Real-time PCR Research Articles

Anti-fibrotic effects of the sirt6-activator mdl-800 in cardiac stromal cells

Abstract Background Epigenetic changes are among the molecular substrates of cellular stress responses and tissue remodeling in heart failure progression. The sirtuin family of NAD+-dependent histone and non-histone protein deacetylases exert a protective anti-fibrotic role by negatively modulating the inflammatory response and by positive regulation of autophagy-related genes. Sirtuin6 (SIRT6) stands out as a key cardiac protector that can mitigate aging-induced cardiomyocyte senescence and cardiac hypertrophy. Cardiac stromal cells (CSCs) play a pivotal role in fibrosis and remodeling, regulating the composition and stiffness of the extracellular matrix (ECM). Autophagy enhancement has anti-fibrotic effects in CSCs, but the specific role of SIRT6 modulation in CSCs remains unexplored. Purpose To investigate the biological effects of MDL-800, a synthetic allosteric SIRT6 activator, on the stress response and fibrotic activation of CSCs. Methods CSCs were isolated from 4-week-old C57Bl6J mice by explant culture and stimulated with 10ng/ml of TGF-beta1 (TGFb1) up to 72h to induce fibrotic activation. CSCs were treated with MDL-800 up to 10µM and up to 72h, analyzed for cell viability, gene and protein expression levels. Results TGFb1 treatment led to a 0.64-fold reduction in SIRT6 gene expression after 24h. The MTS assay was used to assess a viability dose-response to MDL-800 up to 72h of treatment. The non-toxic concentrations of 2.5µM and 5µM were selected for further experiments (2.5µM vs CTR 0.97-fold and 5µM vs CTR 0.92-fold, normalized OD vs t0). Co-treatment of 5µM MDL-800 with TGFb1 for 72h resulted in a dose-dependent decrease in the expression of TGFb1-induced fibroblast activation markers, as confirmed by immunofluorescence staining for aSMA (TGFb1 vs CTR 2.21-fold, p<0.05; 5µM vs TGFb1 0.46-fold, p<0.05, N=4; mean fluorescence intensity). The anti-fibrotic effect mediated by MDL-800 was further validated by real-time PCR indicating a reduction in the mRNA expression of multiple fibrotic markers, including ACTA2 (TGFb1 vs CTR 1.67-fold, 5µM vs TGFB 0.29-fold; N=2), COL1a1 (TGFb1 vs CTR 1.32-fold, 5µM vs TGFb1 0.87-fold; N=2) COL3a1 (TGFb1 vs CTR 1.06-fold, 5µM vs TGFb1 0.43-fold; N=2) and POSTN (TGFb1 vs CTR 6.37-fold, 5µM vs TGFb1 0.23-fold; N=2). Treatment with 5µM MDL-800 demonstrated an increase in SIRT6 gene expression, suggesting a transcriptional modulation (TGFb1 vs CTR 0.95-fold, 5µM vs TGFb1 1.68-fold; N=2). Western Blot analyses showed an increase in protein levels of the autophagosome marker LC3-II following treatment with 5µM MDL-800 (TGFb1 vs CTR 0.95-fold; 5µM vs TGFb1 3.01-fold), suggesting that autophagy enhancement may be a potential mechanism for the observed anti-fibrotic effects. Conclusion MDL-800 treatment in CSCs is associated with a dose-dependent reduction of TGFb1-induced fibrotic markers and to increased levels of the autophagy marker, suggesting its potential use as an anti-fibrotic molecule for the heart.

Diesel exhaust particles activate macrophages and IRF5 expression in atherosclerosis

Abstract Introduction Over 20% of global deaths related to cardiovascular disease have been attributed in part to air pollution, particularly fine particulate matter (PM2.5) (1). In highly trafficked urban areas, smaller ultrafine diesel exhaust particles (DEP) represent a significant proportion of PM2.5. DEP is associated with the progression of atherosclerosis and increased plaque susceptibility to rupture (2). We have previously shown that interferon regulatory factor 5 (IRF5) has a key role in necrotic core formation, a distinguishing feature of vulnerable atherosclerotic plaques, by rendering macrophages defective at efferocytosis (3). Pronounced elevation of IRF5 is also seen in symptomatic carotid plaques, particularly in the rupture-prone shoulder regions (4). Purpose We examined the effect of DEP on macrophage phenotype and IRF5 expression in atherosclerosis. Methods Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice, differentiated with GM-CSF (to mimic inflammatory macrophages) or M-CSF (mimicking homeostatic resident macrophages) were incubated with PBS or DEP (10 µg/mL) in vitro for 18 hours. Expression of inflammatory cytokines (IL-6, IL-12α), macrophage polarisation markers CD11c (itgax, inflammatory) and CD206 (mrc1, resident anti-inflammatory) and IRF5 was quantified by real-time PCR. In addition, high fat-fed ApoE-/- mice were exposed to saline or DEP (35 µL at 1 mg/mL) twice weekly for 5 weeks by pulmonary administration. The pan-macrophage marker CD64, CD206 and IRF5 were quantified in paraffin-embedded sections of brachiocephalic artery lesions by immunohistochemistry. Results In M-CSF-differentiated murine macrophages incubated with DEP, significant fold increases in gene expression (relative to PBS control) were observed: IRF5 (1.4-fold), IL-6 (31.5-fold) and IL-12α (5.8-fold) while CD206 (0.8-fold) decreased (Figure 1a, n = 5, p<0.05, two-tailed paired t-tests). CD11c expression was unchanged. No statistically significant differences were detected in GM-CSF-differentiated macrophages. In the brachiocephalic arteries of DEP-exposed ApoE-/- mice we observed increases in CD64+ macrophages (28.2±7.4 vs 15.5±5.6) and IRF5 (16.6±3.6 vs 10.1±3.4) expression, while CD206 expression decreased (4.1±0.9 vs 11.0±1.2) in comparison to saline-exposed mice (Figure 1b mean±SD, n = 5, p<0.05, two-tailed unpaired t-tests, data expressed as % of intima area). Conclusion DEP promotes CD64+ macrophages and IRF5 expression in murine atherosclerosis, while inflammation and IRF5 is induced in vitro, but only in M-CSF-differentiated murine BMDMs. This suggests that DEP has no impact on inflammatory macrophages but could switch homeostatic resident macrophages to a more inflammatory phenotype via the activation of IRF5. Therefore, IRF5 is a promising candidate for further investigation into the initial macrophage response to inhaled particles that leads to the exacerbation of vascular disease.

