Geranylgeranyl isoprenoids and hepatic Rap1a regulate basal and statin-induced expression of PCSK9.

LDL-C lowering is the main goal of atherosclerotic cardiovascular disease prevention, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is now a validated therapeutic strategy that lowers serum LDL-C and reduces coronary events. Ironically, the most widely used medicine to lower cholesterol, statins, has been shown to increase circulating PCSK9 levels, which limits their efficacy. Here, we show that geranylgeranyl isoprenoids and hepatic Rap1a regulate both basal and statin-induced expression of PCSK9 and contribute to LDL-C homeostasis. Rap1a prenylation and activity is inhibited upon statin treatment, and statin-mediated PCSK9 induction is dependent on geranylgeranyl synthesis and hepatic Rap1a. Accordingly, treatment of mice with a small-molecule activator of Rap1a lowered PCSK9 protein and plasma cholesterol and inhibited statin-mediated PCSK9 induction in hepatocytes. The mechanism involves inhibition of the downstream RhoA-ROCK pathway and regulation of PCSK9 at the post-transcriptional level. These data further identify Rap1a as a novel regulator of PCSK9 protein and show that blocking Rap1a prenylation through lowering geranylgeranyl levels contributes to statin-mediated induction of PCSK9.

Comprehensive Study on Solomonamides: Total Synthesis, Stereochemical Revision, and SAR Studies toward Identification of Simplified Lead.

Solomonamides, a pair of macrocyclic peptide natural products originating from marine sources, have garnered significant attention within the synthetic community owing to their marked anti-inflammatory efficacy and intricate molecular architectures. In this paper, we present a very detailed investigation into solomonamides, including the challenges associated with the total synthesis, the evolution of our synthetic strategies, structural reassignment, synthesis of all possible stereoisomeric macrocycles, biological assessment, structure-activity relationship (SAR) studies, etc. Within the ambit of this total synthesis, diverse strategies for macrocyclization were rigorously explored, encompassing the Friedel-Crafts acylation, cyclization involving the aniline NH2 moiety, macrolactamization utilizing Gly-NH2, and Heck macrocyclization methodologies. In addition, an array of intriguing chemical transformations were devised, including but not limited to photo-Fries rearrangement, Wacker oxidation, ligand-free Heck macrocyclization, oxidative cleavage of indole, synthesis of contiguous stereocenters via substrate/reagent-controlled protocols, and simultaneous making and breaking of olefinic moieties. The findings of this investigation revealed a structurally simplified lead compound. Remarkably, the lead compound, while possessing structural simplification in comparison to the intricate solomonamide counterparts, demonstrates equipotent in vivo anti-inflammatory efficacy.

Rap1 Activation Protects Against Fatty Liver and Non-Alcoholic Steatohepatitis Development.

We previously demonstrated that hepatic activation of a small G protein of the Ras family, Rap1a, is suppressed in obesity, which results in increased hepatic glucose production and glucose intolerance in obese mice. Here, we show that Rap1a inhibition in obese mice liver also results in fatty liver formation, which is characteristic of the diabetic liver. Specifically, we report that Rap1a activity is decreased in the livers of patients with non-alcoholic steatohepatitis (NASH) and mouse models of non-alcoholic fatty liver disease (NAFLD) and NASH. Restoring hepatic Rap1a activity by overexpressing a constitutively active mutant form of Rap1a lowered the mature, processed form of lipogenic transcription factor, Srebp1, without an effect on the unprocessed Srebp1 and suppressed hepatic TG accumulation, whereas liver Rap1a deficiency increased Srebp1 processing and exacerbated steatosis. Mechanistically, we show that mTORC1, which promotes Srebp1 cleavage, is hyperactivated upon Rap1a deficiency despite disturbed insulin signaling. In proof-of-principle studies, we found that treatment of obese mice with a small molecule activator of Rap1a (8-pCPT) or inhibiting Rap1a's endogenous inhibitor, Rap1Gap, recapitulated our hepatic gain-of-function model and resulted in improved hepatic steatosis and lowered lipogenic genes. Thus, hepatic Rap1a serves as a signaling molecule that suppresses both hepatic gluconeogenesis and steatosis, and inhibition of its activity in the liver contributes to the pathogenesis of glucose intolerance and NAFLD/NASH development.

Geranylgeranyl Isoprenoids and Hepatic Rap1a Regulate Basal and Statin-Induced Expression of PCSK9.

