Nuclear Activity Research Articles

SMYD2-Methylated PPARγ Facilitates Hypoxia-Induced Pulmonary Hypertension by Activating Mitophagy.

Hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs) and consequent pulmonary vascular remodeling are the crucial pathological features of pulmonary hypertension (PH). Protein methylation has been shown to be critically involved in PASMC proliferation and PH, but the underlying mechanism remains largely unknown. PH animal models were generated by treating mice/rats with chronic hypoxia for 4 weeks. SMYD2-vTg mice (vascular smooth muscle cell-specific suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 (deformed epidural auto-regulatory factor-1) domain-containing protein 2 transgenic) or wild-type rats and mice treated with LLY-507 (3-cyano-5-{2-[4-[2-(3-methylindol-1-yl)ethyl]piperazin-1-yl]-phenyl}-N-[(3-pyrrolidin-1-yl)propyl]benzamide) were used to investigate the function of SMYD2 (suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 domain-containing protein 2) on PH development in vivo. Primary cultured rat PASMCs with SMYD2 knockdown or overexpression were used to explore the effects of SMYD2 on proliferation and to decipher the underlying mechanism. We demonstrated that the expression of the lysine methyltransferase SMYD2 was upregulated in the smooth muscle cells of pulmonary arteries from patients with PH and hypoxia-exposed rats/mice and in the cytoplasm of hypoxia-induced rat PASMCs. More importantly, targeted inhibition of SMYD2 by LLY-507 significantly attenuated hypoxia-induced pulmonary vascular remodeling and PH development in both male and female rats in vivo and reduced rat PASMC hyperproliferation in vitro. In contrast, SMYD2-vTg mice exhibited more severe PH phenotypes and related pathological changes than nontransgenic mice after 4 weeks of chronic hypoxia treatment. Furthermore, SMYD2 overexpression promoted, while SMYD2 knockdown suppressed, the proliferation of rat PASMCs by affecting the cell cycle checkpoint between S and G2 phases. Mechanistically, we revealed that SMYD2 directly interacted with and monomethylated PPARγ (peroxisome proliferator-activated receptor gamma) to inhibit the nuclear translocation and transcriptional activity of PPARγ, which further promoted mitophagy to facilitate PASMC proliferation and PH development. Furthermore, rosiglitazone, a PPARγ agonist, largely abolished the detrimental effects of SMYD2 overexpression on PASMC proliferation and PH. Our results demonstrated that SMYD2 monomethylates nonhistone PPARγ and inhibits its nuclear translocation and activation to accelerate PASMC proliferation and PH by triggering mitophagy, indicating that targeting SMYD2 or activating PPARγ are potential strategies for the prevention of PH.

The EBI2 receptor is coexpressed with CCR5 in CD4 + T cells and boosts HIV-1 R5 replication.

CCR5, a G protein-coupled receptor (GPCR), is used by most HIV strains as a coreceptor. In this study, we looked for other GPCR able to modify HIV-1 infection. We analyzed the effects of one GPCR coexpressed with CCR5, EBI2, on HIV-1 replicative cycle. We identified GPCR expressed in primary CD4 + CCR5 + T cells by multi-RT-qPCR. We studied GPCR dimerization by FRET technology. Cell lines expressing EBI2 were established by transduction with HIV vectors. HIV-1 entry was quantified with virions harboring β-lactamase fused to the viral protein vpr, early and late HIV-1 transcriptions by qPCR, NFkB nuclear activation by immunofluorescence and transfection, and viral production by measuring p24 concentration in culture supernatant by ELISA. We showed that EBI2 is naturally expressed in primary CD4 + CCR5 + T cells, and that CCR5 and EBI2 heterodimerize. We observed that this coexpression reduced viral entry by 50%. The amount of HIV reverse transcripts was similar in cells expressing or not EBI2. Finally, the presence of EBI2 induced the translocation of NFkB and activated HIV-1 genome expression. Globally, the result was a drastic HIV-1 R5, but not X4, overproduction in EBI2 -transduced cells. EBI2 expression in CD4 + CCR5 + cells boosts HIV-1 R5 productive infection. As the natural ligand for EBI2 is present in blood and lymphoid tissues, the constant EBI2 activation might increase HIV replication in CD4 + T cells. It might be of interest to test the effect of EBI2 antagonists on the residual viral production persisting in patients aviremic under treatment.

