In this work, we investigated the impacts of atmospheric pressure dielectric barrier discharge (DBD), i.e., plasma treatment, on pearl millet seeds germination and plant growth. The effect of plasma ...discharge on water activation, by introducing the reactive species, was explored. We evidenced that about 30 min plasma treated pearl millet seeds exhibited 20% higher germination rate than the control seed watered with tap water. The HR-SEM study revealed that the plasma treatment increased the roughness and FTIR study showed that new oxygen functional groups were introduced on the seed surface. Moreover, it was observed that the water contact angle decreased for plasma treated seeds (50%) and the water uptake also increased considerably as compared to control seeds. These findings indicate that the seed surface has turned more hydrophilic after plasma treatment. A cylindrical double dielectric barrier discharge (D-DBD) reactor was employed for water activation, and 30 min of treatment under air has decreased the pH of deionized water from 7.4 to 4.5 and produced about 1.78 ppm of nitrate (NO
3
−
) and 4.2 ppm of hydrogen peroxide (H
2
O
2
). Interestingly, the plasma activated water (PAW) improved the pearl millet seed germination by 30% (after 24 h of sowing) and plant growth as compared to tap water and deionized water. Remarkably, when PAW and plasma-treated seeds were combined, a beneficial impact in seed germination (95 ± 2%) and seedling growth have been evidenced owing to synergistic effect. We evidenced that among the long-lived species in PAW, NO
3
−
enhanced the seed germination and plant growth under similar conditions. These findings demonstrate that the proposed cold plasma reactors could be utilized to boost seed germination and plant growth.Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 4 Given name: Amine Aymen Last name Assadi. Also, kindly confirm the details in the metadata are correct.Yes, all author names are correctly editted.
Background and Aims
Parenteral nutrition (PN)–associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal ...injury and IL‐1β derived from activated hepatic macrophages to suppress hepatocyte farnesoid X receptor (FXR) signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model.
Approach and Results
To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sulfate sodium DSS for 4 days) followed by central venous catheterization and 14‐day infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, and biliary and sterol transporter expression were determined. GW4064 (30 mg/kg/day) added to PN on days 4–14 prevented hepatic injury and cholestasis; reversed the suppressed mRNA expression of nuclear receptor subfamily 1, group H, member 4 (Nr1h4)/FXR, ATP‐binding cassette subfamily B member 11 (Abcb11)/bile salt export pump, ATP‐binding cassette subfamily C member 2 (Abcc2), ATP binding cassette subfamily B member 4(Abcb4), and ATP‐binding cassette subfamily G members 5/8(Abcg5/8); and normalized serum bile acids. Chromatin immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS‐PN‐induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll‐1b, C‐C motif chemokine receptor 2, integrin subunit alpha M, lymphocyte antigen 6 complex locus C), and hepatic macrophage cytokine transcription in response to lipopolysaccharide in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL‐1β exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15, and organic solute transporter alpha) were not different among groups, supporting a liver‐specific effect of GW4064 in this model.
Conclusions
GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling, resulting in increased expression of canalicular bile and of sterol and phospholipid transporters and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.
The farnesoid X receptor (FXR) belongs to a family of ligand-activated transcription factors that regulate many aspects of metabolism including bile acid homeostasis. Here we show that FXR is ...covalently modified by the small ubiquitin-like modifier (Sumo1), an important regulator of cell signaling and transcription. Well conserved consensus sites at lysine 122 and 275 in the AF-1 and ligand binding domains, respectively, of FXR were subject to SUMOylation in vitro and in vivo. Chromatin immunoprecipitation (ChIP) analysis showed that Sumo1 was recruited to the bile salt export pump (BSEP), the small heterodimer partner (SHP), and the OSTα-OSTβ organic solute transporter loci in a ligand-dependent fashion. Sequential chromatin immunoprecipitation (ChIP-ReChIP) verified the concurrent binding of FXR and Sumo1 to the BSEP and SHP promoters. Overexpression of Sumo1 markedly decreased binding and/or recruitment of FXR to the BSEP and SHP promoters on ChIP-ReChIP. SUMOylation did not have an apparent effect on nuclear localization of FXR. Expression of Sumo1 markedly inhibited the ligand-dependent, transactivation of BSEP and SHP promoters by FXR/retinoid X receptor α (RXRα) in HepG2 cells. In contrast, mutations that abolished SUMOylation of FXR or siRNA knockdown of Sumo1 expression augmented the transactivation of BSEP and SHP promoters by FXR. Pathways for SUMOylation were significantly altered during obstructive cholestasis with differential Sumo1 recruitment to the promoters of FXR target genes. In conclusion, FXR is subject to SUMOylation that regulates its capacity to transactivate its target genes in normal liver and during obstructive cholestasis.
Background: Small ubiquitin-like modifiers (SUMO) are covalently conjugated to other proteins including nuclear receptors leading to modification of various cellular processes.
Results: Ligand-dependent SUMOylation of farnesoid X receptor (FXR) negatively regulates the expression of its target genes.
Conclusion: SUMO modification attenuates the capacity of FXR to function as a transcriptional activator.
Significance: Defining post-translation modification of FXR by SUMO is important to understanding how this nuclear receptor functions in health and disease.
In infants intolerant of enteral feeding because of intestinal disease, parenteral nutrition may be associated with cholestasis, which can progress to end-stage liver disease. Here we show the ...function of hepatic macrophages and phytosterols in parenteral nutrition-associated cholestasis (PNAC) pathogenesis using a mouse model that recapitulates the human pathophysiology and combines intestinal injury with parenteral nutrition. We combine genetic, molecular, and pharmacological approaches to identify an essential function of hepatic macrophages and IL-1β in PNAC. Pharmacological antagonism of IL-1 signaling or genetic deficiency in CCR2, caspase-1 and caspase-11, or IL-1 receptor (which binds both IL-1α and IL-1β) prevents PNAC in mice. IL-1β increases hepatocyte NF-κB signaling, which interferes with farnesoid X receptor and liver X receptor bonding to respective promoters of canalicular bile and sterol transporter genes (Abcc2, Abcb11, and Abcg5/8), resulting in transcriptional suppression and subsequent cholestasis. Thus, hepatic macrophages, IL-1β, or NF-κB may be targets for restoring bile and sterol transport to treat PNAC.
The Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a transcriptional co-activator that plays a central role in adapted metabolic responses. PGC-1α is dynamically ...methylated and unmethylated at the residue K779 by the methyltransferase SET7/9 and the Lysine Specific Demethylase 1A (LSD1), respectively. Interactions of methylated PGC-1αK779me with the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, the Mediator members MED1 and MED17, and the NOP2/Sun RNA methytransferase 7 (NSUN7) reinforce transcription, and are concomitant with the m5C mark on enhancer RNAs (eRNAs). Consistently, loss of Set7/9 and NSun7 in liver cell model systems resulted in depletion of the PGC-1α target genes Pfkl, Sirt5, Idh3b, and Hmox2, which was accompanied by a decrease in the eRNAs levels associated with these loci. Enrichment of m5C within eRNA species coincides with metabolic stress of fasting in vivo. Collectively, these findings illustrate the complex epigenetic circuitry imposed by PGC-1α at the eRNA level to fine-tune energy metabolism.
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•K779 of PGC-1α is methylated by SET7/9 and demethylated by LSD1•PGC-1αK779me recruits SAGA and Mediator at enhancers of target genes•PGC-1αK779me corresponds with NSUN7 and m5C eRNAs at PGC1α-regulated loci•Enrichment of m5C within eRNA coincides with fasting in vivo
Aguilo et al. find that PGC-1α can regulate metabolic networks that integrate the modification of PGC-1α, NSUN7 and m5C-modified eRNAs. These findings illustrate how complex epigenetic circuitry might function to fine-tune metabolic responses through the PGC-1α co-activator.
Prior loss‐of‐function analyses revealed that ATPase class I type 8B member 1 familial intrahepatic cholestasis 1 (FIC1) posttranslationally activated the farnesoid X receptor (FXR). Mechanisms ...underlying this regulation were examined by gain‐of‐function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild‐type and mutated FIC1 expression constructs with a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques. FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR‐dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler disease FIC1 mutants did not activate BSEP, whereas benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent on the presence of the FXR ligand, chenodeoxycholic acid. The effect of FIC1 on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C zeta (PKC ζ) inhibitors (pseudosubstrate or small interfering RNA silencing). Recombinant PKC ζ directly phosphorylated immunoprecipitated FXR. The mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, whereas the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKC ζ in Caco‐2 cells resulted in activation of the human apical sodium‐dependent bile acid transporter promoter. Conclusion: These results demonstrate that FIC1 signals to FXR via PKC ζ. FIC1‐related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. Benign recurrent intrahepatic cholestasis emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity. (HEPATOLOGY 2008;48:1896‐1905.)
