytic adenovirus in combination with radiation improves survival without unwanted toxicity. Based on the rationale that, during infection, the early viral protein E1A binds P300, we hypothesize that combining oncolytic adenoviruses with p300 inhibitors will induce a robust modification of the post-translational landscape of DMGs and increase the anti-cancer effect of the oncolytic virus. METHODS Studies on Delta-24-RGD infectivity and replication were performed in a panel of clinically significant DMG cells. Modification of the acetylation levels of acid-extracted histones after Delta-24-RGD was analyzed using Western blot. Cell Titer Blue was utilized to measure cell viability, and ZIP-synergy scores were calculated using SynergyFinder. RESULTS We observed a time-dependent reduction in H3K27ac in human DMG cells following Delta-24-RGD infection with an 89% reduction in H3K27ac at 48h post-infection. Delta-24-RGD treatment of DMG cells resulted in optimum viral infectivity and replication. Combining Delta-24-RGD with the p300 inhibitor C646 resulted in enhanced cell death in vitro. Using a panel of oncolytic viruses that induce differential enzymatic and binding functions of P300, we showed that Delta-24-RGD mediated alteration of P300 activity is required for hypoacetylation in DMGs. We characterized the in vivo dynamics of K27M-H3.1 and K27M-H3.3 DMG-derived tumors in the brain stem of syngeneic animal models. In vivo experiments are in progress to examine the combinatorial anti-glioma effect of Delta-24-RGD and P300 inhibitors. CONCLUSION Collectively, our studies provide a strong mechanistic rationale for combining oncolytic adenoviruses and p300 inhibitors and should propel the eventual clinical testing of this approach in DMG patients.

Sensory Nerve Regulation via H3K27 Demethylation Revealed in Akermanite Composite Microspheres Repairing Maxillofacial Bone Defect.

Maxillofacial bone defects exhibit intricate anatomy and irregular morphology, presenting challenges for effective treatment. This study aimed to address these challenges by developing an injectable bioactive composite microsphere, termed D-P-Ak (polydopamine-PLGA-akermanite), designed to fit within the defect site while minimizing injury. The D-P-Ak microspheres biodegraded gradually, releasing calcium, magnesium, and silicon ions, which, notably, not only directly stimulated the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) but also activated sensory nerve cells to secrete calcitonin gene-related peptide (CGRP), a key factor in bone repair. Moreover, the released CGRP enhanced the osteogenic differentiation of BMSCs through epigenetic methylation modification. Specifically, inhibition of EZH2 and enhancement of KDM6A reduced the trimethylation level of histone 3 at lysine 27 (H3K27), thereby activating the transcription of osteogenic genes such as Runx2 and Osx. The efficacy of the bioactive microspheres in bone repair is validated in a rat mandibular defect model, demonstrating that peripheral nerve response facilitates bone regeneration through epigenetic modification. These findings illuminated a novel strategy for constructing neuroactive osteo-inductive biomaterials with potential for further clinical applications.

Histone deacetylase inhibition mitigates fibrosis-drivendisease progression in recessive dystrophicepidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disease caused by mutations in the gene encoding type VII collagen (C7). RDEB is associated with fibrosis, which is responsible for severe complications. The phenotypic variability observed in siblings with RDEB suggests that epigenetic modifications contribute to disease severity. Identifying epigenetic changes may help to uncover molecular mechanisms underlying RDEB pathogenesis and new therapeutic targets. To investigate histone acetylation in RDEB skin and to explore histone deacetylase inhibitors (HDACi) as therapeutic molecules capable of counteracting fibrosis and disease progression in RDEB mice. Acetylated histone levels were detected in human skin by immunofluorescence and in RDEB fibroblasts by enzyme-linked immunosorbent assay (ELISA). The effects of givinostat and valproic acid (VPA) on RDEB fibroblast fibrotic behaviour were assessed by a collagen-gel contraction assay, Western blot and immunocytofluorescence for α-smooth muscle actin, and ELISA for released transforming growth factor (TGF)-β1. RNA sequencing was performed in HDACi- and vehicle-treated RDEB fibroblasts. VPA was systemically administered to RDEB mice and effects on overt phenotype were monitored. Fibrosis was investigated in the skin using histological and immunofluorescence analyses. Eye and tongue defects were examined microscopically. Mass spectrometry proteomics was performed on skin protein extracts from VPA-treated RDEB and control mice. Histone acetylation decreases in RDEB skin and primary fibroblasts. RDEB fibroblasts treated with HDACi lowered fibrotic traits, including contractility, TGF-β1 release and proliferation. VPA administration to RDEB mice mitigated severe manifestations affecting the eyes and paws. These effects were associated with fibrosis inhibition. Proteomic analysis of mouse skin revealed that VPA almost normalized protein sets involved in protein synthesis and immune response, processes linked to the increased susceptibility to cancer and bacterial infections seen in people with RDEB. Dysregulated histone acetylation contributes to RDEB pathogenesis by facilitating the progression of fibrosis. Repurposing of HDACi could be considered for disease-modifying treatments in RDEB.

