Chromatin Decondensation Research Articles

Alterations in the chromatin packaging, driven by transcriptional activity, revealed by AFM

BackgroundThe gene expression differs in the nuclei of normal and malignant mammalian cells, and transcription is a critical initial step, which defines the difference. The mechanical properties of transcriptionally active chromatin are still poorly understood. Recently we have probed transcriptionally active chromatin of the nuclei subjected to mechanical stress, by Atomic Force Microscopy (AFM) [1]. Nonetheless, a systematic study of the phenomenon is needed. MethodsNuclei were deformed and studied by AFM. Non-deformed nuclei were studied by fluorescence confocal microscopy. Their transcriptional activity was studied by RNA electrophoresis. ResultsThe malignant nuclei under the study were stable to deformation and assembled of 100–300 nm beads-like units, while normal cell nuclei were prone to deformation. The difference in stability to deformation of the nuclei correlated with DNA supercoiling, and transcription-depended units were responsive to supercoils breakage. The inhibitors of the topoisomerases I and II disrupted supercoiling and made the malignant nucleus prone to deformation. Cell nuclei treatment with histone deacetylase inhibitors (HDACIs) preserved the mechanical stability of deformed malignant nuclei and, at the same time, made it possible to observe chromatin decondensation up to 20–60 nm units. The AFM results were supplemented with confocal microscopy and RNA electrophoresis data. ConclusionsSelf-assembly of transcriptionally active chromatin and its decondensation, driven by DNA supercoiling-dependent rigidity, was visualized by AFM in the mechanically deformed nuclei. General significanceWe demonstrated that supercoiled DNA defines the transcription mechanics, and hypothesized the nuclear mechanics in vivo should depend on the chromatin architecture.

MYSM1 acts as a novel co-activator of ERα to confer antiestrogen resistance in breast cancer

Endocrine resistance is a crucial challenge in estrogen receptor alpha (ERα)-positive breast cancer (BCa). Aberrant alteration in modulation of E2/ERα signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Herein, we demonstrate that MYSM1 as a deubiquitinase participates in modulating ERα action via histone and non-histone deubiquitination. MYSM1 is involved in maintenance of ERα stability via ERα deubiquitination. MYSM1 regulates relevant histone modifications on cis regulatory elements of ERα-regulated genes, facilitating chromatin decondensation. MYSM1 is highly expressed in clinical BCa samples. MYSM1 depletion attenuates BCa-derived cell growth in xenograft models and increases the sensitivity of antiestrogen agents in BCa cells. A virtual screen shows that the small molecule Imatinib could potentially interact with catalytic MPN domain of MYSM1 to inhibit BCa cell growth via MYSM1-ERα axis. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ERα action and provide a potential therapeutic target for endocrine resistance in BCa.

High-capacity sample multiplexing for single cell chromatin accessibility profiling

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.

Gsdme Promotes PAD4 Activation and DNA Externalization from Apoptotic Neutrophils

Introduction Neutrophil extracellular traps (NETs) neutralize bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. However, non-apoptotic cell death signaling pathways mediated by MLKL and GSDMD can also engage PAD4 in neutrophils to trigger NET formation. Additionally, while PAD4 is hypothesized to neutralize charge on histones to facilitate chromatin decondensation, a genetic deficiency in Padi4 fails to prevent chromatin decondensation but does prevent NET formation following MLKL activation. Together, these data suggest more complex roles for PAD4 in the extrusion of nuclear DNA. Here we explored the role of GSDME - another pore-forming protein activated by apoptotic caspases - in controlling PAD4 activation, DNA externalization, and NET formation in apoptotic neutrophils. Methods Cell viability was quantified by flow cytometry and live-cell imaging using Hoechst, Cell Tracker Green, Annexin V, and propidium iodide. Histone citrullination was studied at the population level by immunoblot, at the single cell level by lattice SIM super-resolution microscopy, and at the molecular level by an in-situ ChIP-Seq methodology CUT&Tag. Calcium signaling linked to PAD4 activation was monitored by flow cytometry. Ultrastructural changes occurring in the absence of PAD4 and GSDME were investigated by transmission electron microscopy and super-resolution microscopy. Results Intrinsic and extrinsic apoptosis promote GSDME-dependent calcium mobilization and membrane permeabilization, leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit distribution on chromatin is developmentally controlled by PAD4 in neutrophils, with H3Cit found concentrated at promoter regions during neutrophil development. Lattice SIM super-resolution microscopy revealed clustered distribution of H3Cit in the nuclei of neutrophils, reflecting a unique spatial organization and coordination of multiple promoter elements in neutrophils. Following apoptotic stimulation, H3Cit redistributes in a coordinated process from promoter to intergenic and intronic regions. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils, but also blocks calcium signaling that activates PAD4, ultimately preventing NET formation. During apoptosis in GSDME-deficient neutrophils, early-apoptotic cellular changes to the chromatin and cytoplasmic granules are prolonged, generating highly atypical cellular states that fail to extrude nuclear DNA. Conclusion Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end-of-life for neutrophils.