Type 17 immune response promotes oral epithelial cell proliferation in periodontitis

ObjectivesThis study aims to investigate the effects of type 17 immune response on the proliferation of oral epithelial cells in periodontitis. DesignA time-dependent ligature induced periodontitis mouse model was utilized to explore gingival hyperplasia and the infiltration of interleukin 17A (IL-17A) positive cells. Immunohistochemistry and flow cytometry were employed to determine the localization and expression of IL-17A in the ligature induced periodontitis model. A pre-existing single-cell RNA sequencing dataset, comparing individuals affected by periodontitis with healthy counterparts, was reanalyzed to evaluate IL-17A expression levels. We examined proliferation markers, including proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription (STAT3), Yes-associated protein (YAP), and c-JUN, in the gingival and tongue epithelium of the periodontitis model. An anti-IL-17A agent was administered daily to observe proliferative changes in the oral mucosa within the periodontitis model. Cell number quantification, immunofluorescence, and western blot analyses were performed to assess the proliferative responses of human normal oral keratinocytes to IL-17A treatment in vitro. ResultsThe ligature induced periodontitis model exhibited a marked infiltration of IL-17A-positive cells, alongside significant increase in thickness of the gingival and tongue epithelium. IL-17A triggers the proliferation of human normal oral keratinocytes, accompanied by upregulation of PCNA, STAT3, YAP, and c-JUN. The administration of an anti-IL-17A agent attenuated the proliferation in oral mucosa. ConclusionsThese findings indicate that type 17 immune response, in response to periodontitis, facilitates the proliferation of oral epithelial cells, thus highlighting its crucial role in maintaining the oral epithelial barrier.

Function of NEK2 in clear cell renal cell carcinoma and its effect on the tumor microenvironment.

Previous studies have revealed the critical functions of NEK2 in controlling the cell cycle which is linked to poor prognosis in multiple tumor types, but less research has been devoted to clear cell renal cell carcinoma (ccRCC). We downloaded clinical data from the gene expression omnibus (GEO) and TCGA databases together with transcriptional and mutational datasets. Strongly coexpressed genes with NEK2 were extracted from TCGA-KIRC cohort, and were submitted to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional analyses. According to NEK2 levels, the survival status, mutational characteristics, response to immunotherapy and sensitivity to drugs of the patients were studied. The potential correlations between NEK2 levels and immune cell state as well as immune cell infiltration were examined using the GEPIA, TIMER and TISIDB databases. Double immunofluorescence (IF) was performed to identify the NEK2 overexpression and relationship with CD8 in ccRCC. The NEK2 gene was overexpressed and would enhance the nuclear division and cell cycle activities in ccRCC. ccRCC patients with high NEK2 expression had worse clinical outcomes, higher mutation burden and better therapeutic response. Moreover, NEK2 gene overexpression was positively related to various immune cell marker sets, which was also proved by validation cohort, and more infiltration of various immune cells. ccRCC patients with NEK2 high expression have a poorer prognosis than those with NEK2 low expression, resulting from its function of promoting proliferation, accompanied by increased infiltration of CD8 + T cells and Tregs and T-cell exhaustion and will respond better to proper treatments.

Two peptides LLRLTDL and GYALPCDCL inhibit foam cell formation through activating PPAR-γ/LXR-α signaling pathway in oxLDL-treated RAW264.7 macrophages.

Foam cell formation plays a pivotal role in atherosclerosis-associated cardiovascular diseases. Bioactive peptides generated from marine sources have been found to provide multifunctional health advantages. In the present study, we investigated the anti-atherosclerotic effects of LLRLTDL (Bu1) and GYALPCDCL (Bu2) peptides, isolated from ark shell protein hydrolysates by assessing their inhibitory effect on oxidized LDL (oxLDL)-induced foam cell formation. The two peptides showed a promising anti-atherosclerotic effect by inhibiting foam cell formation, which was evidenced by inhibiting lipid accumulation in oxLDL-treated RAW264.7 macrophages and oxLDL-treated primary human aortic smooth muscle cells (HASMC). Two peptides effectively reduced total cholesterol, free cholesterol, cholesterol ester, and triglyceride levels by upregulating cholesterol efflux and downregulating cholesterol influx. Expression of cholesterol influx-related proteins such as SR-A1 and CD36 were reduced, whereas cholesterol efflux-related proteins such as ATP-binding cassette transporter ABCA-1 and ABCG-1 were highly expressed. In addition, Bu1 and Bu2 peptides increased PPAR-γ and LXR-α expression. However, PPAR-γ siRNA transfection reversed the foam cell formation inhibitory activity of Bu1 and Bu2 peptides. Furthermore, the synergistic effect of Bu1 and Bu2 peptides on foam cell formation inhibition was observed with PPAR-γ agonist thiazolidinediones, indicating that PPAR-γ signaling pathway plays a key role in foam cell formation of macrophages. Beyond their impact on foam cell formation, Bu1 and Bu2 peptides demonstrated anti-inflammatory potential by inhibiting the generation of pro-inflammatory cytokines and nitric oxide and NF-κB nuclear activation. Taken together, these results suggest that Bu1 and Bu2 peptides may be useful for atherosclerosis and associated anti-inflammatory therapies.

Perinuclear damage from nuclear envelope deterioration elicits stress responses that contribute to LMNA cardiomyopathy.

Mutations in the LMNA gene encoding lamins A/C cause an array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the perturbations emanating from LMNA mutations, an integrative understanding of the pathogenesis underlying cardiac dysfunction remains elusive. Using a novel conditional deletion model capable of translatome profiling, we observed that cardiomyocyte-specific Lmna deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Before cardiac dysfunction, Lmna-deleted cardiomyocytes displayed nuclear abnormalities, Golgi dilation/fragmentation, and CREB3-mediated stress activation. Translatome profiling identified MED25 activation, a transcriptional cofactor that regulates Golgi stress. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the Golgi. Systemic administration of modulators of autophagy or ER stress significantly delayed cardiac dysfunction and prolonged survival. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the LMNA cardiomyopathy development.

MpNAC1, a transcription factor from the mangrove associate Millettia pinnata, confers salt and drought stress tolerance in transgenic Arabidopsis and rice

Pongamia (Millettia pinnata Syn. Pongamia pinnata), a mangrove associate plant, exhibits good stress tolerance, making it a treasure of genetic resources for crop improvement. NAC proteins are plant-specific transcription factors, which have been elucidated to participate in the regulation and tolerance of abiotic stresses (such as salt and drought). Here, we identified a salt-induced gene from Pongamia, MpNAC1, which encodes an NAC factor sharing five highly conserved domains with other NACs and exhibits close homology to AtNAC19/AtNAC55/AtNAC72 in Arabidopsis. MpNAC1 showed nuclear localization and transcriptional activator activity. MpNAC1-overexpressing Arabidopsis exhibited significantly stronger salt and drought tolerance compared with wild-type plants. The expression levels of stress-responsive genes were activated in transgenic Arabidopsis. Furthermore, the heterologous expression of MpNAC1 also enhanced the salt and drought tolerance of transgenic rice. The major agronomic traits, such as plant height and tiller number, panicle length, grain size, and yield, were similar between the transgenic lines and wild type under normal field growth conditions. RNA-Seq analysis revealed that MpNAC1 significantly up-regulated stress-responsive genes and activated the biosynthesis of secondary metabolites such as flavonoids, resulting in increased stress tolerance. Taken together, the MpNAC1 increased salt and drought stress tolerance in transgenic plants and did not retard the plant growth and development under normal growth conditions, suggesting the potential of MpNAC1 in breeding stress-resilient crops.

The actin-binding protein CAP1 represses MRTF-SRF-dependent gene expression in mouse cerebral cortex.

Serum response factor (SRF) is an essential transcription factor for brain development and function. Here, we explored how an SRF cofactor, the actin monomer-sensing myocardin-related transcription factor MRTF, is regulated in mouse cortical neurons. We found that MRTF-dependent SRF activity in vitro and in vivo was repressed by cyclase-associated protein CAP1. Inactivation of the actin-binding protein CAP1 reduced the amount of actin monomers in the cytoplasm, which promoted nuclear MRTF translocation and MRTF-SRF activation. This function was independent of cofilin1 and actin-depolymerizing factor, and CAP1 loss of function in cortical neurons was not compensated by endogenous CAP2. Transcriptomic and proteomic analyses of cerebral cortex lysates from wild-type and Cap1 knockout mice supported the role of CAP1 in repressing MRTF-SRF-dependent signaling in vivo. Bioinformatic analysis identified likely MRTF-SRF target genes, which aligned with the transcriptomic and proteomic results. Together with our previous studies that implicated CAP1 in axonal growth cone function as well as the morphology and plasticity of excitatory synapses, our findings establish CAP1 as a crucial actin regulator in the brain relevant for formation of neuronal networks.

Rcn1, the fission yeast homolog of human DSCR1, regulates arsenite tolerance independently from calcineurin.

Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent phosphoprotein phosphatase that plays a key role in Ca2+ signaling. Regulator of calcineurin 1 (RCAN1), also known as Down syndrome critical region gene 1 (DSCR1), interacts with calcineurin and inhibits calcineurin-dependent signaling in various organisms. Ppb1, the fission yeast calcineurin regulates Cl--homeostasis, and Ppb1 deletion induces MgCl2 hypersensitivity. Here, we characterize the conserved and novel roles of the fission yeast RCAN1 homolog rcn1+. Consistent with its role as an endogenous calcineurin inhibitor, Rcn1 overproduction reproduced the calcineurin-null phenotypes, including MgCl2 hypersensitivity and inhibition of calcineurin signaling upon extracellular Ca2+ stimuli as evaluated by the nuclear translocation and transcriptional activation of the calcineurin substrate Prz1. Notably, overexpression of rcn1+ causes hypersensitivity to arsenite, whereas calcineurin deletion induces arsenite tolerance, showing a phenotypic discrepancy between Rcn1 overexpression and calcineurin deletion. Importantly, although Rcn1 deletion induces modest sensitivities to arsenite and MgCl2 in wild-type cells, the arsenite tolerance, but not MgCl2 sensitivity, associated with Ppb1 deletion was markedly suppressed by Rcn1 deletion. Collectively, our findings reveal a previously unrecognized functional collaboration between Rcn1 and calcineurin, wherein Rcn1 not only negatively regulates calcineurin in the Cl- homeostasis, but also Rcn1 mediates calcineurin signaling to modulate arsenite cytotoxicity.

Abstract PS17-09: An FGFR1/2 degrader overcomes antiestrogen resistance in ER+/FGFR1-amplified breast cancer

Abstract We previously reported that nuclear FGFR1 promotes antiestrogen resistance in ER+/FGFR1-amplified breast cancer. Nuclear FGFR1 activity was not affected by FGFR tyrosine kinase inhibitors (TKIs). Furthermore, pan-FGFR TKIs are not well tolerated. Therefore, new therapeutic strategies that inhibit nuclear FGFR1 while sparing FGFR3 and FGFR4 would be required to overcome antiestrogen resistance and limit toxicity. For this study, we used the proteolysis-targeting chimera (PROTAC) DGY-09-192, consisting of the FGFR TKI BCJ398 linked with a VHL recruiting ligand, to degrade FGFR1 and FGFR2. We aimed to determine whether selective degradation of FGFR1/2 overcomes endocrine resistance. Treatment with 50-100 nM DGY-09-192 strongly induced FGFR1 degradation and suppressed phosphorylation of FGFR1 and its downstream targets FRS2, AKT, and ERK1/2 in ER+/FGFR1-amplified CAMA1 and MDA-MB-134 breast cancer cells. FGFR1 degradation was evident within 4 h and lasted 48-96 h after drug washout. The proteasome inhibitor MG132 (10 µM) rescued both FGFR degradation and phosphorylation of downstream signaling molecules induced by DGY-09-192, confirming that FGFR1 degradation is dependent on ubiquitination and proteasomal degradation. At equivalent concentrations, DGY-09-192 suppressed phosphorylation of FGFR1 and its downstream targets more potently than BGJ398. Subcellular fractionation showed treatment with DGY-09-192 (100 nM) degraded FGFR1 in membrane, nuclear, and chromatin-bound fractions in both CAMA1 and MDA-MB-134 cells. Treatment of >CAMA1 cells with DGY-09-192 (>100 nM) downregulated nuclear FGFR1 target genes and canonical ERα target genes, such as CCND1, VEGFA, and CDK12 as measured by qRT-PCR whereas treatment with BGJ398 (100 nM) did not. Treatment with DGY-09-192 (500 nM) reversed resistance to the ER degrader fulvestrant in CAMA1 cells as measured by colony formation assays and using the Incucyte Live-Cell Analysis System. Each drug alone showed a modest effect on cell proliferation whereas the combination completed blocked cell growth (p < 0.0001). Finally, treatment of NOD scid gamma (NSG) mice with established ER+/FGFR1-amplified HCI-011 patient-derived xenografts with DGY-09-192 (40 mg/kg daily x2-6) induced FGFR1 degradation and blocked phosphorylation of downstream targets in the tumors. Studies using the combination of DGY-09-192 and fulvestrant in mice bearing established HCI-011 xenografts are in progress. We next investigated the effect of DGY-09-192 on cancer cells harboring FGFR1 and FGFR2 activating mutations. DGY-09-192 treatment induced FGFR2 degradation and blocked downstream signaling in MFE296 endometrial cancer and EFM-19 breast cancer cells that harbor the FGFR2N549K and FGFR2K659E hotspot mutations, respectively. In addition, DGY-09-192 strongly suppressed MFE296 cell proliferation. We are currently investigating whether DGY-09-192 blocks signaling and cell proliferation induced by the N546K and K656E hotspot FGFR1 mutations in ER+ breast cancer cells. Notably, a recent clinical study showed that tumors harboring these FGFR1 activating mutations are refractory to the pan-FGFR TKI pemigatinib. Conclusions: The FGFR1/2 PROTAC DGY-09-192 induced degradation of nuclear and membrane-bound FGFR1 and blocked FGFR1-induced signaling and nuclear activity in ER+/FGFR1-amplified breast cancer cells. The combination of DGY-09-192 and fulvestrant synergistically suppressed proliferation of these cells. Therefore, FGFR1 degradation represents a promising therapeutic strategy to block FGFR1 activity more completely and selectively in ER+ breast cancers harboring activating FGFR1 alterations. Citation Format: Yasuaki Uemoto, Chang-Ching Lin, Alberto Servetto, Bingnan Wang, Dan Ye, Fabiana Napolitano, Hima Milan Patel, Luigi Formisano, M Rosario Chica-Parrado, Emmanuel Bikorimana, Saurabh Mendiratta, Chuo Chen, Ariella Hanker, Carlos Arteaga. An FGFR1/2 degrader overcomes antiestrogen resistance in ER+/FGFR1-amplified breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS17-09.

A Novel Mechanism Underlying Apoptosis Resistance in Pulmonary Arterial Smooth Cuscle Cells (PASMCs)

RATIONALE: Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, with PASMC hyperplasia and formation of vaso-occlusive lesions. New data show PASMCs isolated from robust animal models of PH are resistant to apoptosis, a feature which may propagate disease as abnormal PASMCs evade death. Apoptosis is a highly regulated pathway resulting in caspase-3 (casp3) activation and translocation to the nucleus, where cleavage of proteins results in cell death. In several cancers the membrane protein aquaporin 1 (AQP1) plays a role in apoptosis resistance, although the mechanism remains unknown. We showed AQP1 is expressed in PASMCs and increased in rat models of PH. Therefore, we hypothesized increased AQP1 contributes to apoptosis resistance in PASMCs. METHODS: The Sugen-hypoxia (SuHx) rat model of PH was used. Rats were injected with vascular endothelial growth factor receptor type 2 inhibitor Sugen-5416 and placed in 10% O2 for 3 wk, then returned to 21% O2 for 2 wk. Control animals received vehicle injection and were kept in 21% O2. Distal PASMCs were isolated via enzymatic digestion. AQP1 was depleted in SuHx PASMCs with small interfering RNA (siAQP1), with non-targeting siRNA as control. AQP1 was overexpressed in control PASMCs with adenoviral constructs containing wild type AQP1 (AdAQP1) or green fluorescent protein (control). Cells were treated with PBS or pro-apoptotic hydrogen peroxide (500 μM; 24 h). Apoptosis was measured by Hoechst staining. Caspase activity was measured via luminescent caspase-3/7 activity assay (Caspase-Glo® 3/7) in whole cell lysates and nuclear fractions. RESULTS: Depleting AQP1 via siAQP1 restored apoptosis susceptibility in SuHx PASMCs, and overexpressing AQP1 via AdAQP1 reduced susceptibility to apoptosis in control PASMCs. At baseline, there was no difference in casp3 activity between SuHx and control total cell lysates. However, casp3 activity was lower in SuHx nuclear fractions. Under stimulated conditions, SuHx PASMCs exhibited significantly less total and nuclear casp3 activity, with nuclear activity similar to levels observed in unstimulated controls. To explore an underlying mechanism for these findings, in silico analysis of AQP1 was performed revealing 3 potential casp3 cleavage sites, suggesting the possibility these proteins could interact. To investigate a potential protein-protein interaction, proximity-based biotinylation assays were performed in PASMCs using AdAQP1 (control) or AQP1 fused to biotin ligase. Cells were incubated with cell-permeable biotin (50 μM), lysed, and biotinylated proteins precipitated and immunoblotted for casp3 and other cytosolic proteins not expected to interact with AQP1. To confirm bidirectionality, similar biotinylation assays were performed using adenoviral constructs with wild-type (control) or biotin ligase-fused casp3 and immunoblotting for AQP1. Results showed a specific AQP1/casp3 interaction in PASMCs. Furthermore, the predominant form of casp3 interacting with AQP1 appears to be inactive (37kD). CONCLUSION: We speculate that increased AQP1 in SuHx PASMCs interacts with casp3 preventing its activation and/or nuclear translocation, conferring apoptosis resistance. Future studies further characterizing the interaction between AQP1 and casp3 could provide the basis for novel therapeutic targets focused on restoring PASMC apoptosis susceptibility. F32 HL165766-01, T32 HL007534, Bauernschmidt Fellowship. 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.

Design of a novel ECR ion trap facility for nuclear physics and fundamental plasma processes studies

An innovative ECR ion trap facility, called PANDORA (Plasma for Astrophysics, Nuclear Decay Observation and Radiation for Archaeometry), was designed for fundamental plasma processes and nuclear physics investigations. The overall structure consists of three subsystems: a) a large (70 cm in length, 28 cm in inner diameter) ECR plasma trap with a fully superconducting B-minimum magnetic system (Bmax = 3.0 T) and an innovative design to host detectors and diagnostic tools; b) an advanced non-invasive plasma multidiagnostics system to locally characterize the plasma thermodynamic properties; c) an array of 14 HPGe detectors. The PANDORA facility is conceived to measure, for the first time, in-plasma β-decaying isotope rates under stellar-like conditions. The experimental approach consists in a direct correlation of plasma parameters and nuclear activity by disentangling - by means of the multidiagnostic system that will work in synergy with the γ-ray array - the photons emitted by the plasma (from microwave to hard X-ray) and γ-rays emitted after the isotope β-decay. In addition to nuclear physics research, fundamental plasma physics studies can be conducted in this unconventional ion source equipped with tens of detection and diagnostic devices (RF polarimeter, optical emission spectroscopy (OES), X-ray imaging, space and time-resolved spectroscopy, RF probes, scope), with relevant implications for R&D of ion sources for accelerator physics and technology. Several studies have already been performed in downsized nowadays operating ECRIS. Stable and turbulent plasma regimes have been described quantitatively, studying the change of plasma morphology, confinement, and dynamics of losses using space resolved X-ray spectroscopy.

ANGPTL3 accelerates atherosclerotic progression via direct regulation of M1 macrophage activation in plaque

IntroductionThe N-terminal domain of angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity. Its C-terminal fibrinogen-like (FBN) domain is a ligand of macrophage integrin αvβ3. ObjectivesANGPTL3 might home to plaque where it directly regulates macrophage function via integrin αvβ3 for atherosclerosis progression. MethodsLdlr-/- mice on a high-fat diet and ApoE-/- mice on a chow diet were received adeno-associated virus (AAV)-mediated Angptl3 gene transfer and followed up for 12 weeks. ApoE-/- mice were injected AAV containing FLAG-tagged Angptl3 cDNA for tracing. Atherosclerotic features were compared between Angptl3-/-ApoE-/- mice and ApoE-/- littermates. THP-1 cells were exposed to 0 or 50 μg/ml ANGPTL3 FBN domain for 24 h to evaluate Toll-like receptor (TLR)4 expression using western blot analysis and circulating cytokine and chemokine profiles by the MILLIPLEX MAP assay. Phospho-proteomic profile was established in ANGPTL3-treated macrophages. Integrin β3 deficient THP-1 cells were obtained by sgRNAs targeting RGD sequence using Lentivirus-Cas9 system. ResultsAngptl3 overexpression increased atherosclerotic progression and CD68+ macrophages in plaque (p < 0.05 for all). By immunostaining, FLAG+ cells were identified in plaque of gene transferred ApoE-/- mice. Fluorescent immunostaining detected co-localisation of Angptl3 and CD68 in plaque macrophages. Phospho-proteomic analysis revealed that Angptl3 induced phosphorylation of proteins that were involved in the IL-17 signalling pathway in THP-1 cells. In vitro, ANGPTL3 treatment increased the production of interleukin (IL)-1β and tumour necrosis factor-α in THP-1 cells (p < 0.05 for both). Exposure of ANGPTL3 to THP-1 cells induced Akt phosphorylation which was weakened in integrin β3 deficient ones. ANGPTL3 elevated TLR4 expression via Akt phosphorylation. In response to lipopolysaccharide, nuclear factor-κB activity was 2.2-fold higher in THP-1 cells pre-treated with ANGPTL3 than in untreated cells (p < 0.05). ConclusionsTargeting ANGPTL3 could yield a dual benefit of lowering lipid levels in the blood and suppressing macrophage activation in plaque.

Formyl-Peptide Receptor 2 Signaling Modulates SLC7A11/xCT Expression and Activity in Tumor Cells.

Cancer cells exhibit high levels of oxidative stress and consequently require a high amount of cysteine for glutathione synthesis. Solute Carrier Family 7 Member 11 (SLC7A11), or xCT, mediates the cellular uptake of cystine in exchange for intracellular glutamate; imported extracellular cystine is reduced to cysteine in the cytosol through a NADPH-consuming reduction reaction. SLC7A11/xCT expression is under the control of stress-inducing conditions and of several transcription factors, such as NRF2 and ATF4. Formyl-peptide receptor 2 (FPR2) belongs to the FPR family, which transduces chemotactic signals mediating either inflammatory or anti-inflammatory responses according to the nature of its ligands and/or FPR2 binding with other FPR isoforms. The repertoire of FPR2 agonists with anti-inflammatory activities comprises WKYMVm peptide and Annexin A1 (ANXA1), and the downstream effects of the intracellular signaling cascades triggered by FPR2 include NADPH oxidase (NOX)-dependent generation of reactive oxygen species. Herein, we demonstrate that stimulation of CaLu-6 cells with either WKYMVm or ANXA1: (i) induces the redox-regulated activation of SLC7A11/xCT; (ii) promotes the synthesis of glutathione; (iii) prevents lipid peroxidation; and (iv) favors NRF2 nuclear translocation and activation. In conclusion, our overall results demonstrate that FPR2 agonists and NOX modulate SLC7A11/xCT expression and activity, thereby identifying a novel regulative pathway of the cystine/glutamate antiport that represents a new potential therapeutical target for the treatment of human cancers.

The IL-17 pathway intertwines with neurotrophin and TLR/IL-1R pathways since its domain shuffling origin

The IL-17 pathway displays remarkably diverse functional modes between different subphyla, classes, and even orders, yet its driving factors remains elusive. Here, we demonstrate that the IL-17 pathway originated through domain shuffling between a Toll-like receptor (TLR)/IL-1R pathway and a neurotrophin-RTK (receptor-tyrosine-kinase) pathway (a Trunk-Torso pathway). Unlike other new pathways that evolve independently, the IL-17 pathway remains intertwined with its donor pathways throughout later evolution. This intertwining not only influenced the gains and losses of domains and components in the pathway but also drove the diversification of the pathway's functional modes among animal lineages. For instance, we reveal that the crustacean female sex hormone, a neurotrophin inducing sex differentiation, could interact with IL-17Rs and thus be classified as true IL-17s. Additionally, the insect prothoracicotropic hormone, a neurotrophin initiating ecdysis in Drosophila by binding to Torso, could bind to IL-17Rs in other insects. Furthermore, IL-17R and TLR/IL-1R pathways maintain crosstalk in amphioxus and zebrafish. Moreover, the loss of the Death domain in the pathway adaptor connection to IκB kinase and stress-activated protein kinase (CIKSs) dramatically reduced their abilities to activate nuclear factor-kappaB (NF-κB) and activator protein 1 (AP-1) in amphioxus and zebrafish. Reinstating this Death domain not only enhanced NF-κB/AP-1 activation but also strengthened anti-bacterial immunity in zebrafish larvae. This could explain why the mammalian IL-17 pathway, whose CIKS also lacks Death, is considered a weak signaling activator, relying on synergies with other pathways. Our findings provide insights into the functional diversity of the IL-17 pathway and unveil evolutionary principles that could govern the pathway and be used to redesign and manipulate it.

Nuclear Rac1 controls nuclear architecture and cell migration of glioma cells

Rac1 (Ras-related C3 botulinum toxin substrate 1) protein has been found in the cell nucleus many years ago, however, its nuclear functions are still poorly characterized but some data suggest its nuclear accumulation in cancers. We investigated nuclear Rac1 in glioma cancer cells nuclei and compared its levels and activity to normal astrocytes, and also characterized the studied cells on various nuclear properties and cell migration patterns.Nuclear Rac1 indeed was found accumulated in glioma cells, but only a small percentage of the protein was in active, GTP-bound state in comparison to healthy control. Altering the nuclear activity of Rac1 influenced chromatin architecture and cell motility in GTP-dependent and independent manner. This suggests that the landscape of Rac1 nuclear interactions might be as complicated and wide as its well-known, non-nuclear signaling.

A 20 kiloparsec bipolar Lyman alpha outflow from a radio galaxy at z=2.95.

The study of ionized gas kinematics in high-z active galaxies plays a key part in our understanding of galactic evolution, in an age where nuclear activity was widespread and star formation close to its peak. We present a study of a radio galaxy at z=2.95, using VLT/MUSE integral field optical spectroscopy as part of a project aimed studying of the properties of ionized gas in high redshift radio galaxies (HzRGs). The line profile of this object presents various emission and absorption components. By utilizing Voronoi binning, we obtained a comprehensive map of the kinematic properties of these components. These observations revealed the presence of a redshifted, high velocity (v $ bipolar structure of emission, most likely corresponding to an outflow of ionized gas. The outflow extends beyond the compact radio source on both sides, with a total size of sim 21 kpc. Its kinetic power $ erg s$^ $) is about five orders of magnitude smaller than its radio power. Additional ionized lines, including HeIIlambda 1640, CIVlambda 1550 and CIII

Screening and mechanistic study of antioxidant peptides from Bangia fusco-purpurea targeting the Keap1–Nrf2 pathway

Using 2236 peptides derived from Bangia fusco-purpurea as ligands and the Kelch domain of Kelch-like ECH-associated protein 1 as the receptor, molecular docking was employed to screen peptides with strong inhibitory effects on Kelch-like ECH-associated protein 1–nuclear erythroid 2-related factor interactions. Subsequently, an H2O2-induced oxidative damage model was established using human umbilical vein endothelial cells to validate the results. The most active antioxidant peptides, IAY and TIL, induced nuclear erythroid 2-related factor translocation from the cytoplasm to the nuclei of human umbilical vein endothelial cells when subjected to oxidative damage. Nuclear erythroid 2-related factor activation resulted in the upregulation of the downstream target proteins, including nicotinamide adenine dinucleotide phosphate, quinone oxidoreductase 1, heme oxygenase-1, glutathione, and superoxide dismutase. This cascade reduced the malondialdehyde and reactive oxygen species levels, thereby alleviating oxidative stress, and inhibited H2O2-induced cell apoptosis by regulating apoptosis-related proteins. This study successfully identified IAY and TIL as antioxidant peptides that capable of interfering with Kelch-like ECH-associated protein 1–nuclear erythroid 2-related factor interactions, and elucidated the underlying cellular and molecular mechanisms, laying the groundwork for further research and application of antioxidant peptides.

Long-timescale atomistic simulations uncover loss-of-function mechanisms of uncharacterized Angiogenin mutants associated with ALS

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive degeneration of motor neurons, resulting in respiratory failure and mortality within 3–5 years. Mutations in the Angiogenin (ANG) cause loss of ribonucleolytic and nuclear translocation activities, contributing to ALS pathogenesis. This study focused on investigating two uncharacterized ANG mutations, T11S and R122H, newly identified in the Project Mine consortium. Using extensive computational analysis, including structural modeling and microsecond-timescale molecular dynamics (MD) simulations, we observed conformational changes in the catalytic residue His114 of ANG induced by T11S and R122H mutations. These alterations impaired ribonucleolytic activity, as inferred through molecular docking and binding free energy calculations. Gibbs free energy landscape and residue-residue interaction network analysis further supported our findings, revealing the energetic states and allosteric pathway from the mutated site to His114. Additionally, we assessed the binding of NCI-65828, an inhibitor of ribonucleolytic activity of ANG, and found reduced effectiveness in binding to T11S and R122H mutants when His114 assumed a non-native conformation. This highlights the crucial role of His114 and its association with ALS. Elucidating the relationship between physical structure and functional dynamics of frequently mutated ANG mutants is essential for understanding ALS pathogenesis and developing more effective therapeutic interventions.

The MCM2-7 Complex: Roles beyond DNA Unwinding.

The MCM2-7 complex is a hexameric protein complex that serves as a DNA helicase. It unwinds the DNA double helix during DNA replication, thereby providing the single-stranded replication template. In recent years, it has become clear that the MCM2-7 complex has additional functions that extend well beyond its role in DNA replication. Through physical and functional interactions with different pathways, it impacts other nuclear events and activities, including folding of the genome, histone inheritance, chromosome segregation, DNA damage sensing and repair, and gene transcription. Collectively, the diverse roles of the MCM2-7 complex suggest it plays a critical role in maintaining genome integrity by integrating the regulation of DNA replication with other pathways in the nucleus.