Association of ethanolamine desaturase TMEM189-plasmalogen axis with the stability of atherosclerotic plaque and cardiovascular events after acute coronary syndrome

Abstract Background Patients with acute coronary syndrome (ACS) are still at high risk of recurring major adverse cardiovascular events (MACE). Disorders of lipid metabolism play an important role in the pathogenesis and progression of ACS. However, whether there are lipid components that can play an early warning role on the recurrence of MACE in patients with ACS and its potential mechanism is not yet known. Purpose To explore the changes of lipid profile before recurrent MACE in ACS patients, screen lipid molecules related to recurrent MACE, and explore their effects on pathological changes of atherosclerotic plaque. Methods We selected 44 Chinese ACS patients from the ‘Peking and Rotterdam on Mission to Reduce Coronary Artery Disease’ (PRoMISS) study. Among them, patients with recurrent MACE within 1 year after first onset of ACS were defined as MACE group, and the control group was recruited through 1:1 matching. The lipid profile of patients was analyzed by liquid chromatography-mass spectrometry. The effects of the screened potential lipid biomarkers and their synthesis enzymes were evaluated by real-time PCR, Western blot, IHC staining and ELISA. The enzyme knockout mice and ApoE knockout mice were hybridized and bred to obtain double gene knockout mice to determine whether the enzyme deficiency affects atherosclerotic plaque. Results Lipomic analysis showed that in the MACE group, plasmalogens and their 12 subtypes continued to decrease three months before the MACE recurrence. The most critical ethanolamine desaturase in the synthesis pathway of plasmalogen, transmembrane protein 189 (TMEM189) was also significantly lower in the MACE group than in the control group, and the level of TMEM189 was positively correlated with plasmalogen. Analysis of single-cell RNA sequencing results in human carotid artery plaques shows that TMEM189 is enriched in endothelial cells and smooth muscle cells (VSMC). In VSMC, treatment with oxLDL reduced TMEM189 expression. Overexpression of TMEM189 significantly improved oxLDL induced reduction in collagen synthesis, expression of alpha-smooth muscle actin (alpha-SMA) and smooth muscle protein 22 (SM22). Overexpression of TMEM189 in VSMC, significantly reduced the content of plasmalogens in the cells. After 12 weeks of feeding with high-fat and high-cholesterol diet, it was found that the content of smooth muscle cells and vulnerability index in the aortic plaque in TMEM189 -/-ApoE -/- mice were significantly decreased than in ApoE -/- mice. Conclusions Before the recurrence of MACE in ACS patients, the synthesis pathway of plasmalogen was gradually damaged, and the reduction of plasmalogen was a potential marker for predicting the recurrence of MACE in ACS patients. The deficiency of TMEM189 - plasmalogen axis aggravates atherosclerosis, it can regulate the phenotype transformation of smooth muscle cells, inhibit their collagen synthesis, and thus affect plaque stability.

Epicardial carbohydrate antigen 125 (CA-125) levels, associated with neutrophils activity in heart failure: No differences regarding sex

Abstract Background Carbohydrate antigen 125 (CA125), is the name of the soluble part of mucin-16, after being cleaved, which is detected and increased into circulation in congestive heart failure (HF) patients. Purpose We aimed to study plasma and epicardial fat markers related to inflammation, fibrosis, adiposity, and its association with plasma and epicardial CA125 for understanding the underlying mechanisms of heart failure and the differences between men and women. Methods We have included women and men undergoing open heart surgery (n=134). Epicardial fat biopsy and blood were obtained before the procedure under signed consent. Immunohistochemistry, ELISA, multiplex-Luminex, and real-time PCR were used for analyzing protein or mRNA expression levels of CA125 and markers. Results Out of 135, 57 patients suffered heart failure (HF). CA125 was expressed on the epicardium. Their levels were positively related to neutrophils, monocytes, and fibroblasts-related markers and negatively to adiposity markers. Both, plasma and epicardial CA125 levels were increased in HF patients. In this group of patients, epicardial CA125 levels were positively associated with circulating and local neutrophil activity, measured by defensin 3 levels. After testing all markers between women and men in HF patients, we observed similar levels in plasma and epicardial CA125. However, higher plasma and epicardial adiposity markers and lower mesothelial marker ITLN-1 were detected in women than in men. Conclusion CA125 is expressed in epicardial cells and its higher levels in HF might explain the higher detected levels in plasma. Its association with local and peripheral neutrophils-related markers with CA125 suggests its common pathophysiological mechanism in women and men. However, high plasma and epicardial adiposity and low mesothelial marker ITNL-1 in epicardium in women might explain the differential mechanism in HF.