Peptidyl Arginine Deiminase (PAD) 4 Modulates the GI Microbiome and Baseline Inflammation in Mice: Implications for Sepsis

Introduction: As part of the innate immune response, neutrophils release neutrophil extracellular traps (NETs), webs of decondensed chromatin coated with histones that capture bacteria but cause collateral tissue damage when produced in a dysregulated manner. Deficiency of PAD4, a protein that converts histone arginine to citrulline to promote chromatin decondensation essential to NET release (NETosis), has been associated with improved outcomes in murine sepsis models, leading some to propose that inhibiting NETosis may be protective. This conclusion has primarily been drawn from studies using the murine cecal-ligation and puncture (CLP) model of polymicrobial sepsis, in which the cecum is perforated to disseminate GI microbiota. However, NET release is an evolutionarily conserved function, present in protozoa and retained in all studied animal species, suggesting that it plays a crucial role in the immune response. In this study, we seek to characterize the baseline immunophenotype of NET-deficient, PAD4 knockout (PAD4 -/-) mice, to determine how PAD4 influences the GI microbiome to impact outcomes in GI models of polymicrobial sepsis. Methods: Whole blood samples were collected from wild type (WT) C57BL/6 mice, PAD4 -/- mice generated by our group through deletion of exon II in the PAD4 gene (PAD4D ΔII), and PAD4 -/- mice with deletion of exon IX and X (PAD4D ΔIX+X) obtained from Jackson laboratory. Complete blood counts (CBCs) and weights were obtained from PAD4 -/- and healthy WT mice at 9-12 weeks. The Olink target 96 mouse exploratory panel was used to quantify variation in levels of 96 plasma proteins. Next generation sequencing was used to assess the abundance of different bacterial species in stool samples from WT, PAD4D ΔII, and PAD4D ΔIX+X animals. Polymicrobial sepsis was then induced through peritoneal injections of cecal slurry (CS), using cecal samples from WT, PAD4D ΔII, and PAD4D ΔIX+X donor mice. 24 hours post injection, mean sepsis score (MSS) was assessed, and blood was obtained for CBC measurement, plasma proteomic analysis, and quantification of bacterial colony forming units (CFUs). Relative abundance of bacterial species was quantified in blood and liver homogenates. Survival outcome was performed on parallel subsets of WT and PAD4 -/- mice. Results: At comparable ages, both strains of PAD4 -/- animals had lower weights and significantly higher baseline neutrophil counts than WT controls bred in the same colony. Proteomic analysis revealed that PAD4D ΔII mice had baseline elevation in the inflammatory markers IL6 and Plin1, while PAD4D ΔIX+X mice, the strain with more pronounced neutrophilia, had higher GM-CSF and decreased IL23r, proteins crucial to neutrophil mobilization. In CS sepsis studies, WT animals that received WT CS were sicker with higher MSS, lower platelet counts, higher inflammatory markers, and a 30% mortality rate. In contrast, PAD4D ΔII mice that received PAD4D ΔII CS had a milder clinical course, lower bacterial dissemination, and 100% survival (Fig2). To assess the basis for this unexpected outcome, we compared bacterial colony-forming units (CFU) in WT to PAD4 -/- CS and found lower levels of bacteria in PAD4D ΔII CS. Deep sequencing of stool from WT, PAD4D ΔII, and PAD4D ΔIX+X animals revealed marked differences in the relative abundance of bacterial species. Sequencing of blood and liver samples obtained 24 hrs after CS injection showed that the predominant proliferating organism in WT mice was Enterococcaceae species while there was an abundance of Lactobacillaceae species in the blood and liver of PAD4D ΔII mice (Fig1). Sepsis studies repeated with genotype switched CS showed that WT mice treated with PAD4D ΔIX+X CS had a milder disease course with 100% survival, whereas PAD4D ΔIX+X mice injected with WT CS had elevated MSS, a trend towards higher plasma inflammatory markers, and a 50% mortality rate, similar to that observed in WT mice injected with WT CS (Fig2). Conclusions: These studies show that NETosis influences baseline inflammation and modulates the GI microbiome in mice. Differences in disease severity and survival between PAD4 -/- and WT mice observed in the CS model of polymicrobial sepsis is primarily due to greater pathogenicity of the WT CS inoculum rather than collateral tissue damage secondary to dysregulated NETosis. The basis for differences in inflammation and microbiome composition in PAD4 -/- mice needs to be examined.