Low-density lipoprotein cholesterol (LDL-C) lowering is the main goal of atherosclerotic cardiovascular disease prevention, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is now a validated therapeutic strategy that lowers serum LDL-C and reduces coronary events. Ironically, the most widely used medicine to lower cholesterol, statins, has been shown to increase circulating PCSK9 levels, which limits their efficacy. Here, we show that geranylgeranyl isoprenoids and hepatic Rap1a regulate both basal and statin induced expression of PCSK9 and contribute to LDL-C homeostasis. Rap1a prenylation and activity is inhibited upon statin treatment, and statin mediated PCSK9 induction is dependent on geranylgeranyl synthesis and hepatic Rap1a. Accordingly, treatment of mice with a small molecule activator of Rap1a lowered PCSK9 protein and plasma cholesterol and inhibited statin mediated PCSK9 induction in hepatocytes. The mechanism involves inhibition of the downstream RhoA-ROCK pathway and regulation of PCSK9 at the post transcriptional level. These data further identify Rap1a as a novel regulator of PCSK9 protein and show that blocking Rap1a prenylation through lowering geranylgeranyl levels contributes to statin-mediated induction of PCSK9.

Rap1 in the Context of PCSK9, Atherosclerosis, and Diabetes.

PURPOSE OF REVIEW: The focus of this article is to highlight the importance of the small GTPase, Ras-associated protein 1 (Rap1), in proprotein convertase subtilisin/kexin type 9 (PCSK9) regulation and atherosclerosis and type 2 diabetes etiology and discuss the potential therapeutic implications of targeting Rap1 in these disease areas. REVIEW FINDINGS: Cardiometabolic disease characterized by obesity, glucose intolerance, dyslipidemia, and atherosclerotic cardiovascular disease remain an important cause of mortality. Evidence using mouse models of obesity and insulin resistance indicates that Rap1 deficiency increases proatherogenic PCSK9 and low-density lipoprotein cholesterol levels and predisposes these mice to develop obesity- and statin-induced hyperglycemia, which highlights Rap1's role in cardiometabolic dysfunction. Rap1 may also contribute to cardiovascular disease through its effects on vascular wall cells involved in the atherosclerosis progression. Rap1 activation, specifically in the liver, could be beneficial in the prevention of cardiometabolic perturbations, including type 2 diabetes, hypercholesterolemia, and atherosclerosis.

Standardized fraction of Xylocarpus moluccensis inhibits inflammation by modulating MAPK-NFκB and ROS-HIF1α-PKM2 activation.

OBJECTIVE: Present study investigates the effect of Xylocarpus moluccensis (Lamk.) M. Roem fruit fraction (CDR) on endotoxemia and explores the underlying mechanisms. MATERIALS AND METHODS: The effect of CDR (1-100 µg/ml) was assessed on cytokines, MAPKs, ROS, and metabolic reprogramming in LPS-induced cells (J774.2 and THP-1) by the conventional methodology of ELISA, PCR, and Western blotting. The effect of CDR (1-50 mg/kg, p.o.) was also evaluated in the mice model of endotoxemia and sepsis. RESULTS: CDR prevents LPS-induced cytokine production from murine and human whole blood and cell lines. CDR suppressed total cellular and mitochondrial superoxide generation and preserved mitochondrial function in LPS-stimulated phagocytes. Additionally, CDR abrogated LPS-induced MAPK's phosphorylation and IκBα degradation in J774.2 cells. Moreover, CDR suppressed LPS-induced glycolytic flux as indicated from PKM2, HK-2, PDK-2, and HIF-1α expression in J774.2 cells. In vivo, CDR pre-treatment inhibited pro-inflammatory cytokines release, metabolic reprogramming from oxidative phosphorylation to glycolysis in both LPS-induced endotoxemia and cecal slurry-induced sepsis mice model. CONCLUSION: Present study demonstrates the protective effect of CDR on LPS-induced inflammation and sepsis and identifies MAPK-NFκB and ROS-HIF1α-PKM2 as the putative target axis.

Evaluation of seed associated endophytic bacteria from tolerant chilli cv. Firingi Jolokia for their biocontrol potential against bacterial wilt disease.