Epigenetic modifications in transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

Abstract Background Adipose tissue-derived stromal vascular fraction (adipose SVF) contains pluripotent mesenchymal stem cells and rarely transdifferentiate into beating cardiomyocytes. We developed a simple culture protocol under which the adult murine inguinal adipose SVF reproductively transdifferentiates into beating cardiomyocyte-like cells (beating CM) without inductions in the primary culture. We also reported that Mef2c is a crucial factor in this conversion. However, the characteristics of beating CM and the factors that regulate the differentiation of adipose SVF toward the cardiac lineage are unknown. Purpose To investigate sequential changes in global gene expression profiles of adipose SVF in primary culture and determine the mechanism of differentiation into beating CM. Methods Adipose SVF cells were isolated from adult mice's inguinal subcutaneous fat pad and cultured using the previously established beating CM induction method (Sci Rep. 2021). At 6 time points (days 0, 3, 7, 14, 21, and 28) in primary culture, total RNA was extracted, and RNA-sequencing was performed (n = 3). The data was analyzed using Subio Platform and MetaCore software. To assess the candidate gene and proteins, adipose SVF cells were transduced with the gene using a lentivirus vector and supplemented with the specific inhibitors. The cardiac differentiation was evaluated by quantitative PCR on day 28. Results The beating CM was confirmed on days 14, 21, and 28 in the primary culture. Among prefiltered 14,574 genes, the expression level of 749 genes were significantly changed (153 genes upregulated vs. 596 genes downregulated) between days 7 and 14 (2-fold, P < 0.05), in which beating CM appeared. The GO molecular functions analysis showed that O-GluNAC transferase and histone deacetylase (HDAC)-associated genes were expressed dominantly before day 7, and KLF4-associated genes were expressed prominently after day 14 in primary culture. The expression of cardiac troponin T (Tnnt2) and myosin light chain ventricular type (Myl2) increased on day 3 and day 14, respectively. The HDAC7, an epigenetic regulator, decreased the expression on day 14 or later. Treatment with the HDAC inhibitor (150 nmol/L) increased the expression of Tnnt2 (5-fold vs. control) on day 28 in adipose SVF. Furthermore, treatment of Mef2c-transduced adipose SVFs with HDAC inhibitors increased expression of cardiac troponin T (29-fold vs. control, P < 0.05). Conclusion The time course analysis demonstrated that a remarkable change occurred in the regulation of transcription on day 14 in adipose SVF primary culture. Alterations in epigenetic modifications increased beating CM in adipose SVFs. Adipose SVF could be applied to new cardiac regeneration therapies by increasing the efficiency of introduction into the beating CM.

Metabolome and Transcriptome Analyses Reveal Metabolomic Variations and Key Transcription Factors Involved in Lipid Biosynthesis During Seed Development in Carya illinoinensis

Plant oils are a large group of neutral lipids that play a vital role in the food and oleochemical industries. The pecan (Carya illinoinensis) is a promising woody oil crop known for its high-quality sources of essential fatty acids and various bioactive compounds that may aid in preventing heart diseases. However, there is still a lack of understanding regarding the accumulation of lipids and the molecular mechanism of lipid biosynthesis during seed development. This study aims to analyze the metabolite variations and molecular mechanisms of lipid biosynthesis by integrating untargeted metabolomics and transcriptomics during pecan seed development. A total of 293 differentially accumulated metabolites were identified and further categorized into 13 groups, with lipids and lipid-like molecules constituting the largest group. The oil content and fatty acid compositions of pecan embryos were assessed at three stages of seed development. Oleic acid (c18:1) and linoleic acid (c18:2n6) were found to be the most abundant unsaturated fatty acid components in pecan embryos. Additionally, a comprehensive analysis revealed 15,990 differentially expressed genes, with a focus on the key genes related to lipid metabolism. Furthermore, the study identified 1201 transcription factors from differentially expressed genes. These transcription factors were divided into 65 families, with different members in the same family exhibiting different expression patterns during seed development. The expression patterns of ten transcription factor genes during seed development were verified by qRT–PCR. Two key genes, CiABI3 and CiFUS3 were further cloned and found to be localized in the nucleus. This study used metabolome and transcriptome analysis during key periods of pecan seed development to identify the key genes associated with seed development and fatty acid biosynthesis.

RACK1 contributes to the upregulation of embryonic genes in a model of cardiac hypertrophy

Receptors for activated C kinases (RACKs) have been shown to coordinate PKC-mediated hypertrophic signalling in mice. However, little information is available on its participation in embryonic gene expression. This study investigated the involvement of RACK1 in the expression of embryonic genes in a zebrafish (ZF) ex vivo heart culture model by using phenylephrine (PE) or a growth factors cocktail (GFs) as a prohypertrophic/regeneration stimulus. Blebbistatin (BL) inhibition has also been studied for its ability to block the signal transduction actions of some PEs. qRT‒PCR and immunoblot analyses confirmed the upregulation of RACK1 in the PE- and GFs-treated groups. BL administration counteracted PE-induced hypertrophy and downregulated RACK1 expression. Immunohistochemical analyses of the heart revealed the colocalization of RACK1 and embryonic genes, namely, Gata4, Wt1, and Nfat2, under stimulation, whereas these genes were expressed at lower levels in the BL treatment group. Culturing ZF heart cells activated via GFs treatment increased the expression of RACK1. The overexpression of RACK1 induced by the transfection of recombinant RACK1 cDNA in ZF heart cells increased the expression of embryonic genes, especially after one week of GFs treatment. In summary, these results support the involvement of RACK1 in the induction of embryonic genes during cardiac hypertrophy/GFs stimulation in a fish heart model, which can be used as an alternative study model for mammals.