Neutrophil elastase activates the release of extracellular traps from COPD blood monocyte-derived macrophages.

Neutrophil elastase (NE), a major inflammatory mediator in chronic obstructive pulmonary disease (COPD) airways, impairs macrophage function, contributing to persistence of airway inflammation. We hypothesized that NE activates a novel mechanism of macrophage-induced inflammation: release of macrophage extracellular traps (METs). The METs are composed of extracellular DNA decorated with granule proteinases and oxidants and may trigger persistent airway inflammation in COPD. To test the hypothesis, human blood monocytes were isolated from whole blood of subjects with COPD recruited following informed written consent. Patient demographics and clinical data were collected. Cells were cultured in media with GM-CSF to differentiate into blood monocyte derived macrophages (BMDMs). The BMDMs were treated with FITC-NE and unlabeled NE to determine intracellular localization by confocal microscopy and intracellular proteinase activity by DQ-Elastin assay. After NE exposure, released extracellular traps were quantified by abundance of extracellular DNA in conditioned media using the Pico Green assay. BMDM cell lysates were analyzed by Western analysis for proteolytic degradation of histone H3 or H4 or upregulation of peptidyl arginine deiminase (PAD) 2 and 4, two potential mechanisms to mediate extracellular trap DNA release. We observed that NE was taken up by COPD BMDM, localized to the cytosol and nucleus, and retained proteinase activity in the cell. NE induced MET release at doses as low as 50 nM. NE treatment caused histone H3 clipping but no effect on histone H4 nor PAD 2 or 4 abundance or activity. In summary, NE activated COPD MET release by clipping histone H3, a prerequisite for chromatin decondensation.

Pathogenetic role of neutrophil extracellular traps in coronary flow impairment following percutaneous coronary intervention in acute myocardial infarction patients -A multicenter study-

Abstract Background A suicidal function of neutrophils to ensnare and kill microbes by expelling neutrophil extracellular traps (NETs) via histone citrullination and chromatin decondensation has emerged as it enhances local inflammation and thrombogenicity at the culprit lesion of acute myocardial infarction (AMI). Purpose This multicenter study was aimed to examine 1) the histopathological feature of coronary thrombus with the NETs formation in AMI patients, and 2) the association of NETs with impaired coronary flow after percutaneous coronary intervention (PCI) for AMI patients. Methods Coronary thrombi were obtained from 44 patients with AMI [male; 77%, age; 69±12(SD) y-o)]who underwent PCI within 24 hours from the onset. Aspirated thrombi were immediately bisected and frozen, and 5-µm thickness unstained sections were processed for polarized light microscopy (PLM) imaging and immunofluorescence staining. NETs were immunohistochemically defined when objects were co-stained with anti-myeloperoxidase and anti-citrullinated histone H3 antibodies (Figure A,B). Adjacent sections were stained with Masson’s trichrome to measure the whole thrombus area. The ImageJ1 software was utilized for the automatic segmentation on the digitized histology images. Segmented NETs area was divided by the thrombus area as a measure of NETs. Clinical characteristics, including AMI subset, post PCI final TIMI flow grade and TIMI frame count, were collected. Results All aspirated thrombi exhibited NETs (Figure A), and then subjects were split into 2 groups with a median value of NETs (62.8 x10-4 um2/um2); high-NETs and low-NETs groups. Patient characteristics were all comparable between the 2 groups. PLM results showed that birefringent crystals, suggestive of cholesterol crystals, were more frequently noted in the thrombi with high-NETs compared with those with low-NETs (50.0% vs. 13.6%, P=0.010). In the immunofluorescence, NETs were seen in the proximity of the crystals (Figure B). The multivariable analysis with covariates of age, sex, major coronary risk factors, MI related to left anterior descending artery and the onset to aspiration time revealed that the presence of birefringent crystals remained an independent predictor for high-NETs formation [OR=5.78, 95%CI=1.13-29.66, P=0.035]. In the high-NETs group, the prevalence of the final TIMI flow grade 3 was significantly lower than the low-NETs group (63.6% vs. 90.9 %, P=0.031). Likewise, there was a significant positive correlation between NETs area and TIMI frame count, defined as the number of cineframes required for contrast to reach a standardized distal coronary landmark (R=0.681, P<0.001). Conclusion The present study demonstrated that NETs were highly expressed in coronary thrombus aspirated from AMI patients in association with crystalline components, relating to coronary flow impairment following PCI in AMI patients.