In this study, the seed endosphere of a bacterial wilt tolerant chilli cv. Firingi Jolokia was explored in order to find effective agents for bacterial wilt disease biocontrol. A total of 32 endophytic bacteria were isolated from freshly collected seeds and six isolates were selected based on R. solanacearum inhibition assay. These isolates were identified as Bacillus subtilis (KJ-2), Bacillus velezensis (KJ-4), Leuconostoc mesenteroides (KP-1), Lactococcus lactis (LB-3), Bacillus amyloliquefaciens (WK-2), and Bacillus subtilis (WK-3) by 16S rRNA gene sequencing. In the in planta R. solanacearum inhibition assay carried out by seedling root bacterization method, Bacillus subtilis (KJ-2) exhibited highest biocontrol efficacy of 86.6 % on 7th day post R. solanacearum inoculation and a minimum biocontrol efficacy of 52.9 % was noted for Leuconostoc mesenteroides (KP-1). GC-HRMS analysis detected several known antimicrobial compounds in the extract of the culture supernatant of Bacillus subtilis (KJ-2); which may contribute to inhibition of R. solanacearum. In the growth promotion assay conducted using these isolates, only two of them namely Bacillus subtilis (KJ-2) and Bacillus amyloliquefaciens (WK-2) showed growth promotion in true leafed tomato plants. All the selected seed endophytic isolates were able to control bacterial wilt of tomato at the seedling stage and Bacillus subtilis (KJ-2) was found to be most effective in controlling the disease. The results of the present study highlighted that seed endosphere of bacterial wilt tolerant cultivar is a rich source of R. solanacearum antagonizing bacterial isolates.

Macrophage p47phox regulates pressure overload-induced left ventricular remodeling by modulating IL-4/STAT6/PPARγ signaling.

NADPH oxidase (Nox) mediates ROS production and contributes to cardiac remodeling. However, macrophage p47phox, a Nox subunit regulating cardiac remodeling, is unclear. We aimed to investigate the role of macrophage p47phox in hypertensive cardiac remodeling. Pressure-overload induced by Angiotensin II (AngII) for two weeks in young adult male p47phox deficient (KO) mice showed aggravated cardiac dysfunction and hypertrophy as indicated from echocardiographic and histological studies in comparison with wild-type littermates (WT). Additionally, LV of AngII-infused KO mice showed augmented interstitial fibrosis, collagen deposition and, myofibroblasts compared to AngII-infused WT mice. Moreover, these changes in AngII-infused KO mice correlated well with the gene analysis of hypertrophic and fibrotic markers. Similar results were also found in the transverse aortic constriction model. Further, AngII-infused KO mice showed elevated circulating immunokines and increased LV leukocytes infiltration and CD206+ macrophages compared to AngII-infused WT mice. Likewise, LV of AngII-infused KO mice showed upregulated mRNA expression of anti-inflammatory/pro-fibrotic M2 macrophage markers (Ym1, Arg-1) compared to AngII-infused WT mice. AngII and IL-4 treated bone marrow-derived macrophages (BMDMs) from KO mice showed upregulated M2 macrophage markers and STAT6 phosphorylation (Y641) compared to AngII and IL-4 treated WT BMDMs. These alterations were at least partly mediated by macrophage as bone marrow transplantation from KO mice into WT mice aggravated cardiac remodeling. Mechanistically, AngII-infused KO mice showed hyperactivated IL-4/STAT6/PPARγ signaling and downregulated SOCS3 expression compared to AngII-infused WT mice. Our studies show that macrophage p47phox limits anti-inflammatory signaling and extracellular matrix remodeling in response to pressure-overload.

Identification of putative miRNAs from expressed sequence tags of Gnetum gnemon L. and their cross-kingdom targets.

Wild edible plants are often found to be rich sources of nutrients and medicinally beneficial compounds with pharmacological activities. Gnetum gnemon is a nutritionally important plant and a popular food source in parts of Assam and North-East India. Various microRNAs (miRNAs) have been recently identified in many plants; however, there are no records of identification of miRNAs in any species of Gnetum. The prediction of miRNA-target associations in G. gnemon is an important step to facilitate functional genomics studies in this species. In the present study, all known miRNAs from plants available in public domain were used to search for the conserved G. gnemon miRNA homologues in publicly accessible expressed sequence tags (ESTs) in NCBI database. An aggregate of 20 new potential miRNAs belonging to two diverse miRNA families (miR399 and miR5021) were identified through a homology-based search by following stringent filtering criteria. To investigate the potential cross-kingdom effects of the identified miRNAs, we further identified the putative target genes of G. gnemon miRNAs in human transcriptome and analyzed them against the NCBI non-redundant protein database. The KEGG analysis of the target genes indicated that these genes were involved in different metabolic pathways such as caffeine metabolism, drug metabolism, and nitrotoluene degradation. The target genes of G. gnemon miRNAs in humans were found to be associated with various disorders of both hereditary and non-hereditary origin. These results could help to shed new light on understanding of miRNA-mRNAs functional networks in this species and its potential use as a small RNA-based therapy against some human diseases.