Epigenetic modulation of vascular calcification: Looking for comprehending the role of sirt1 and histone acetylation in VSMC phenotypic transition

In light of the complex origins of ectopic vascular calcification and its significant health implications, this study offers a comprehensive exploration of the molecular dynamics governing vascular smooth muscle cells (VSMCs). Focusing on epigenetic modulation, we investigate the transition from a contractile to a calcifying phenotype in VSMCs, with an emphasis on understanding the role of SIRT1. For this purpose, a single batch of human aortic SMCs, used at a specified passage number to maintain consistency, was subjected to calcium and phosphate overload for up to 72 h. Our findings, validated through RT q-PCR, Western blot, immunofluorescence, and DNA methylation analyses, reveal a complex interplay between acetyltransferases and deacetylases during this phenotypic transition. We highlight HAT1A's critical role in histone acetylation regulation and the involvement of HDACs, as evidenced by subcellular localization studies. Moreover, we demonstrate the modulation of SIRT1 expression, a class III deacetylase, during VSMC calcification, underscoring the influence of DNA methylation in this process. Importantly, the study addresses previously unexplored aspects of the dynamic protein expression patterns observed, providing insight into the counterintuitive expressions of key proteins such as Runx2 and osterix. This research underscores the significant impact of epigenetic mechanisms, particularly the modulation of SIRT1, in the transition from a contractile to a calcifying phenotype in VSMCs, offering potential avenues for further exploration in the context of vascular calcification.

L-Securinine Induces ROS-Dependent Apoptosis on Pancreatic Cancer Cells via the PI3K/AKT/mTOR Signaling Pathway.

Accumulating evidence has shown that l-securinine can, in certain circumstances, suppress tumor development by elevating reactive oxygen species (ROS) levels. The current work set out to examine l-securinine's apoptotic effects on HuP-T3 cells as well as any potential underlying molecular mechanism(s) that could explain its action as an anticancer agent. In this study, we used 1.2B4 cells as a control human cell line to verify our findings. Hup-T3 and 1.2B4 cells were cultured with a medium containing the following dilutions of l-securinine: 1-10 μΜ for up to 72 h. We examined the viability and proliferation levels of cells in both cell lines. Then, we measured only 1.2B4 insulin levels and content. We also quantified cell apoptosis, cell cycle levels, and the intracellular reactive oxygen species on HuP-T3 and 1.2B4. Afterwards, we performed a real-time quantitative polymerase chain reaction and western blot analysis. Our results demonstrated that l-securinine inhibited both proliferation and growth of Hup-T3 cells, showing inhibitory and antiproliferative activity in comparison with the control group. In addition, l-securinine had no impact on the proliferation and growth of 1.2B4 cells, nor on their insulin levels and content. By boosting ROS production, and inhibiting the PI3K/AKT/mTOR signaling pathway, l-securinine induced apoptosis on HuP-T3 cells. Pancreatic cancer was successfully inhibited by l-securinine in vitro. l-securinine triggers ROS-dependent apoptosis on pancreatic cancer cells while inhibiting the PI3K/AKT/mTOR signaling pathway. These findings suggest that l-securinine holds promise as a potential lead for future drug development in the fight against pancreatic adenocarcinoma.

Association of genetically predicted human metabolomic gene expression with incident heart failure

Abstract Background Heart failure (HF) represents a significant challenge both for patients and healthcare systems worldwide. Despite ongoing efforts, therapeutic options are limited. The underlying pathophysiology involves intricate changes in cardiac metabolism that strongly influence the clinical phenotype, thus potentially offering novel options for therapeutic targeting. Systematic prioritisation of metabolic genes regarding their phenotypic impact on HF is outstanding. Purpose We investigate the association of genetically predicted metabolic gene expression with the onset of HF within UK Biobank (UKB). We followed up on identified candidate genes using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) models of cardiomyocyte hypertrophy. Methods Utilising genomic data of 484,372 UKB participants and left ventricle-specific expression quantitative trait loci (eQTL) information from Genotype-Tissue Expression (GTEx) project, we predicted genetically regulated gene expression using established methods (PrediXcan) for all metabolic genes. Subsequently, we employed per-gene Cox proportional hazards (COX-PH) models to assess the association of genetically predicted metabolic gene expression with incident HF. We adjusted models for clinical risk using established risk models (Pooled Cohort Equations to Prevent HF (PCP-HF)). To validate our findings in vitro, we subjected hiPSC-CMs to a sympathomimetic stimulus via treatment with endothelin-1 (ET-1; 10 nM). We evaluate alterations in expression via real-time quantitative polymerase chain reaction (RT-qPCR) and establish methods for assessing hypertrophic remodelling. Results COX-PH models revealed the predicted expression of 104/1515 metabolic genes to be independently associated with the onset of HF. Subsequent refinement with a meta-analysis of commonly dysregulated genes in end-stage HF narrowed our findings to 46 candidate genes, of which several were known for their role in HF pathophysiology. We further traced nine genes for validation in hiPSC-CM models and showed significant dysregulation of several genes following the treatment with ET-1. Flow cytometry measured hiPSC-CM hypertrophy following sympathomimetic stimulation (p=0.036). Extracellular flux analysis revealed canonical changes following treatment with ET-1. Conclusions The study revealed several metabolic genes associated with incident HF, thus confirming previous findings and highlighting novel, putative therapeutic target genes. We confirmed transcriptional changes in an in vitro model. Ongoing work includes further validation via silencing candidate genes in hiPSC-CMs and subjection to the established phenotypic in vitro models.

Ammopiptanthus nanus (M. Pop.) Cheng f. stem ethanolic extract ameliorates rheumatoid arthritis by inhibiting PI3K/AKT/NF-κB pathway-mediated macrophage infiltration

Ethnopharmacological relevanceAmmopiptanthus nanus (M. Pop.) Cheng f. (A. nanus), a traditional Kirgiz medicinal plant, its stem has shown potential in treating rheumatoid arthritis (RA) in China, either through oral medication or by topical application directly to the affected joints, but its underlying mechanism of action remains unexplored. Aim of the studyThe purpose of this study is to elucidate pharmacological mechanism of A. nanus in ameliorating RA using a comprehensive approach that combines network pharmacology, molecular docking and experimental evaluations. Materials and methodsFirstly, the major constituents of A. nanus stem ethanolic extract were identified and quantified by High-Performance Liquid Chromatography (HPLC). Disease target data from Gene Cards database was then used to define RA-associated targets. A protein-protein interaction (PPI) network was created via STRING database. The DAVID database powered gene ontology (GO) function and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to gain functional insights. In vitro, RAW264.7 cells were treated with A. nanus to investigate the roles of target proteins and pathways during lipopolysaccharide (LPS) - induced inflammation. Immunofluorescence assays were performed to assess the effects of A. nanus on macrophage infiltration. The key targets and signalling pathways were validated using enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), molecular docking, immunohistochemical analysis, western blotting and immunofluorescence. Finally, the therapeutic potential of A. nanus in RA was evaluated in a carrageenan-induced rat model. ResultsNetwork analysis identified 31 potential targets of A. nanus associated with RA, including 10 hub targets. KEGG analysis highlighted the involvement of PI3K/AKT signaling pathway. In vivo experiments demonstrated that A. nanus treatment significantly protected against carrageenan-induced inflammatory paw tissue and attenuated macrophage infiltration. Both in vivo and in vitro experiments confirmed that A. nanus significantly downregulated the protein expression of COX-2, iNOS and IL-1β, and inhibited PI3K/AKT/NFκB pathway, which are closely linked to RA. Furthermore, molecular docking and cellular thermal shift assay revealed that licoflavanone showed a strong binding affinity with key targets. ConclusionIn summary, this study provides the first evidence of the potent anti-inflammatory activity of A. nanus in experimental RA. The mechanism of action appears to involve inactivation of the PI3K/AKT/NF-κB pathway-mediated macrophage infiltration. These findings indicate that A. nanus has significant potential as a therapeutic potential agent for RA treatment and offer novel insights for future research and drug development in this field.

Ubiquitin-Specific Protease 15 Plays an Important Role in Controlling the Tolerance to Salt, Drought and Abscisic Acid in Arabidopsis thaliana

Ubiquitin-specific proteases (UBPs), the largest subfamily of deubiquitinating enzymes (DUBs), are critical for plant growth and development as well as abiotic-stress responses. In this study, we discovered that the expression of the ubiquitin-specific protease 15 (UBP15) gene of the gene ubiquitin-specific protease 15 (UBP15) was induced by salt, mannitol and abscisic acid (ABA) treatments. Further research revealed that UBP15 is involved in modulation of salt, drought tolerance and ABA signaling during seed germination, early seedling development, post-germination root growth or adult-plant stage. Enrichment analysis showed that many genes related to abiotic stresses and metabolic pathways were altered in the ubp15-1 mutant. Through the joint analysis of the quantitative real-time polymerase chain reaction (qRT-PCR) and differentially-expressed gene relationship network, we found that UBP15 may mainly regulate salt-stress tolerance by modulating the dwarf and delayed flowering 1 (DDF1) pathway through a cascade reaction. In the regulation of drought-stress responses, ring domain ligase1 (RGLG1) may be a direct substrate of UBP15. Moreover, we cannot exclude the possibility that UBP15 acts in a feed-forward loop mechanism in the regulation of drought-stress responses via ethylene response factor 53 (ERF53) and its ubiquitin (Ub) ligase RGLG1. In ABA signal transduction, UBP15 may play a role in at least three aspects of the ABA signaling pathway: ABA synthesis, stomatal closure regulated by ABA signaling, and transcription factors in the ABA pathway. Taken together, our results suggest that UBP15 is involved in salt, osmotic, and drought-stress tolerance and the ABA signaling pathway by directly regulating the stability of key substrates or indirectly affecting the expression of genes related to abiotic stresses in Arabidopsis thaliana. Our research provides new germplasm resources for stress-resistant crops cultivation. These results demonstrate that UBP15 is a key regulator of salt, drought and ABA tolerance in Arabidopsis.

CircSEC24A induces KLF8 expression to promote the malignant progression of non-small cell lung cancer by regulating miR-1253.

This study aimed to analyze the role of circSEC24A in non-small cell lung cancer (NSCLC) and its underlying mechanism. RNA levels of circSEC24A, microRNA-1253 (miR-1253), and KLF transcription factor 8 (KLF8) were detected by quantitative real-time polymerase chain reaction. Protein expression was analyzed by western blot or immunohistochemistry assay. Cell proliferation and apoptosis were investigated by colony formation assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry analysis. Glycolysis was evaluated by commercial kits. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted to identify the associations among circSEC24A, miR-1253, and KLF8. Xenograft mouse model assay was used to evaluate the effect of circSEC24A on tumor tumorigenesis. CircSEC24A and KLF8 were upregulated, while miR-1253 was downregulated in NSCLC. CircSEC24A knockdown inhibited proliferation and glycolysis but induced the apoptosis of NSCLC cells. CircSEC24A acted as a miR-1253 sponge and regulated NSCLC cell malignancy by targeting miR-1253. KLF8 was identified as a target of miR-1253, and its overexpression attenuated miR-1253-induced effects in NSCLC cells. Besides, circSEC24A upregulated KLF8 by sponging miR-1253. Further, circSEC24A knockdown suppressed NSCLC cell tumorigenesis in vivo. CircSEC24A silencing inhibited NSCLC cell malignancy through the miR-1253/KLF8 pathway, providing a potential therapeutic target for NSCLC.

Ginsenoside Rg3 activates the immune function of CD8+ T cells via circFOXP1-miR-4477a-PD-L1 axis to induce ferroptosis in gallbladder cancer.

Gallbladder cancer (GBC) is the most common and leading cause of cancer-associated mortality among biliary tract carcinomas worldwide and there is no specific drug for treatment. Activation of CD8+ T cell immune activity is one of the strategies to improve GBC treatment. This study is aimed to investigate the role of Ginsenoside Rg3 on CD8+ T cell activation and pathogenesis of GBC. In GBC cells, Rg3 administration led to the significant reduction of circFOXP1 and PD-L1 as measured by Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting. Mechanistically, circFOXP1 acted as the sponge of miR-4477a to regulate PD-L1 expression as demonstrated by RNA pull-down assay and dual luciferase reporter assay. Rg3 treatment enhanced the activity of CD8+ T cells by inhibiting thecircFOXP1/miR-4477a/PD-L1 signaling axis. Besides, Rg3 administration induced lipid oxidation and ROS reduction as detected by Flow cytometry, resulting in ferroptosis via theinactivation of circFOXP1/miR-4477a/PD-L1 axis. Ferroptosis inhibitor Fer-1 administration could reverse the beneficial effects caused by Rg3 treatment while ferroptosis inducer Erastin treatment enhanced the effects. Moreover, Rg3 gavage alleviated tumor growth and elevated ferroptosis and apoptosis in tumor tissues, which were prevented by PD-L1 overexpression. Furthermore, Rg3 was demonstrated to activate the function of CD8+ T cells via regulating thecircFOXP1-miR-4477a-PD-L1 signaling axis in vivo. Rg3 inactivated thecircFOXP1-miR-4477a-PD-L1 signaling axis to activate the immune function of CD8+ T cells, thereby inducing ferroptosis and apoptosis in GBC cells. This research recognizes the mechanism of Rg3-mediated anti-cancer effect and offers evidence for the potentiality of Rg3 in clinical application for GBC therapy.

Transcriptomic analysis of the gonads of Locusta migratoria (Orthoptera: Acrididae) following infection with Paranosema locustae.

Paranosema locustae is an environmentally friendly parasitic predator with promising applications in locust control. In this study, transcriptome sequencing was conducted on gonadal tissues of Locusta migratoria males and females infected and uninfected with P. locustae at different developmental stages. A total of 18,635 differentially expressed genes (DEGs) were identified in female ovary tissue transcriptomes, with the highest number of DEGs observed at 1 day post-eclosion (7141). In male testis tissue transcriptomes, a total of 32,954 DEGs were identified, with the highest number observed at 9 days post-eclosion (11,245). Venn analysis revealed 25 common DEGs among female groups and 205 common DEGs among male groups. Gene ontology and Kyoto Encyclopaedia of Genes and Genome analyses indicated that DEGs were mainly enriched in basic metabolism such as amino acid metabolism, carbohydrate metabolism, lipid metabolism, and immune response processes. Protein-protein interaction analysis results indicated that L. migratoria regulates the expression of immune- and reproductive-related genes to meet the body's demands in different developmental stages after P. locustae infection. Immune- and reproductive-related genes in L. migratoria gonadal tissue were screened based on database annotation information and relevant literature. Genes such as Tsf, Hex1, Apolp-III, Serpin, Defense, Hsp70, Hsp90, JHBP, JHE, JHEH1, JHAMT, and VgR play important roles in the balance between immune response and reproduction in gonadal tissues. For transcriptome validation, Tsf, Hex1, and ApoLp-III were selected and verified by quantitative real-time polymerase chain reaction (qRT-PCR). Correlation analysis revealed that the qRT-PCR expression patterns were consistent with the RNA-Seq results. These findings contribute to further understanding the interaction mechanisms between locusts and P. locustae.

The bacterial strains JAM1T and GP59 of the species Methylophaga nitratireducenticrescens differ in their expression profiles of denitrification genes in oxic and anoxic cultures.

Strain JAM1T and strain GP59 of the methylotrophic, bacterial species Methylophaga nitratireducenticrescens were isolated from a microbial community of the biofilm that developed in a fluidized-bed, methanol-fed, marine denitrification system. Despite of their common origin, both strains showed distinct physiological characters towards the dynamics of nitrate ( ) reduction. Strain JAM1T can reduce to nitrite ( ) but not to nitric oxide (NO) as it lacks a NO-forming reductase. Strain GP59 on the other hand can carry the complete reduction of to N2. Strain GP59 cultured under anoxic conditions shows a 24-48h lag phase before reduction occurs. In strain JAM1T cultures, reduction begins immediately with accumulation of . Furthermore, is reduced under oxic conditions in strain JAM1T cultures, which does not appear in strain GP59 cultures. These distinct characters suggest differences in the regulation pathways impacting the expression of denitrification genes, and ultimately growth. Both strains were cultured under oxic conditions either with or without , or under anoxic conditions with . Transcript levels of selected denitrification genes (nar1 and nar2 encoding reductases, nirK encoding reductase, narK12f encoding / transporter) and regulatory genes (narXL and fnr) were determined by quantitative reverse transcription polymerase chain reaction. We also derived the transcriptomes of these cultures and determined their relative gene expression profiles. The transcript levels of nar1 were very low in strain GP59 cultured under oxic conditions without . These levels were 37 times higher in strain JAM1T cultured under the same conditions, suggesting that Nar1 was expressed at sufficient levels in strain JAM1T before the inoculation of the oxic and anoxic cultures to carry reduction with no lag phase. Transcriptomic analysis revealed that each strain had distinct relative gene expression profiles, and oxygen had high impact on these profiles. Among denitrification genes and regulatory genes, the nnrS3 gene encoding factor involved in NO-response function had its relative gene transcript levels 5 to 10 times higher in strain GP59 cultured under oxic conditions with than those in both strains cultured under oxic conditions without . Since NnrS senses NO, these results suggest that strain GP59 reduced to NO under oxic conditions, but because of the oxic environment, NO is oxidized back to by flavohemoproteins (NO dioxygenase; Hmp), explaining why reduction is not observed in strain GP59 cultured under oxic conditions. Understanding how these two strains manage the regulation of the denitrification pathway provided some clues on how they response to environmental changes in the original biofilm community, and, by extension, how this community adapts in providing efficient denitrifying activities.

Diacylglycerol kinase γ facilitates the proliferation and migration of neural stem cells in the developing neural tube.

In this study, we aim to investigate diacylglycerol kinase (DGK) γ expression in developing neural tubes (NTs) and its effects on neural stem cell (NSC) proliferation and migration. Whole-mount in situ hybridization (WMISH) and immunohistochemistry are performed to explore DGKγ localization in developing NTs in vivo. NSCs are treated with sh-DGKγ, R59949, or PMA in vitro. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and neurosphere formation assay are utilized to evaluate NSC proliferation. Neurosphere migration assay and a transwell chamber assay are used to assess NSC migration. The diacylglycerol (DAG) content is detected via enzyme-linked immunosorbent assay (ELISA). The mRNA expression of DGKγ is detected via quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of DGKγ, protein kinase C (PKC) and phosphorylated PKC (p-PKC) are detected via western blot analysis. The results show that DGKγ mRNA is expressed predominantly in developing NTs. The neuroepithelium in developing NTs is positive for NSC markers, including Nestin, glial fibrillary acidic protein (GFAP), and DGKγ. DGKγ is expressed in the cytoplasm and nucleus of the neuroepithelium and is coexpressed with p-PKCγ and p-PKCδ. The proliferation of NSCs, the number of EdU-positive NSCs, and the number of neurospheres are decreased by sh-DGKγ and R59949 but increased by PMA. There is a shorter migration distance of NSCs and fewer migrated NSCs in the sh-DGKγ, R59949 and PMA groups. DAG content and the p-PKCδ/PKCδ ratio are increased by sh-DGKγ, R59949 and PMA, whereas the p-PKCγ/PKCγ ratio is decreased by PMA. Taken together, our findings indicate that DGKγ facilitates NSC proliferation and migration, which is responsible for the participation of DGK in NT development. DGKγ facilitates NSC migration via the DAG/PKCδ pathway.

Biomimetic porphyrin-lipid nanoparticles - novel nanoscale theranostics for multimodal imaging and therapy in atherosclerotic cardiovascular disease

Abstract Background High-density lipoprotein (HDL) mimetic nanoagents have unrealized potential for atherosclerosis theranostics. Porphyrin-lipid HDL mimetic nanoparticles (Por-HDL-NPs) incorporate a porphyrin-lipid conjugate which permits near infrared fluorescence imaging and positron emission tomography (PET) through chelation of Copper-64 (64Cu). The outer shell contains apolipoprotein A-I mimetic peptide R4F designed to interact with scavenger receptor class B type I (SR-BI), enabling macrophage-targeting and therapeutic effects. Purpose To investigate the multimodal imaging, macrophage-targeting and therapeutic properties of Por-HDL-NPs and applications in atherosclerotic cardiovascular disease. Methods In vitro, the cholesterol efflux capacity of PBS (control), reconstituted HDL (rHDL) and Por-HDL-NPs was determined in immortalized bone marrow-derived macrophages (iBMDMs) loaded with [³H]-cholesterol. Real-time quantitative PCR (RT-qPCR) assessed mRNA levels of inflammatory mediators. ELISAs and Western blotting measured changes in protein levels. Apolipoprotein (Apo)e-/- mice were fed high-cholesterol diet (HCD) for 6 weeks, for early-stage plaque, and 13 weeks for the unstable plaque model in which two partial stenoses were made in the right carotid artery. PET and fluorescence (IVIS, flow cytometry, microscopy) imaging identified Por-HDL-NPs in plaques, circulating and aortic cells. Histological techniques assessed plaque area and composition in aortic sinus and carotid artery sections. Results Por-HDL-NPs were internalized by iBMDMs, visualized via fluorescence microscopy and flow cytometry. Por-HDL-NPs increased cholesterol efflux (49%, P<0.05), compared to rHDL. Por-HDL-NPs reduced macrophage mRNA levels of Il-1b (88%), Il-18 (54%) and Ccl5 (75%), and protein secretion of IL-1β (69%) and CCL5 (82%), P<0.05. Por-HDL-NPs suppressed inflammasome components Nlrp3 (69%) and Asc (36%), and activation of inflammatory transcription factor p65-NF-κB (53%), P<0.05. SR-BI siRNA knockdown and methyl-β-cyclodextrin, revealed that the anti-inflammatory properties of Por-HDL-NPs were independent of SR-BI and cholesterol efflux. In Apoe-/- mice, PET imaging showed 64Cu-Por-HDL-NPs localized in hearts and could detect increases in plaque size over time with HCD feeding. Por-HDL-NP fluorescence was detected within aortic sinus and unstable carotid plaques, co-localized with CD68+ macrophages. Por-HDL-NP-treated mice had smaller early-stage (22%) and unstable plaques (52%) than control PBS-treated mice, P<0.05. Por-HDL-NP mice had fewer circulating (32%) and aortic monocytes (81%), and a reduction in aortic arch Rela (26%), P<0.05. Conclusions Por-HDL-NPs present as potential nanoscale theranostics for atherosclerotic cardiovascular disease. Por-HDL-NPs can detect plaques using multiple imaging modalities and exhibit atheroprotective effects.Atherosclerosis imaging with Por-HDL-NPs