Integrative Analysis of Histone Acetylation Regulated CYP4F12 in Esophageal Cancer Development.

Current therapeutic strategies for esophageal cancer (EC) patients have yielded limited improvements in survival rates. Recent research has highlighted the influence of drug metabolism enzymes on both drug response and EC development. Our study aims to identify specific drug metabolism enzymes regulated by histone acetylation and to elucidate its molecular and clinical features. CYP4F12 exhibited a notable upregulation subsequent to trichostatin A treatment as evidenced by RNA sequencing analysis conducted on the KYSE-150 cell line. The change in gene expression was associated with increased acetylation level of histone 3 K18 and K27 in the promoter. The regulation was dependent on p300. In silicon analysis of both The Cancer Genome Atlas esophageal carcinoma and GSE53624 dataset suggested a critical role of CYP4F12 in EC development, because CYP4F12 was downregulated in tumor tissues and predicted better disease-free survival. Gene ontology analysis has uncovered a robust correlation between CYP4F12 and processes related to cell migration, as well as its involvement in cytosine-mediated immune activities. Further investigation into the relationship between immune cells and CYP4F12 expression has indicated an increased level of B cell infiltration in samples with high CYP4F12 expression. CYP4F12 was also negatively correlated with the expression of inhibitory checkpoints. An accurate predictive nomogram model was established combining with clinical factors and CYP4F12 expression. In conclusion, CYP4F12 was crucial in EC development, and targeting CYP4F12 may improve the therapeutic efficacy of current treatment in EC patients. SIGNIFICANCE STATEMENT: CYP4F12 expression was downregulated in esophageal cancer (EC) patients and could be induced by trichostatin A. During EC development, CYP4F12 was linked to reduced cell migration and increased infiltration of B cells. CYP4F12 also is a biomarker as prognostic predictors and therapeutic guide in EC patients.

Towards monitoring of critical illness via the detection of histones with extended gate field-effect transistor sensors

Extracellular histone proteins in the blood indicate a heightened risk of morbidity after trauma or in major illnesses such as sepsis. We present the development of an aptasensor for histone detection with an extended gate field-effect transistor (EGFET) configuration, which benefits from low power consumption, rapid response, and compatibility with miniaturized gold electrodes. Histones have a high isoelectric point and charge density, which cause them to physically adsorb to non-specific elements of the sensor that have available electrostatic charges. To combat this, the sensing surface is formed with a thiol-modified, high-affinity and histone-specific RNA aptamer sequence and by co-immobilizing with poly(ethylene glycol) methyl ether thiol (PEG) as a blocking agent. Surface plasmon resonance (SPR) is used to analyze aptamer and PEG immobilization strategies, confirm histone binding, and calculate kinetic binding constants. Through comparison of different blocking agents and time-dependent preparation, the ideal equilibrium dissociation constant (KD) is estimated to be below 200 pM, which is the upper range of extracellular histone concentrations in critically ill patients with high mortality. The EGFET sensitivity of the optimized aptasensor is 6.65 mV/decade concentration change for histone H4 with a physiologically relevant 5 pM limit of detection. Selectivity tests with 100 nM bovine serum albumin (BSA) demonstrate a signal response that is 13-fold smaller than for histones. This EGFET aptasensor platform is suitable for future point-of-care monitoring of histone levels in critically ill patients, thus permitting the early detection of increased risk and the need for more aggressive interventional measures to prevent mortality.

Extracellular histones promote TWIK2-dependent potassium efflux and associated NLRP3 activation in alveolar macrophages during sepsis-induced lung injury.