Endophytes from Gnetum gnemon L. can protect seedlings against the infection of phytopathogenic bacterium Ralstonia solanacearum as well as promote plant growth in tomato.

Endophytes are beneficial plant microbes which help the plants by producing various plant growth promoting substances and also by acting as biocontrol agents against various plant pathogens. In the present study, evaluation of endophytic bacteria isolated from Gnetum gnemon, an ethnomedicinal plant was carried out for their plant growth promoting (PGP) activity and antagonistic potential against bacterial wilt pathogen Ralstonia solanacearum. Initially a total of 40 endophytic bacteria were isolated which were clustered into 13 groups based on RFLP and BOX-PCR fingerprinting. These 13 representative isolates belonged to different genera of Bacillus (9), Solibacillus (1), Staphylococcus (2) and Caballeronia (1). Among them, six isolates were positive for production of IAA, the value of which ranged from 11.16 to 27.63 µg mL-1. Phosphate solubilisation in the range of 106.4 to -212.7 µg mL-1 was shown by three isolates. Eight isolates producedammonia, the value of which ranged from 1.3 to 6.1 µmol mL-1. All tested isolates were positive for siderophore production. For extracellular enzyme production, 7 isolates were positive for protease, 8 for cellulase and 10 for amylase production. The isolates were also tested for their antagonistic activity against R. solanacearum in vitro and in planta assay using tomato seedlings. Staphylococcus warneri GL1 showed the highest biocontrol efficacy of 77.67 % followed by Bacillus velezensis GL3 i.e. 70.1 %. R. solanacearum antagonistic isolates were analysed for the presence of antimicrobial peptide biosynthesis genes bmyB, srfAA, fenD and ituC. All the antagonistic isolates showed the presence of all four genes, except the isolate Bacillus velezensis GMC2, where the gene for fengycin synthetase (fenD) was absent. Based on in vitro PGP traits, three isolates Bacillus velezensis GL3, Bacillus atrophaeus GMC1 and Bacillus megaterium GS2 were selected, these three endophytic bacteria individually and their consortia were tested for in planta PGP activities in tomato plants. Application of Bacillus velezensis GL3 alone and consortia of three isolates showed significant improvement in growth parameters such as shoot length, fresh weight and dry weight in a pot experiment. Colonization of endosphere of treated tomato seedlings by the endophytic isolate Bacillus velezensis GL3 was confirmed by visualization of colony morphology and BOX-PCR fingerprinting. Our study highlights the potential of endophytes associated with unexplored plants like G. gnemon for development of bioformulation aimed at enhancing plant growth and bacterial wilt disease control.

Mining of miRNAs from EST data in Dendrobium nobile.

Dendrobium nobile is an orchid species highly popular for its therapeutic properties and is often used as a medicinal herb. Documenting miRNA-target associations in D. nobile is an important step to facilitate functional genomics studies in this species. Therefore, it is of interest to identify miRNA sequences from EST data available in public databases using known techniques and tools. We report 14 potential miRNAs from three ESTs of D. nobile. They belong to 3 miRNA families (miR390, miR528 and miR414) linking to transcription factor regulation, signal transduction, DNA and protein binding, and various cellular processes covering 34 different metabolic networks in KEGG. These results help in the understanding of miRNA-mRNAs functional networks in Dendrobium nobile.

MicroRNA 27a Is a Key Modulator of Cholesterol Biosynthesis.