Azacitidine Combination with Lenalidomide in Treating Higher-Risk Myelodysplastic Syndromes: A Single-Center, Phase 2 Trial

Background: Azacitidine, which works through the proteosomic destruction of DNA methyltransferase and resultant chromatin decondensation, is used primarily in patients with higher-risk myelodysplastic syndromes (MDS). Lenalidomide has been approved by the US Food and Drug Administration for the treatment of MDS patients with del(5q) cytogenetic abnormality. It has also shown activity in patients with higher-risk MDS and acute myeloid leukemia. In this trial, we assessed the efficacy and safety of combining azacytidine and lenalidomide in higher-risk MDS. Methods: This is a single-center, phase 2 trial. Aza (75mg/m 2/d on days 1-7 of a 28-day cycle) was administered in combination with lenalidomide (10 mg/day on days 1 to 21 every 28 days) for patients. The primary endpoint was the overall response rate (ORR). Secondary endpoints were overall survival (OS), progression-free survival (PFS), and adverse events (AEs). An MDS targeted-exome-seq panel including 37 driver genes was used for screening. Results: Between Oct 2020 to Apr 2023, a total of 41 MDS patients were enrolled. 32 patients were evaluable for efficacy. 23 (71.875%) responded to therapy: 7 achieved a complete response (CR) (21.875%), 12 a marrow CR (mCR) (37.5%), and 4 a hematologic improvement (HI) (12.5%), of whom 10 of the mCR patients also had a HI. The median follow-up was 5.6 months (1 to 27.4), the median overall survival (OS) was not reached, and the median progression-free survival was 12.2 months. The time to reach CR was 2 months (range, 1 to 4.5), and the median CR duration was 8+ months (range, 5 to 27.4) ( Table 1). 27 patients had at least one mutation, the most common being TET2 (n = 12), ASXL1 (n = 12), TP53 (n = 7), SRSF2 (n = 6), and RUNX1 (n = 6) ( Fig.1A). Compared with patients without mutations, CR/mCR rate was significantly higher for those with mutations in TP53 (100% v 48%; P =0 .025) ( Fig. 1B), ORR were numerically higher for those with TP53 (100% v 64%; P = 0.149) and TET2 (85% v 63%; P = 0.422) ( Fig. 1C). We also observed a reduction of VAF in TP53 and TET2 mutation after the combination treatment ( Fig. 1D & 1E). Common grade 3/4 nonhematologic adverse events included thrombocytopenia (18.75%), infection (12.5%), and febrile neutropenia (9.4%). Conclusion This trial demonstrated that the combination of azacytidine with a lenalidomide regimen is an effective therapy for higher-risk MDS patients, with high efficacy and well-tolerance. Patients with TP53 mutation appear more likely to achieve a CR/mCR.

A Novel Natural Derivative GL-V9 Inducing Mitotic Catastrophe and Displaying a Synergistic Anti-Tumor Effect with EZH2 Inhibitor in Acute Myeloid Leukemia