Comparative analysis of the NF-Y transcription factor family identifies VaNF-YA6 as a positive regulator of salt and drought tolerance in grapevine

Nuclear factor Y transcription factors (NF-Y TFs) play crucial roles in plant responses to abiotic stress. However, there is a lack of research on the comparative analysis of evolutionary relationships, real-time quantitative fluorescence PCR (RT-qPCR), and functions of NF-Y TFs to screen key NF-Y TFs that are resistant to salt and drought stresses between Vitis vinifera (V. vinifera) and Vitis amurensis (V. amurensis). In this study, 27 and 26 NF-Y TFs were identified in V. vinifera and V. amurensis, respectively, and were divided into three subgroups. Subcellular localization prediction revealed that NF-Ys TFs were mainly located in the nucleus. Interestingly, the NF-YA protein sequence of ‘NTKKLDWEFWGCCDDCEKWFGGCC’ was lost in the V. vinifera compared to V. amurensis, whereas the sequence ‘SSVYSQPWWGHSIVCVA’ was gained, thus, these sequences might be closely related to the functions performed. RT-qPCR analysis of ‘Pinot Noir’ (cultivated variety) and ‘Zuoyouhong’ (wild variety) plantlets demonstrated that the expression levels of VaNF-YA6, VaNF-YB5, VvNF-YA3, VvNF-YA5, and VvNF-YC2 were significantly upregulated under 400 mmol·L-1 NaCl and 10% PEG treatments for 24 h Subcellular localization showed that the VaNF-YA6-GFP fusion protein was functioned primarily in the nucleus. Overexpression of VaNF-YA6 in grapevine leaves and Arabidopsis thaliana (Arabidopsis) could significantly enhance tolerance to salt and drought stresses by improving VvSOS2, VvSOS3, VvABF3, VvCPK6 expression levels, enzyme activities, and other protective substances. In summary, our study provides a theoretical basis for the further use of VaNF-YA6 to improve salt and drought resistance in grapevines.

VEGFB ameliorates insulin resistance in NAFLD via the PI3K/AKT signal pathway

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of the most universal liver diseases with complicated pathogenesis throughout the world. Insulin resistance is a leading risk factor that contributes to the development of NAFLD. Vascular endothelial growth factor B (VEGFB) was described by researchers as contributing to regulating lipid metabolic disorders. Here, we investigated VEGFB as a main target to regulate insulin resistance and metabolic syndrome.MethodsIn this study, bioinformatics, transcriptomics, morphological experiments, and molecular biology were used to explore the role of VEGFB in regulating insulin resistance in NAFLD and its molecular mechanism based on human samples, animal models, and cell models. RNA-seq was performed to analyze the signal pathways associated with VEGFB and NAFLD; Palmitic acid and High-fat diet were used to induce insulin-resistant HepG2 cells model and NAFLD animal model. Intracellular glucolipid contents, glucose uptake, hepatic and serum glucose and lipid levels were examined by Microassay and Elisa. Hematoxylin-eosin staining, Oil Red O staining, and Periodic acid-schiff staining were used to analyze the hepatic steatosis, lipid droplet, and glycogen content in the liver. Western blot and quantitative real-time fluorescent PCR were used to verify the expression levels of the VEGFB and insulin resistance-related signals PI3K/AKT pathway.ResultsWe observed that VEGFB is genetically associated with NAFLD and the PI3K/AKT signal pathway. After VEGFB knockout, glucolipids levels were increased, and glucose uptake ability was decreased in insulin-resistant HepG2 cells. Meanwhile, body weight, blood glucose, blood lipids, and hepatic glucose of NAFLD mice were increased, and hepatic glycogen, glucose tolerance, and insulin sensitivity were decreased. Moreover, VEGFB overexpression reduced glucolipids and insulin resistance levels in HepG2 cells. Specifically, VEGFB/VEGFR1 activates the PI3K/AKT signals by activating p-IRS1Ser307 expression, inhibiting p-FOXO1pS256 and p-GSK3Ser9 expressions to reduce gluconeogenesis and glycogen synthesis in the liver. Moreover, VEGFB could also enhance the expression level of GLUT2 to accelerate glucose transport and reduce blood glucose levels, maintaining glucose homeostasis.ConclusionsOur studies suggest that VEGFB could present a novel strategy for treating NAFLD as a positive factor.Graphical