Hypercholesterolemia is a strong predictor of cardiovascular diseases. The 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (Hmgcr) coding for the rate-limiting enzyme in the cholesterol biosynthesis pathway is a crucial regulator of plasma cholesterol levels. However, the posttranscriptional regulation of Hmgcr remains poorly understood. The main objective of this study was to explore the role of microRNAs (miRNAs) in the regulation of Hmgcr expression. Systematic in silico predictions and experimental analyses reveal that miRNA 27a (miR-27a) specifically interacts with the Hmgcr 3' untranslated region in murine and human hepatocytes. Moreover, our data show that Hmgcr expression is inversely correlated with miR-27a levels in various cultured cell lines and in human and rodent tissues. Actinomycin D chase assays and relevant experiments demonstrate that miR-27a regulates Hmgcr by translational attenuation followed by mRNA degradation. Early growth response 1 (Egr1) regulates miR-27a expression under basal and cholesterol-modulated conditions. miR-27a augmentation via tail vein injection of miR-27a mimic in high-cholesterol-diet-fed Apoe-/- mice shows downregulation of hepatic Hmgcr and plasma cholesterol levels. Pathway and gene expression analyses show that miR-27a also targets several other genes (apart from Hmgcr) in the cholesterol biosynthesis pathway. Taken together, miR-27a emerges as a key regulator of cholesterol biosynthesis and has therapeutic potential for the clinical management of hypercholesterolemia.

Total synthesis of the potent anti-inflammatory natural product solomonamide A along with structural revision and biological activity evaluation.

Herein, we report the total synthesis of solomonamide A along with its structural revision for the first time. The natural product possesses very potent anti-inflammatory activity, and it contains a macrocyclic peptide having four consecutive stereocenters on an unnatural amino acid component. The key features in the present synthesis include the application of an Evans aldol reaction, ligand-free Heck macrocyclization and chemoselective oxidations. The challenging task of fixing the stereochemistry of OH at the C5-position was accomplished with the help of DFT calculations, applying a quantum-mechanical (QM)/NMR combined approach. Biological evaluation in a mouse paw edema model revealed that a low dose (0.3 mg kg-1) of the synthesized solomonamide A showed 74% reduction at 6 h, which was comparable to a high dose (10 mg kg-1) standard drug dexamethasone effect (75% at 6 h). Thus, we further confirmed the revised structure of solomonamide A.

Cilostazol ameliorates heart failure with preserved ejection fraction and diastolic dysfunction in obese and non-obese hypertensive mice.

Cilostazol (Ciloz) a potent Type III phosphodiesterase inhibitor is effective against inflammation, insulin resistance and cardiomyopathy. However, the effect of Ciloz on obesity-associated left ventricular diastolic dysfunction has not been explored yet. Hence, we examined the effect of Ciloz on cardiac remodelling and dysfunction in non-obese and obese-insulin resistant mice infused with AngiotensinII (AngII). Male C57BL/6 J mice were initially subjected to 19 weeks of chow or high fat diet (HFD) regimen and thereafter animals were randomised for AngII (1500 ng/kg/min, s.c) infusion or saline and Ciloz (50 mg/kg, p.o) for another 1 week. Obese and non-obese mice infused with AngII exhibited significant diastolic dysfunction and features of heart failure with preserved ejection fraction (HFpEF) since a decrease in fractional shortening and no change in ejection fraction were observed when compared with respective controls. Administration of AngII and Ciloz in HFD fed mice significantly improved the left ventricular function compared with AngII infused HFD mice as evinced from the echocardiographic data. Further, Ciloz treatment significantly reduced cardiomyocyte area, interstitial and perivascular fibrosis; and collagen deposition. Moreover, Ciloz reduced the inflammatory milieu in the heart as evinced by decreased F4/80+ and CD68+ cells; IL-1ß and IL-6 gene transcripts. Quantitative assessment of the expression levels revealed substantial upregulation of MMP-9 (pro- and mature-forms) and α-SMA in the left ventricle of AngII infused HFD-fed mice, which was considerably suppressed by Ciloz regimen. The beneficial effect of Ciloz was associated with the normalization in gene expression of hypertrophic and fibrotic markers. Likewise, Ciloz administration markedly reduced the AngII and HFD induced TGF-ß1/SMAD3 and Akt/mTOR signalling. Additionally, AngII administered and HFD-fed mice showed increased glycolytic flux, which was considerably diminished by Ciloz treatment as indicated from suppressed PKM2, HK-2, PDK-2, HIF-1α mRNA and GLUT-1 protein expression. Taken together, Ciloz might be therapeutically exploited against AngII and obesity-associated diastolic dysfunction thereby preventing overt heart failure.