Sepsis-induced acute lung injury (ALI) is a complex and life-threatening condition lacking specific and efficient clinical treatments. Extracellular histones, identified as a novel type of damage-associated molecular patterns, have been implicated in the inflammatory process of ALI. However, further elucidation is needed regarding the precise mechanism through which extracellular histones induce inflammation. The aim of this study was to investigate whether extracellular histones can activate NLRP3 inflammasome-mediated inflammation in alveolar macrophages (AMs) by affecting TWIK2-dependent potassium efflux. We conducted experiments using cecal ligation and puncture (CLP) C57BL/6 mice and extracellular histone-stimulated LPS-primed MH-Scells. The results demonstrated a significant increase in the levels of extracellular histones in the plasma and bronchoalveolar lavage fluid (BALF) of CLP mice. Furthermore, neutralizing extracellular histone mitigated lung injury and inflammation in CLP-induced ALI mice. In vitro studies confirmed that extracellular histones upregulated the expression of NLRP3 inflammasome activation-related proteins in MH-S cells, and this effect was dependent on increased potassium efflux mediated by the TWIK2 channel on the plasma membrane. Moreover, extracellular histones directly triggered a substantial influx of calcium, leading to increased Rab11 activity and facilitating the trafficking and location of TWIK2 to the plasma membrane. These findings underscore the critical role of extracellular histone-induced upregulation of TWIK2 expression on the plasma membrane of alveolar macrophages (AMs). This upregulation leads to potassium efflux and subsequent activation of the NLRP3 inflammasome, ultimately exacerbating lung inflammation and injury during sepsis.

Sex and Age Differentially Regulate Epigenetic Modifications and Renal Injury Markers in Mice

Elucidation of epigenetic mechanism associated with aging renal physiology may reveal potential biomarkers and therapeutic targets for reversing aging renal pathologies. There is a sex difference in the age of onset of renal and cardiac disease, where females are protected against age related changes, including hypertension and fibrosis, until menopause. We hypothesized that sex and age would differentially regulate epigenetic modifications and renal physiology. To test this hypothesis, we examined kidney tissue and serum from 4-month (4M), 12-month (12M), and 24-month (24M)-old male and female C57BL/6JN mice (National Institute of Aging). Renal histone deacetylase (HDAC) and histone acetyltransferase (HAT) activities (ELISA) were significantly higher in all male mice groups compared with age-matched females. However, HAT activity decreased significantly in 24M males compared with 4M males and exhibited sex differences with 24M males having higher (p < 0.05) activity that 24M females. Levels of histone 3 (H3) acetylation at lysine (K) 9 and 27 and methylation at K9 were comparable in all male and female groups. Nonetheless, age and sex differences were observed in H3K27 methylation levels with 24M males having significantly higher (p < 0.05) levels as compared with 24M female mice. Serum creatinine (sCr) levels exhibited significant interaction between age and sex (p < 0.0001). Aging significantly increased sCr levels in 24M mice compared with 4M mice in both sexes. However, sex-differences were evident in sCr levels with 24M female mice having higher levels compared with their age matched male counterparts. Of note, serum levels of kidney injury molecule-1 (KIM-1), a tubule injury biomarker were significantly higher in 24M mice compared with 4M and 12M sex-matched mice (male 24M, 410.1 ± 94.6 vs. 12M, 53.6 ± 17.2 and 4M, 27.9 ± 2.0; pg/ml, p < 0.001; female 24M, 358.3 ± 56.1 vs. 12M, 63.8 ± 13.3 and 4M, 28.2 ± 5.0; pg/ml, p < 0.001). Histones are released in the extracellular space during disease state, act as damage-associated molecular pattern molecules and activate inflammatory pathways. In the serum, circulating histone H3 levels showed significant interaction with age and sex (p < 0.05) and increased significantly with age in both sexes. The data demonstrate that aged female mice exhibited higher levels of serum Cr and KIM-1 whereas aged males have higher KIM-1 levels as compared to sex matched young mice. Sex differences were observed in HDAC and HAT activity with males having higher enzymatic activity as compared to females. These findings will have important implications in age-related renal injury as aging is a primary risk factor for hypertension and related renal disease in men and women. Supported by the NIH grant R03AG075396. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extracellular histones: a unifying mechanism driving platelet-dependent extracellular vesicle release and thrombus formation in COVID-19

BackgroundCOVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state. ObjectivesFrom the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis. MethodsWe utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice. ResultsCompared with controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell-free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo. ConclusionWe conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial.