Cytoplasmic Accumulation of Histones Induced by BET Inhibition Protects Cells from C9orf72 Poly(PR)-Induced Cell Death.

Repeat dipeptides such as poly(proline-arginine) (polyPR) are generated from the hexanucleotide GGGGCC repeat expansions in the C9orf72 gene. These dipeptides are often considered as the genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In the study, fluorescein isothiocyanate (FITC) labeled PR20 is used to investigate PR20-induced cell death. The findings reveal that the cell death induced by PR20 is dependent on its nuclear distribution and can be blocked by a nuclear import inhibitor called importazole. Further investigation reveals that BRD4 inhibitors, such as JQ-1 and I-BET762, restrict cytoplasmic localization of PR20, thereby reducing its cytotoxic effect. Mechanistically, the inhibition of BRD4 leads to an increase in the expression of numerous histones, resulting in the accumulation of histones in the cytoplasm. These cytoplasmic histones associate with PR20 and limit its distribution within the nucleus. Notably, the ectopic expression of histones alone is enough to confer protection to cells treated with PR20. In addition, phenylephrine (PE) induces cellular hypertrophy and cytoplasmic distribution of histone, which also helps protect cells from PR20-induced cell death. The research suggests that temporarily inducing the presence of cytoplasmic histones may alleviate the neurotoxic effects of dipeptide repeat proteins.

RING1 Inhibition Has a Cell-Specific Antitumoral Role by Promoting Autophagy in Endometrial Cancer Cells.

BACKGROUND/AIMS: Factors influencing gene expression through chemical modifications of histones may play an important role in the regulation of the autophagy process in cancers. RING1A or RING1B are responsible for the catalytical activity of Polycomb repressive complex 1 (PRC1) which monoubiquitylate histone H2A. The aim of the study was to determine the effect of the RING1A/B protein inhibition on the autophagy process in endometrial cancer cells and the anticancer effectiveness of RING1 inhibitor PRT4165 in combination with autophagy inhibitors. METHODS: The expression of autophagy genes and proteins were analyzed in endometrial cancer cells HEC-1A and Ishikawa grown in different glucose concentrations and treated with PRT4165. To assess the effectiveness of PRT4165 used alone or in combination with HCQ or Lys05, IC50 and the combination index (CI) were calculated. Flow cytometry method was used to estimate apoptotic cells after treatment. RESULTS: The results confirm the impact of RINGs on autophagy and apoptosis in endometrial cancer cells. PRT4165 inhibitor causes changes in the expression of ATG genes and autophagy markers and the effect depends on glucose concentration and cell types. However, the anticancer effectiveness of PRT4165 was lower when it was used in combination with autophagy inhibitors, suggesting that such a combination is not a promising anticancer strategy. CONCLUSION: The results indicate the importance of the RINGs in the process of autophagy and apoptosis. Further potentially more effective combinations of PRT4165 with autophagy modulators should be sought.

The hydroxamic acid derivative YPX-C-05 alleviates hypertension and vascular dysfunction through the PI3K/Akt/eNOS pathway.

BACKGROUND: Hypertension is a prevalent cardiovascular disease characterized by elevated blood pressure and increased vascular resistance. HDAC inhibitors have emerged as potential therapeutic agents due to their ability to modulate gene expression and cellular processes. YPX-C-05, a novel hydroxamic acid-based HDAC inhibitor, shows promise in its vasodilatory effects and potential targets for hypertension treatment. In this study, we aimed to elucidate the mechanisms underlying YPX-C-05's vasodilatory effects and explore its therapeutic potential in hypertension. METHODS: To determine the ex vivo vasodilatory effects of YPX-C-05, isolated aortic rings precontracted with phenylephrine were used. We assessed YPX-C-05's inhibitory effects on HDACs and its impact on histone H4 deacetylation levels in endothelial cells. Network pharmacology analysis was employed to predict putative targets of YPX-C-05 for hypertension treatment. To investigate the involvement of the PI3K/Akt/eNOS pathway, we employed enzyme-linked immunosorbent assay and to assess the levels of NO, ET-1, BH2, and BH4 in human umbilical vein endothelial cells. And we also analyzed the mRNA expression of eNOS and ET-1. Furthermore, Western blotting was conducted to quantify the phosphorylated and total Akt and eNOS levels in human umbilical vein endothelial cell lysates following treatment with YPX-C-05. In order to elucidate the vasodilatory mechanism of YPX-C-05, we employed pharmacological inhibitors for evaluation purposes. Furthermore, we evaluated the chronic antihypertensive effects of YPX-C-05 on N-omega-nitro-L-arginine-induced hypertensive mice in an in vivo model. Vascular remodeling was assessed through histological analysis. RESULTS: Our findings demonstrated that YPX-C-05 exerts significant vasodilatory effects in isolated aortic rings precontracted with phenylephrine. Furthermore, YPX-C-05 exhibited inhibitory effects on HDACs and increased histone H4 acetylation in endothelial cells. Network pharmacology analysis predicted YPX-C-05 might activate endothelial eNOS via PI3K/Akt signaling pathway. Inhibition of the PI3K/Akt/eNOS pathway attenuated the vasodilatory effects of YPX-C-05, as evidenced by reduced levels of phosphorylated Akt and eNOS in human umbilical vein endothelial cell lysates. The chronic administration of YPX-C-05 in N-omega-nitro-L-arginine-induced hypertensive mice resulted in significant antihypertensive effects. Histological analysis demonstrated a reduction in vascular remodeling, further supporting the therapeutic potential of YPX-C-05 in hypertension. CONCLUSION: This study demonstrates for the first time that the novel hydroxamic acid-based HDAC inhibitor YPX-C-05 produces significant antihypertensive and vasodilatory effects through the PI3K/Akt/eNOS pathway. Our findings support the developing prospect of YPX-C-05 as a novel antihypertensive drug.

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.

Early detection of hepatocarcinogens in rats by immunohistochemistry of γ-H2AX.

We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of γ-H2AX, a well-known marker of DNA damage. To investigate the potential application of γ-H2AX as a biomarker for early detection of hepatocarcinogens, we examined γ-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitrosodiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3'-dimethylbenzidine dihydrochloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for γ-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in γ-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Brip1, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with γ-H2AX formation. These results suggest that γ-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether γ-H2AX immunostaining should be combined with other markers to increase sensitivity. γ-H2AX immunostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.

Resveratrol exhibits diverse anti-cancer activities through epigenetic regulation of E-cadherin and p21 in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) has an aggressive phenotype and poor outcome, however no specific targeted therapy has been established for TNBC lacking germline BRCA1/2 pathogenic variants. To develop a novel therapeutic strategy, we explored the potential of resveratrol (RSV) for TNBC treatment. METHODS: We investigated the effects of RSV on malignant phenotypes of TNBC cells as well as on apoptosis induced by ABT263, a specific inhibitor of BCL-2 and BCL-xL, using morphological observation, migration assay, ß-galactosidase staining, and Hoechst staining. To elucidate the underlying mechanisms of RSV-mediated effects, expression levels and histone acetylation levels of cadherin 1 (CDH1, E-cadherin) and cyclin dependent kinase inhibitor 1A (CDKN1A, p21) were determined by RT-qPCR, western blotting, and chromatin immunoprecipitation. Furthermore, knockdown analysis was conducted to evaluate the involvement of E-cadherin and/or p21 in RSV potentiation on cytotoxic activity of ABT263. RESULTS: RSV treatment induced epithelial-like cellular morphology and suppressed the migration capacity in MDA-MB-231 and BT-549-Luc TNBC cells. ß-galactosidase-positive cells were increased after RSV treatment, indicating the induction of cellular senescence, in MDA-MB-231 cells but not in BT-549-Luc cells. RSV increased the expression and histone acetylation of CDH1 and CDKN1A in both cells. Interestingly, pre-treatment with RSV enhanced the induction of apoptosis in the ABT263-treated MDA-MB-231 and BT-549-Luc cells, and knockdown of CDKN1A decreased ABT263-induced apoptosis in RSV-treated MDA-MB-231 cells. CONCLUSIONS: RSV represses the metastatic capacity and enhances the cytotoxic activity of ABT263 in TNBC cells. Our results suggested that RSV can potentially be used as a repressor of metastasis or a sensitizer to ABT263 for TNBC treatment via up-regulation of CDH1 and CDKN1A through epigenetic mechanisms.

Effect of hubble-bubble smoking on global DNA methylation and transcription levels of protamine and histone genes in human spermatozoa.

This study was conducted to assess the impact of hubble-bubble smoking on global DNA methylation, DNA fragmentation; protamine deficiency of spermatozoa, and to determine whether the transcription levels of the protamine and histone genes are different in hubble-bubble smokers compared to nonsmokers. Five hundred semen samples were collected from males with an average age of 32.2 ± 6.1 years (300 hubble-bubble smokers "60%" and 200 nonsmokers "40%"). The nucleic acid was isolated from purified sperm, then ELISA and qPCR were used to evaluate the global DNA methylation and transcription level of protamine and histone, respectively. A significant elevation in global DNA methylation, protamine deficiency, and DNA fragmentation was found in hubble-bubble smokers compared to nonsmokers (P < 0.0001). A significant decline was shown in transcription levels of protamine and histone genes in hubble-bubble compared to nonsmokers (P < 0.0001). Additionally, a down-regulation in the transcription levels of protamine and histone was revealed in hubble-bubble compared to nonsmokers with fold change (0.0001 and 0.007, respectively). In conclusion, this study provided proof that hubble-bubble smoking has a negative impact on global DNA methylation, DNA fragmentation, protamine deficiency, and the transcription of protamine and histone genes in spermatozoa, and these findings influence negatively males' fecundity.

Histone-deacetylase-inhibitory effects of periodontopathic-bacterial metabolites induce human gingival epithelial Ca9-22 cell death.

Dental plaque bacteria produce high concentrations of short-chain fatty acids (SCFAs), as bacterial metabolites. SCFA-treated gingival epithelial cells undergo cell death. Our previous reports demonstrated that butyrate-induced cell death depends on autophagy and reactive oxygen species (ROS). However, the precise mechanisms underlying SCFA-induced gingival epithelial cell death is poorly understood. Butyrate is a strong histone deacetylase (HDAC) inhibitor. Therefore, we determined the involvement of HDAC inhibitory activity in SCFA-induced gingival epithelial cells. Ca9-22 cells were used as an in vitro counterpart of gingival epithelial cells. Ca9-22 cells were treated with HDAC inhibitors in the presence or absence of C646, a P300 histone acetyltransferase (HAT) inhibitor, and compared the number of dead cells, which are measured using SYTOX Green dye. Acetylation levels of histone H3 were examined using western blotting. Changes in transcriptomes during the butyrate and C646 treatment were examined using RNA sequencing analysis. The butyrate or propionate-treatment of Ca9-22 cells induced acetylation of histone H3, while the C646 treatment strongly reduced the elevated acetylation levels. Accordingly, butyrate or propionate-induced cell death was inhibited by the C646 treatment. Similar results were obtained when other HDAC inhibitors were used. Whole transcriptome analysis revealed that the expression of numerous genes was altered by butyrate-induced histone acetylation. Moreover, some autophagy and ROS-related genes found in the altered genes might induce cell death. This study suggests the need for HDAC-inhibitory activity of bacterial metabolites to induce cell death, and the effects might enhance autophagy and ROS production.

Antitumor Effect of Demethylzeylasteral (T-96) on Triple-Negative Breast Cancer via LSD1-Mediate Epigenetic Mechanisms.

Background and Purpose. Breast cancer ranks first in the incidence of female tumors. Triple-negative breast cancer (TNBC), one type of breast cancer, is more aggressive and has a worse prognosis. Demethylzeylasteral (T-96) is isolated from Tripterygium wilfordii Hook F. Our previous study found that T96 could inhibit TNBC invasion via suppressing the canonical and noncanonical TGF-ß signaling pathways. However, the antitumor effects and mechanisms of T-96 on TNBC have not been studied. This study is aimed at investigating the antitumor effect and mechanism of T-96 on breast cancer. Experimental approach. MTT assay, Live and Dead cell assay, and TUNEL were used to observe the antitumor effect of breast cancer cells treated with T-96. siRNA of LSD1, Co-IP, and molecular docking were used to explore the direct target and mechanism of T-96. Subcutaneous murine xenograft models were used to detect the efficacy of T-96 antitumor activity in vivo. Key Results. T-96 was more susceptible to inducing the apoptosis of highly metastatic TNBC cell lines (SUM-1315). An abnormal level of histone methylation is a crucial characteristic of metastatic cancer cells. LSD1 is a histone demethylase. We found that T-96 could significantly decrease the protein expression of LSD1, increase its target protein PTEN expression and enhance histone methylation. T-96 could also down-regulate the PI3K/AKT signaling pathway, which could be blocked by PTEN. Knockdown of LSD1 by siRNA blocked the pharmacological activity of T-96. And the molecular docking predicted T-96 processed affinity toward LSD1 through hydrogen bonding. Finally, T-96 was evaluated in a murine xenograft model of SUM-1315 cells. And T-96 could significantly inhibit tumor growth without showing marked toxicity. Conclusions & Implications. The results illustrated that T-96 exerted antitumor activity in highly metastatic TNBC by inactivating the LSD1 function.

In vitro and ex vivo anti-myeloma effects of nanocomposite As4S4/ZnS/Fe3O4.

Nanoparticles in medicine can integrate actively targeted imaging agents and drug delivery vehicles, and combining multiple types of therapeutics in a single particle has numerous advantages, especially in multiple myeloma. MM is an incurable hematological disorder characterized by clonal proliferation of plasma cells in the bone marrow. In this study, we evaluated the anti-myeloma activity of 3 nanocomposites (3NPs): As4S4/ZnS/Fe3O4 (1:4:1), As4S4/ZnS/Fe3O4 with folic acid (FA), and As4S4/ZnS/Fe3O4 with FA and albumin with reduced survival MM cell lines and primary MM samples by each of 3NP. Cytotoxic effects of 3NPs were associated with caspase- and mitochondria-dependent apoptosis induction and reduced c-Myc expression. Modulation of cell cycle regulators, such as p-ATM/ATM and p-ATR/ATR, and increases in p-Chk2, cyclin B1, and histones were accompanied by G2/M arrest triggered by 3NPs. In addition, 3NPs activated several myeloma-related signaling, including JNK1/2/3, ERK1/2 and mTOR. To overcome BM microenvironment-mediated drug resistance, nanocomposites retained its anti-MM activity in the presence of stroma. 3NPs significantly decreased the stem cell-like side population in MM cells, even in the context of stroma. We observed strong synergistic effects of 3NPs combined with lenalidomide, pomalidomide, or melphalan, suggesting the potential of these combinations for future clinical studies.

Osteogenic growth peptide is a potent anti-inflammatory and bone preserving hormone via cannabinoid receptor type 2.

The endocannabinoid system consists mainly of 2-arachidonoylglycerol and anandamide, as well as cannabinoid receptor type 1 and type 2 (CB2). Based on previous studies, we hypothesized that a circulating peptide previously identified as osteogenic growth peptide (OGP) maintains a bone-protective CB2 tone. We tested OGP activity in mouse models and cells, and in human osteoblasts. We show that the OGP effects on osteoblast proliferation, osteoclastogenesis, and macrophage inflammation in vitro, as well as rescue of ovariectomy-induced bone loss and prevention of ear edema in vivo are all abrogated by genetic or pharmacological ablation of CB2. We also demonstrate that OGP binds at CB2 and may act as both an agonist and positive allosteric modulator in the presence of other lipophilic agonists. In premenopausal women, OGP circulating levels significantly decline with age. In adult mice, exogenous administration of OGP completely prevented age-related bone loss. Our findings suggest that OGP attenuates age-related bone loss by maintaining a skeletal CB2 tone. Importantly, they also indicate the occurrence of an endogenous peptide that signals via CB2 receptor in health and disease.

Design, synthesis, cytotoxicity, and molecular docking studies of novel thiazolyl-hydrazone derivatives as histone lysine acetyl-transferase inhibitors and apoptosis inducers.

Compounds containing both thiazole and arylsulfone moieties are recognized for their high biological activity and ability to fight a variety of ailments. Thus, in this context, new derivatives of (thiazol-2-yl)hydrazone with an arylsulfone moiety were synthesized as CPTH2 analogs with potent anti-histone lysine acetyl-transferase activity. Compounds 3, 4, 10b, and 11b showed an excellent inhibitory effect on P300 (E1A-associated protein p300), compared to CPTH2. Among all the tested derivatives, compound 10b revealed the highest activity against both P300 and pCAF. In addition, the new hits were tested for anticancer efficacy against two leukemia cell lines. Most of them showed a moderate to potent antitumor effect on the k562 and CCRF-CEM cell lines. Interestingly, the activity of compound 10b against the k562 cell line was found to be higher than that of CPTH2. Furthermore, it showed a good safety profile, better than CPTH2 on normal cells. Molecular docking analysis was carried out to reveal the crucial binding contacts in the inhibition of the P300 and pCAF enzymes.

Ultrasound-stimulated microbubbles enhances radiosensitivity of ovarian cancer.

BACKGROUND: Radiation therapy is regarded as an effective treatment for early ovarian cancer (OC). However, due to radiation resistance caused by DNA double-strand breaks (DSBs) and angiogenesis, the efficacy of radiotherapy for advanced OC is limited and controversial. PURPOSE: To explore whether ultrasound-stimulated microbubbles (USMBs) can enhance the radiosensitivity of OC. MATERIAL AND METHODS: OC cells (ES-2) were respectively irradiated with 5-Gy and 10-Gy radiation doses with or without exposure to USMB. Methyl thiazolyltetrazolium (MTT) and colony-formation assays were conducted to detect the viability and proliferation of ES-2 cells after USMBs and ionizing radiation (IR) treatment. Immunofluorescence assays were conducted to examine levels of gamma-H2A histone family member X (γ-H2AX), an indicator for DSBs. Flow cytometry analyses were carried out to assess the apoptosis of ES-2 cells. The angiogenic activity of human umbilical vein endothelial cells (HUVECs) was measured by tube formation assays. RESULTS: USMBs enhanced IR-induced suppressive effect on the viability and proliferation of OC cells. The protein levels of phosphorylated γ-H2AX and CHK1 were significantly upregulated after IR treatment and further enhanced by USMBs. In addition, USMBs enhanced the promotion of IR-mediated OC cell apoptosis. The inhibitory effect of IR on angiogenesis was further enhanced by USMBs, and protein levels of AT1R, VEGFA, and EGFR were downregulated by IR in a dose-dependent way and then enhanced by USMB treatment in HUVECs. CONCLUSIONS: USMB exposure significantly enhances the radiosensitivity of OC by suppressing cell proliferation, promoting OC cell apoptosis, and inhibiting angiogenesis.

[In vitro pharmacodynamic studies of novel class â and â ¡b selective histone deacetylase inhibitor purinostat mesylate in the treatment of diffuse large B-cell lymphoma and its mechanism].

Objective: To investigate the in vitro inhibitory activity of a novel class Ⅰ and Ⅱb selective histone deacetylase (HDAC) inhibitor, purinostat mesylate (PM) , in diffuse large B-cell lymphoma and its mechanism. Methods: The 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide method was used to detect the effect of PM on cell proliferation. The effects of PM on cell cycle and apoptosis were detected by flow cytometry. The acetylation levels of HDAC substrate, cell cycle protein, apoptosis-related protein, and oncogene protein expression were detected by Western blot. Results: PM significantly inhibited the proliferation of lymphoma SUDHL-4 and SUDHL-6 cells and increased the acetylation levels of HDAC substrates H3, H4, and α-tubulin. In cell cycle experiments, PM induced G(0)/G(1) phase arrest in SUDHL-4 and SUDHL-6 cells. Western blot experiment showed that PM could significantly downregulate the expression of cyclin-dependent kinases Cdk2, Cdk4, Cdk6, cyclin D1, and cyclin E and upregulate the expression of CDK inhibitor protein p21. In the apoptosis experiment, PM could induce the apoptosis of SUDHL-4 and SUDHL-6 cells. Western blot experiment demonstrated that PM promoted endogenous apoptosis by activating caspase-3 kinase and affecting antiapoptotic protein Bcl-2. In addition, PM could downregulate the expression of oncogene marker proteins MYC, IKZF1, and IKZF3. Conclusion: PM has an efficient biological activity in vitro for diffuse large B-cell lymphoma, including double-hit lymphoma, and provides valuable experimental evidence for PM in clinical treatment.

H3K36 trimethylation-mediated biological functions in cancer.

Histone modification is an important form of epigenetic regulation. Thereinto, histone methylation is a critical determination of chromatin states, participating in multiple cellular processes. As a conserved histone methylation mark, histone 3 lysine 36 trimethylation (H3K36me3) can mediate multiple transcriptional-related events, such as the regulation of transcriptional activity, transcription elongation, pre-mRNA alternative splicing, and RNA m6A methylation. Additionally, H3K36me3 also contributes to DNA damage repair. Given the crucial function of H3K36me3 in genome regulation, the roles of H3K36me3 and its sole methyltransferase SETD2 in pathogenesis, especially malignancies, have been emphasized in many studies, and it is conceivable that disruption of histone methylation regulatory network composed of "writer", "eraser", "reader", and the mutation of H3K36me3 codes have the capacity of powerfully modulating cancer initiation and development. Here we review H3K36me3-mediated biological processes and summarize the latest findings regarding its role in cancers. We highlight the significance of epigenetic combination therapies in cancers.

Harnessing Tolerogenic Histone Peptide Epitopes From Nucleosomes for Selective Down-Regulation of Pathogenic Autoimmune Response in Lupus (Past, Present, and Future).

Autoantigen-directed tolerance can be induced by certain nucleosomal histone peptide epitope/s in nanomolar dosage leading to sustained remission of disease in mice with spontaneous SLE. By contrast, lupus is accelerated by administration of intact (whole) histones, or whole nucleosomes in microparticles from apoptotic cells, or by post-translationally acetylated histone-peptides. Low-dose therapy with the histone-peptide epitopes simultaneously induces TGFß and inhibits IL-6 production by DC in vivo, especially pDC, which then induce CD4+CD25+ Treg and CD8+ Treg cells that suppress pathogenic autoimmune response. Both types of induced Treg cells are FoxP3+ and act by producing TGFß at close cell-to-cell range. No anaphylactic adverse reactions, or generalized immunosuppression have been detected in mice injected with the peptides, because the epitopes are derived from evolutionarily conserved histones in the chromatin; and the peptides are expressed in the thymus during ontogeny, and their native sequences have not been altered. The peptide-induced Treg cells can block severe lupus on adoptive transfer reducing inflammatory cell reaction and infiltration in the kidney. In Humans, similar potent Treg cells are generated by the histone peptide epitopes in vitro in lupus patients' PBMC, inhibiting anti-dsDNA autoantibody and interferon production. Furthermore, the same types of Treg cells are generated in lupus patients who are in very long-term remission (2-8 years) after undergoing autologous hematopoietic stem cell transplantation. These Treg cells are not found in lupus patients treated conventionally into clinical remission (SLEDAI of 0); and consequently they still harbor pathogenic autoimmune cells, causing subclinical damage. Although antigen-specific therapy with pinpoint accuracy is suitable for straight-forward organ-specific autoimmune diseases, Systemic Lupus is much more complex. The histone peptide epitopes have unique tolerogenic properties for inhibiting Innate immune cells (DC), T cells and B cell populations that are both antigen-specifically and cross-reactively involved in the pathogenic autoimmune response in lupus. The histone peptide tolerance is a natural and non-toxic therapy suitable for treating early lupus, and also maintaining lupus patients after toxic drug therapy. The experimental steps, challenges and possible solutions for successful therapy with these peptide epitopes are discussed in this highly focused review on Systemic Lupus.

Potential new targets and drugs related to histone modifications in glioma treatment.

Glioma accounts for 40-50% of craniocerebral tumors, whose outcome rarely improves after standard treatment. The development of new therapeutic targets for glioma treatment has important clinical significance. With the deepening of research on gliomas, recent researchers have found that the occurrence and development of gliomas is closely associated with histone modifications, including methylation, acetylation, phosphorylation, and ubiquitination. Additionally, evidence has confirmed the close relationship between histone modifications and temozolomide (TMZ) resistance. Therefore, histone modification-related proteins have been widely recognized as new therapeutic targets for glioma treatment. In this review, we summarize the potential histone modification-associated targets and related drugs for glioma treatment. We have further clarified how histone modifications regulate the pathogenesis of gliomas and the mechanism of drug action, providing novel insights for the current clinical glioma treatment. Herein, we have also highlighted the limitations of current clinical therapies and have suggested future research directions and expected advances in potential areas of disease prognosis. Due to the complicated glioma pathogenesis, in the present review, we have acknowledged the limitations of histone modification applications in the related clinical treatment.

In vivo effectiveness of hybrid membranes with osteogenic growth peptide for bone regeneration.

Guided bone regeneration (GBR) technique helps to restore bone tissue through cellular selectivity principle. Currently no osteoinductive membrane exists on the market. Osteogenic growth peptide (OGP) acts as a hematopoietic stimulator. This association could improve the quality of bone formation, benefiting more than 2.2 million patients annually. The objective of this work was to develop membranes from ureasil-polyether materials containing OGP. The membranes were characterized by differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS). OGP was synthesized by the solid phase method. Sterilization results using gamma radiation at 24 kGy did not change the structure of the material, as confirmed by DSC. The SAXS technique revealed the structural homogeneity of the matrix. OGP was incorporated in 66.25 × 10-10  mol and release results showed that the ureasil-PPO400/PEO500 and ureasil-PPO400/PEO1900 membranes released 7% and 21%, respectively, after 48 h. In vivo results demonstrated that the amount and quality of bone tissue formed in the bone defects in the presence of ureasil-polyether membranes with OGP were similar to commercial collagen material with BMP. The results allow us to conclude that membranes with OGP have characteristics that make them potential candidates for the GBR.

Citrullination of extracellular histone H3.1 reduces antibacterial activity and exacerbates its proteolytic degradation.

BACKGROUND: Cystic fibrosis (CF), involves excessive airway accumulation of neutrophils, often in parallel with severe infection caused by Pseudomonas aeruginosa. Free histones are known to possess bactericidal properties, but the degree of antibacterial activity exerted on specific lung-based pathogens is largely unknown. Neutrophils have a high content of peptidyl deiminase 4 (PADI4), which citrullinate cationic peptidyl-arginines. In histone H3.1, several positions in the NH2-terminal tail are subject to citrullination. METHODS: Full-length and segmented histone subunit H3.1 was investigated for bactericidal activity towards P. aeruginosa (strain PAO1). PADI4-induced citrullination of histone H3.1 was assessed for antibacterial activity towards P. aeruginosa. Next, the effect of neutrophil elastase (NE)-mediated proteolysis of histone H3.1 was investigated. Finally, PADI4, H3.1, and citrullinated H3.1 were examined in healthy control and CF patient lung tissues. RESULTS: Full-length histone H3.1 and sections of the histone H3.1 tail, displayed bactericidal activity towards P. aeruginosa. These antibacterial effects were reduced following citrullination by PADI4 or proteolysis by NE. Interestingly, citrullination of histone H3.1 exacerbated NE-mediated degradation. In CF lung tissue, citrullinated histone H3.1 and PADI4 immunoreactivity was abundant. Degraded histone H3.1 was detected in the sputum of CF patients but was absent in the sputum of healthy controls. CONCLUSIONS: Citrullination impairs the antibacterial activity of histone H3.1 and exacerbates its proteolytic degradation by NE. Citrullination is likely to play an important role during resolution of acute inflammation. However, in chronic inflammation akin to CF, citrullination may dampen host defense and promote pathogen survival, as exemplified by P. aeruginosa.

Histone H1 improves regeneration after mouse spinal cord injury and changes shape and gene expression of cultured astrocytes.

BACKGROUND: We have shown that histone H1 is a binding partner for polysialic acid (PSA) and that it improves functional recovery, axon regrowth/sprouting, and target reinnervation after mouse femoral nerve injury. OBJECTIVE: Here, we analyzed whether histone H1 affects functional recovery, axon regrowth/sprouting, and target reinnervation after spinal cord injury of adult mice. Furthermore, we tested in vitro histone H1's effect on astrocytic gene expression, cell shape and migration as well as on cell survival of cultured motoneurons. METHODS: We applied histone H1 to compressed spinal cord and determined functional recovery and number of fibrillary acidic protein (GFAP)- and neuron-glial antigen 2 (NG2)- positive glial cells, which contribute to glial scarring. Histone H1's effect on migration of astrocytes, astrocytic gene expression and motoneuronal survival was determined using scratch-wounded astroglial monolayer cultures, astrocyte cultures for microarray analysis, and motoneuron cell culture under oxidative stress conditions, respectively. RESULTS: Histone H1 application improves locomotor functions and enhances monoaminergic and cholinergic reinnervation of the spinal cord. Expression levels of GFAP and NG2 around the lesion site were decreased in histone H1-treated mice relative to vehicle-treated mice six weeks after injury. Histone H1 reduced astrocytic migration, changed the shape of GFAP- and NG2-positive glial cells and altered gene expression. Gene ontology enrichment analysis indicated that in particular genes coding for proteins involved in proliferation, differentiation, migration and apoptosis are dysregulated. The up- and down-regulation of distinct genes was confirmed by qPCR and Western blot analysis. Moreover, histone H1 reduced hydrogen peroxide-induced cell death of cultured motoneurons. CONCLUSIONS: The combined observations indicate that histone H1 locally applied to the lesion site, improves regeneration after spinal cord injury. Some of these beneficial functions of histone H1 in vivo and in vitro can be attributed to its interaction with PSA-carrying neural cell adhesion molecule.