Background GL-V9 is a flavonoid derivative from the natural product wogonin with elegant anti-tumor activity, while the effect of GL-V9 in acute leukemia has no report. Enhancer of zeste homolog 2 (EZH2) catalyzes trimethylation on the K27 residue of histone H3 (H3K27Me3), which facilitates non-homologous DNA end joining (NHEJ). EZH2 inhibitor could induce chromatin decondensation and promote the sensitivity of tumor cells towards DNA damage drugs. In the present study, we evaluated the anti-leukemia activity of GL-V9 and its synergy with EZH2 inhibitor DZNeP in acute myeloid leukemia (AML). Methods Cell proliferation and cytotoxicity were measured by Cell Counting Kit-8(CCK-8) in U937, THP-1, and MV4-11 AML cells. RNA-seq was performed in U937 cells treated with 2μM DZNeP, MV4-11 cells treated with 4μM GL-V9, and vehicle for 48 hours. The clone formation rate was measured by a plate clone formation assay. Cell cycle and apoptosis were measured by flow cytometry analysis. Morphology examination was conducted with Fluorescence (IF) microscopy . Expression of target genes was detected by western blot and RT-qPCR. Results GL-V9 had a dose-/time-dependent effect on cell viability with IC50 at 3-4μM in U937 and THP-1 cells (Fig.1A); it also significantly induced the G2/M arrest and apoptosis of the cells (Fig.1B, C). IF staining showed GL-V9 triggers micro-, multi-nucleation, and cytoskeleton abnormalities, the feature of mitotic catastrophe in U937 cells 4hrs after the treatment (Fig1.D). These data indicated that GL-V9 exerts the anti-leukemia effect by inducing mitotic catastrophe. We further analyzed the whole genome transcriptome upon GL-V9 treatment in U937 cells, and GSEA analysis of the differentially expressed genes (DEGs) showed that GL-V9 treatment negatively correlated with genes involved in sister chromatid separation, microtubule cytoskeleton organization, DNA double-strand break repair, mismatch repair, and cell cycle checkpoint signaling (Fig1.E). These data revealed that GL-V9 may induce DNA damage-mediated mitotic catastrophe in the AML cells. H3K27me3 and EZH2 play an important role in the early DNA damage response. We thus carried out the combination therapy of EZH2 inhibitor DZNeP with GL-V9. Results showed that co-treatment of GL-V9 with DZNeP exerted synergistic effects on reduced cell viability and clone formation versus single-drug in U937 and MV4-11 cells (Fig2.AB). DZNeP induced G0/G1 arrest; and GL-V9 induced G2/M arrest; whereas the combination significantly decreased the number of cells in the S-phase (Fig2.C). Also, the percentage of the apoptotic cells significantly increased upon the GL-V9+DZNeP treatment versus the controls in the cells (Fig2.D) as well as in ASXL1 mutant AML-M5 and AML-M2 primary cells (Fig2.E). Consistently, the combination significantly induced the expression changes of cell cycle regulator CCNE2 and p21, and dramatically up-regulated apoptotic effectors PARP, Caspase-3, and DNA damage marker γH2AX versus single-drug (Fig2.F). Furthermore, the overlapped DEGs were identified with 242 genes regulated in homo-direction but 25 regulated in the opposite treated with GL-V9 or DZNeP, these genes mainly influenced DNA replication, chromatin structures, and DNA damage response (Fig2.G). GL-V9 and/or DZNeP down-regulated the EZH2 expression, induced the decrease of p-AKT and c-Myc, and the increase of p53 in U937 cells (Fig. 2H). These data indicate that GL-V9+DZNeP may exert the anti-leukemia effect by inducing DNA damage via targeting EZH2/ PI3K/AKT signaling and the p53-c-Myc axis in the cells. EZH2 inhibition modifies the chromatin structure and decondensed the H3K27me3-marked chromatin, making AML cells more accessible to the nuclear-attacked agent GL-V9. The mechanism model is depicted in Fig. 2I. Conclusions GL-V9 is an effective novel nature compound against AML and exhibited strong synergistic effects with the epigenomic drug EZH2 inhibitor DZNeP potentially by inducing DNA damage-mediated mitotic catastrophe through targeting EZH2/ PI3K/AKT signaling and p53-cMyc axis. Our results provide pre-clinical evidence for the new combination as a promising clinical option for AML patients.

Automated Image Analysis of Transmission Electron Micrographs: Nanoscale Evaluation of Radiation-Induced DNA Damage in the Context of Chromatin.

Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns.

The activation of SIRT1 by resveratrol reduces breast cancer metastasis to lung through inhibiting neutrophil extracellular traps

Neutrophil extracellular traps (NETs) play a crucial role in breast cancer metastasis. However, the therapeutic target of NETs in breast cancer metastasis is still unknown. Using a natural metabolite library and single-cell sequencing data analysis, we identified resveratrol (RES), a polyphenolic natural phytoalexin, and agonist of silent information regulator-1 (SIRT1) that suppressed NETs formation after cathepsin C (CTSC) treatment. In vivo, RES significantly hindered breast cancer metastasis in a murine orthotopic 4T1 breast cancer model. Serum levels of myeloperoxidase-DNA and neutrophil elastase-DNA in mouse breast cancer model were significantly lower after RES treatment. Correspondingly, the tumour infiltrated CD8+T cells in the lungs increased after the treatment. Mechanistically, RES targets SIRT1 in neutrophils and significantly inhibits the citrullination of histones H3, which is essential for chromatin decondensation and NETs formation. Furthermore, we identified that the NETs were suppressed by RES in bone marrow neutrophils after CTSC treatment, while specific deficiency of SIRT1 in neutrophils promoted NETs formation and breast cancer to lung metastasis. Thus, our results revealed that RES could be potentially identified as a viable therapeutic drug to prevent neutrophil cell death and breast cancer metastasis.

In Vitro Study on Spermicidal Action of Hydro-methanol Extract of Tinospora cordifolia (Willd.) Stem in Rat and Human Sperm: a Comparative Analysis.

Human fertility regulation is a major way to control overpopulation. In this perspective, this study emphasized the in vitro effect of hydro-methanol extract of Tinospora cordifolia (TCHME) stem for spermicidal and reproductive hypo-functions using human and rat samples. Control, 0.5-, 1-, and 2-mg TCHME-charged groups were considered to assess the relevant parameters. Levels of spermiological parameters like sperm motility, viability, the integrity of plasma and acrosomal membrane, and nuclear chromatin decondensation were significantly reduced (p < 0.05) in the dose- and duration-dependent TCHME-charged groups compared to the control. The inhibitory concentration 50 (IC50) of TCHME on motile human and rat sperms were 0.8 and 0.4 mg/ml, respectively. Testicular androgenic key enzymes and antioxidant enzymes (human sperm pellet, testes, and epididymis of rat)' activities were significantly diminished (p < 0.05), while antioxidant enzymes' activities were significantly elevated (p < 0.05) in renal and insignificantly (p > 0.05) elevated in hepatic tissues of rat in TCHME-charged groups compared to the control. Significant elevation (p < 0.05) of thiobarbituric acid reactive substances (TBARS)' level in human sperm pellet, testes, and epididymis of rats and significant diminution (p < 0.05) in TBARS levels of liver and kidney were observed in TCHME-charged groups. It focused that TCHME is more potent for stress imposition on reproductive tissues and sperm compared to the other tested tissues. Non-significant alterations (p > 0.05) in glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities in the said organs of rat indicated its non-toxic effect. It highlighted that TCHME possesses spermicidal and reproductive tissue-specific effects which strengthen the possibilities of male contraceptive development from it.

CXCR4 Targeting Nanoplatform for Transcriptional Activation of Latent HIV-1 Infected T Cells.

Antiretroviral drugs are limited in their ability to target latent retroviral reservoirs in CD4+ T cells, highlighting the need for a T cell-targeted drug delivery system that activates the transcription of inactivated viral DNA in infected cells. Histone deacetylase inhibitors (HDACi) disrupt chromatin-mediated silencing of the viral genome and are explored in HIV latency reversal. But single drug formulations of HDACi are insufficient to elicit therapeutic efficacy, warranting combination therapy. Furthermore, protein kinase C activators (PKC) have shown latency reversal activity in HIV by activating the NF-κB signaling pathway. Combining HDACi (SAHA) with PKC (PMA) activators enhances HIV reservoir activation by promoting chromatin decondensation and subsequent transcriptional activation. In this study, we developed a mixed nanomicelle (PD-CR4) drug delivery system for simultaneous targeting of HIV-infected CD4+ T cells with two drugs, suberoylanilide hydroxamic acid (SAHA) and phorbol 12-myristate 13-acetate (PMA). SAHA is a HDACi that promotes chromatin decondensation, while PMA is a PKC agonist that enhances transcriptional activation. The physicochemical properties of the formulated PD-CR4 nanoparticles were characterized by NMR, CMC, DLS, and TEM analyses. Further, we investigated in vitro safety profiles, targeting efficacy, and transcriptional activation of inactivated HIV reservoir cells. Our results suggest that we successfully prepared a targeted PD system with dual drug loading. We have compared latency reversal efficacy of a single drug nanoformulation and combination drug nanoformulation. Final PD-SP-CR4 successfully activated infected CD4+ T cell reservoirs and showed enhanced antigen release from HIV reservoir T cells, compared with the single drug treatment group as expected. To summarize, our data shows PD-SP-CR4 has potential T cell targeting efficiency and efficiently activated dormant CD4+ T cells. Our data indicate that a dual drug-loaded particle has better therapeutic efficacy than a single loaded particle as expected. Hence, PD-CR4 can be further explored for HIV therapeutic drug delivery studies.

Formulation and evaluation of solid lipid nanoparticles loaded with papaya seed chloroform extract for long-term antifertility effects on the male rat, Bandicota bengalensis

The study is the first to formulate and investigate potential of papaya seed chloroform extract based solid lipid nanoparticles (PSCEN) as antifertility agents on male Bandicota bengalensis. The prepared nanoparticles were spherical of size 300–600 nm. The release kinetics showed a controlled release of the drug with major release over 48 h. To assess the antifertility effects of PSCEN, adult male rats were fed a diet containing two different concentrations of PSCEN (5% and 10%) for 15 days under bi-choice conditions. The mean total active ingredient ingestion of the rats in the two treated groups ranged from 2.13–3.31 and 3.92–5.87 g/100g body weight, respectively. No adverse effects of treatment on body weight were observed. Also, no mortality of rats was observed. The treatment had a significant effect on the weight of the testis and the epididymis, but not on the other organs. Sperm motility (%), sperm viability (%), sperm count (millions/ml), sperm mitochondrial activity (%), sperm nuclear chromatin de-condensation (%) and sperm hypo-osmotic swelling (%) were significantly decreased, and sperm abnormality (%) significantly increased compared to the vehicle control group. The reproductive success rates of male rats treated with 5% and 10% PSCEN and mated with untreated female rats were 20.00–66.67% and 16.67%, respectively, while in untreated female rats mated with male rats of vehicle control group, reproductive success rate was 33.33 to 80%. The study found a maximal antifertility effect of the 10% PSCEN containing bait, which was irreversible up to 105 days after stopping treatment, suggesting long-term efficacy.

Lack of eosinophil extracellular trap formation due to failure of plasma membrane breakdown in the absence of elastase

AbstractActivated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell’s DNA covered with granule-derived antimicrobial peptides. Upon stimulation of eosinophils with the known EET-inducers phorbol 12-myristate 13-acetate, monosodium urate crystals, or Candida albicans, we observed that their plasma membrane became compromised, resulting in accessibility of the nuclear DNA for staining with the impermeable DNA dye Sytox Green. However, we did not observe any DNA decondensation or plasma membrane rupture by eosinophils, which sharply contrasts with neutrophil extracellular trap (NET) formation and the subsequent cell death known as NETosis. Neutrophil elastase (NE) activity is thought to be essential for the cleavage of histones and chromatin decondensation during NETosis. We observed that the neutrophils of a patient with a mutation in ELANE, leading to congenital neutropenia and NE deficiency, were unable to undergo NETosis. Taken together, we may suggest that the natural absence of any NE-like proteolytic activity in human eosinophils explains why EET formation is not observed, even when eosinophils become positive for an impermeable DNA dye in response to stimuli that induce NETosis in neutrophils.

Citrullination of actin-ligand and nuclear structural proteins, cytoskeleton reorganization and protein redistribution across cellular fractions are early events in ionomycin-induced NETosis

Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.

PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin

RNA modifications have been known for many years, but their function has not been fully elucidated yet. For instance, the regulatory role of acetylation on N4-cytidine (ac4C) in RNA can be explored not only in terms of RNA stability and mRNA translation but also in DNA repair. Here, we observe a high level of ac4C RNA at DNA lesions in interphase cells and irradiated cells in telophase. Ac4C RNA appears in the damaged genome from 2 to 45 min after microirradiation. However, RNA cytidine acetyltransferase NAT10 did not accumulate to damaged sites, and NAT10 depletion did not affect the pronounced recruitment of ac4C RNA to DNA lesions. This process was not dependent on the G1, S, and G2 cell cycle phases. In addition, we observed that the PARP inhibitor, olaparib, prevents the recruitment of ac4C RNA to damaged chromatin. Our data imply that the acetylation of N4-cytidine, especially in small RNAs, has an important role in mediating DNA damage repair. Ac4C RNA likely causes de-condensation of chromatin in the vicinity of DNA lesions, making it accessible for other DNA repair factors involved in the DNA damage response. Alternatively, RNA modifications, including ac4C, could be direct markers of damaged RNAs.

Mitochondrial ROS and base excision repair steps leading to DNA nick formation drive ultraviolet induced-NETosis.

Reactive oxygen species (ROS) is essential for neutrophil extracellular trap formation (NETosis), and generated either by NADPH oxidases (e.g., during infections) or mitochondria (e.g., sterile injury) in neutrophils. We recently showed that ultraviolet (UV) radiation, a sterile injury-inducing agent, dose-dependently induced mitochondrial ROS generation, and increasing levels of ROS shifted the neutrophil death from apoptosis to NETosis. Nevertheless, how ROS executes UV-induced NETosis is unknown. In this study, we first confirmed that UV doses used in our experiments generated mitochondrial ROS, and the inhibition of mitochondrial ROS suppressed NETosis (Mitosox, SYTOX, immunocytochemistry, imaging). Next, we showed that UV irradiation extensively oxidized DNA, by confocal imaging of 8-oxyguanine (8-oxoG) in NETs. Immunofluorescence microscopy further showed that a DNA repair protein, proliferating cell nuclear antigen, was widely distributed throughout the DNA, indicating that the DNA repair machinery was active throughout the genome during UV-induced NETosis. Inhibition of specific steps of base excision repair (BER) pathway showed that steps leading up to DNA nick formation, but not the later steps, suppressed UV-induced NETosis. In summary, this study shows that (i) high levels of mitochondrial ROS produced following UV irradiation induces extensive oxidative DNA damage, and (ii) early steps of the BER pathway leading to DNA nicking results in chromatin decondensation and NETosis. Collectively, these findings reveal how ROS induces NOX-independent NETosis, and also a novel biological mechanism for UV irradiation- and -mitochondrial ROS-mediated NETosis.

Effect of pesticide exposure on human sperm characteristics, genome integrity, and methylation profile analysis.

According to the United Nations' Food and Agriculture Organization, the quantities of pesticide used around the world have increased regularly since the 1990s. Given that pesticides may be classified as carcinogenic, mutagenic, neurotoxic, or toxic for reproduction, some have endocrine-disrupting properties that might be associated with a decline in sperm parameters in general and sperm DNA integrity in particular. These days, a sperm analysis is not enough to determine the etiology of male infertility. Genome integrity analysis is a key step in clarifying a large proportion of cases of male infertility. The objective of the present retrospective study was to assess the impact of self-reported pesticide exposure on sperm parameters and sperm DNA integrity in men consulting for infertility. In a retrospective study, a population of 671 men living in the Picardy region of France were assessed in a conventional sperm parameter analysis, Shorr staining, a DNA fragmentation assay (terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling), and chromatin decondensation with aniline blue staining. The exposed and the non-exposed groups did not differ significantly in some of the conventional sperm parameters (including volume, sperm count, and percent typical forms). However, vitality, progressive motility, and non-progressive motility were significantly lower in the exposed group. Levels of DNA fragmentation and chromatin decondensation were moderately higher in the exposed group.

Evaluation of DNA integrity and chromatin condensation in cat sperm collected by urethral catheterization and epididymis slicing.

The aim of this study was to evaluate the effectiveness of the original sperm chromatin dispersion (SCD) assay and the toluidine blue (TB) stain to assess DNA fragmentation and chromatin condensation, respectively, in cat sperm obtained by urethral catheterization (CT) and epididymis slicing (EP). CT and EP samples were collected from the same cat, and sperm motility, concentration, morphology, DNA integrity and chromatin condensation were evaluated. As controls, aliquots of the samples were incubated with 0.3 M NaOH and with 1% of dithiothreitol (DTT) to promote DNA fragmentation and chromatin decondensation, respectively. With SCD, four DNA dispersion halo patterns were observed: large, medium, small and no halo. TB staining patterns were as follows: light blue (condensed chromatin), light violet (moderate chromatin decondensation) and dark blue-violet (high chromatin decondensation). Sperm incubations with NaOH and with DTT were effective in inducing DNA fragmentation and chromatin decondensation, respectively. No significant differences were observed in the percentages of the SCD and TB patterns between samples (CT and EP) and no correlation was observed between sperm head abnormalities and the different SCD and TB patterns. The original SCD technique and the TB stain were adapted to evaluate DNA integrity and chromatin condensation in cat sperm obtained by CT and EP.