Adenosine triphosphate–binding cassette subfamily C member 2 (ABCC2/Abcc2) is critically important to biliary excretion of many endobiotic and xenobiotic compounds, and is a major driving force for ...bile acid–independent bile flow. Abcc2 expression is reduced at the messenger RNA (mRNA) and protein levels in various forms of experimental cholestasis. In a microRNA (miRNA) screen of mouse liver after biliary obstruction, we found that miRNA let7a‐5p was significantly up‐regulated approximately 4‐fold. Similarly, ABCC2 mRNA was depleted and miRNA let7a‐5p was elevated over 4‐fold in livers of children with biliary atresia compared with normal livers. In silico analysis predicted that let7a‐5p would target the 3′ untranslated region (3′ UTR) of ABCC2/Abcc2 RNA. The objective of this study was to determine whether let7a‐5p contributes to the depletion of ABCC2/Abcc2 in cholestasis. To demonstrate the functional importance of miRNA let7a‐5p in regulating the expression of ABCC2, co‐transfection of a let7a‐5p mimic and an ABCC2‐3′ UTR luciferase construct into Huh‐7 cells led to a marked inhibition of luciferase activity by about 60%‐70% compared with controls, which was reversed by a let7a‐5p mimic inhibitor. Expression of this mimic led to a significant decrease in endogenous ABCC2 mRNA and protein levels in a Huh‐7 liver cell line, which could be blocked by expression of a let7a‐5p mimic inhibitor. Injection of a lentivirus let7a‐5p inhibitor into normal mouse liver or into mouse liver after common bile duct ligation led to a significant increase in endogenous Abcc2 mRNA and protein levels and a depletion of let7a‐5p mRNA levels compared with untreated, saline‐injected livers or livers treated with an inactive lentivirus control. Conclusion: These studies demonstrate that miR‐let7a‐5p is involved in regulating ABCC2/Abcc2 expression, and is aberrantly up‐regulated in obstructive cholestasis.
ABCC2 is critically important to biliary excretion of many endobiotic and xenobiotic compounds, and is a major driving force for bile acid–independent bile flow. ABCC2 expression is reduced at the mRNA and protein levels in various forms of experimental cholestasis, but the underlying mechanisms for this change have not been defined. Our studies demonstrate that miR‐let7a‐5p is involved in regulating ACCC2/Abcc2 expression, and is aberrantly up‐regulated in obstructive cholestasis.
Proinflammatory cytokines such as TNF-alpha and IL-1beta lead to downregulation of hepatic organic anion transporters in cholestasis. This adapted response is transcriptionally mediated by nuclear ...hormone receptors and liver-specific transcription factors. Because little is known in vivo about cytokine-dependent regulatory events, mice were treated with either TNF-alpha or IL-1beta for up to 16 h. Transporter mRNA expression was determined by Northern blot analysis, nuclear activity, and protein-expression of transactivators by EMSA and Western blotting. TNF-alpha induces a sustained decrease in Ntcp, Oatp1/Oatp1a1, and Bsep mRNA expression but exerts only transient multidrug resistance-associated protein 2 (Mrp2) or no effects (Mrp3) on Mrps. In addition to Ntcp and Oatp1/Oatp1a1, IL-1beta also downregulates Bsep, Mrp2, and Mrp3 mRNAs to some extent. To study transcriptional regulation, Ntcp and Bsep promoters were first cloned from mice revealing a new distal Ntcp hepatocyte nuclear factor 1 (HNF-1) element but otherwise show a conserved localization to known rat regulatory elements. Changes in transporter-expression are preceeded by a reduction in binding activities at IR-1, ER-8, DR-5, and HNF-1alpha sites after 4 h by either cytokine, which remained more sustained by TNF-alpha in the case of nuclear receptors. Nuclear protein levels of retinoid X receptor (RXR)-alpha are significantly decreased by TNF-alpha but only transiently affected by IL-1beta. Minor reductions of retinoic acid receptor, farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor nuclear proteins are restricted to 4 h after cytokine application and paralleled by a decrease in mRNA levels. Basolateral and canalicular transporter systems are downregulated by both cytokines, TNF-alpha and IL-1beta. Activity of HNF-1alpha as regulator of mNtcp is suppressed by both cytokines. Decreased binding activities of nuclear receptor heterodimers may be explained by a reduction of the ubiquitous heterodimerization partner RXR-alpha.
In this study we demonstrate that the class II nuclear hormone receptor, farnesoid X-receptor (FXR), incorporates histone methyltransferase activity within the gene locus for bile salt export pump ...(BSEP), a well established FXR target gene that functions as an ATP-dependent canalicular bile acid transporter. This methyltransferase activity is directed specifically to arginine 17 of histone H3. We demonstrate that FXR is directly associated with co-activator-associated arginine methyltransferase 1 (CARM1) activity. Furthermore, we show by chromatin immunoprecipitation that the ligand-dependent activation of the human BSEP locus is associated with a simultaneous increase of FXR and CARM1 occupation. The increased occupation of the BSEP locus by CARM1 also corresponds with the increased deposition of Arg-17 methylation and Lys-9 acetylation of histone H3 within the FXR DNA-binding element of BSEP. Consistent with these findings, CARM1 led to increased BSEP promoter activity with an intact FXR regulatory element, whereas CARM1 failed to transactivate the BSEP promoter with a mutated FXRE. Induction of endogenous BSEP mRNA and Arg-17 methylation by FXR regulatory element ligand, CDCA, requires CARM1 activity. Therefore, histone methylation at Arg-17 by CARM1 is a downstream target of signaling through ligand-mediated activation of FXR. Our studies provide evidence that FXR directly recruits specific chromatin modifying activity of CARM1 necessary for full potentiation of the BSEP locus in vivo.
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