Ameliorative effects of elderberry (Sambucus nigra L.) extract and extract-derived monosaccharide-amino acid on H2O2-induced decrease in testosterone-deficiency syndrome in a TM3 Leydig cell.

With aging, men develop testosterone-deficiency syndrome (TDS). The development is closely associated with age-related mitochondrial dysfunction of Leydig cell and oxidative stress-induced reactive oxygen species (ROS). Testosterone-replacement therapy (TRT) is used to improve the symptoms of TDS. However, due to its various side effects, research on functional ingredients derived from natural products that do not have side effects is urgently needed. In this study, using the mitochondrial dysfunction TM3 (mouse Leydig) cells, in which testosterone biosynthesis is reduced by H2O2, we evaluated the effects of elderberry extract and monosaccharide-amino acid (fructose-leucine; FL) on mRNA and protein levels related to steroidogenesis-related enzymes steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1(CYP11A1, cytochrome P450 17A1(CYP17A1), cytochrome P450 19A1(CYP19A1, aromatase), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and 17ß-hydroxysteroid dehydrogenase(17ß-HSD). We analyzed elderberry extract and extract-derived FL for changes in ROS scavenging activity and testosterone secretion. Elderberry extract and FL significantly reduced H2O2-induced intracellular ROS levels, improved testosterone secretion, and increased the mRNA and protein expression levels of steroidogenesis-related enzymes (StAR, 3b-HSD, 17b-HSD, CYP11A1, CYp17A1). However, the conversion of testosterone to estradiol was inhibited by elderberry extract and extract-derived FL, which reduced the mRNA and protein expression of CYP19A1. In conclusion, elderberry extract and FL are predicted to have value as novel functional ingredients that may contribute to the prevention of TDS by ameliorating reduced steroidogenesis.

The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Integrative proteomic and phosphoproteomic analysis in the female goat hypothalamus to study the onset of puberty.

BACKGROUND: Puberty marks the end of childhood and achieve sexual maturation and fertility. The role of hypothalamic proteins in regulating puberty onset is unclear. We performed a comprehensive differential proteomics and phosphoproteomics analysis in prepubertal and pubertal goats to determine the roles of hypothalamic proteins and phosphoproteins during the onset of puberty. RESULTS: We used peptide and posttranslational modifications peptide quantification and statistical analyses, and identified 69 differentially expressed proteins from 5,057 proteins and 576 differentially expressed phosphopeptides from 1574 phosphorylated proteins. Combined proteomic and phosphoproteomics, 759 correlated proteins were identified, of which 5 were differentially expressed only at the protein level, and 201 were only differentially expressed at the phosphoprotein level. Pathway enrichment analyses revealed that the majority of correlated proteins were associated with glycolysis/gluconeogenesis, Fc gamma R-mediated phagocytosis, focal adhesion, GABAergic synapse, and Rap1 signaling pathway. These pathways are related to cell proliferation, neurocyte migration, and promoting the release of gonadotropin-releasing hormone in the hypothalamus. CTNNB1 occupied important locations in the protein-protein interaction network and is involved in focal adhesion. CONCLUSION: The results demonstrate that the proteins differentially expression only at the protein level or only differentially expressed at the phosphoprotein level and their related signalling pathways are crucial in regulating puberty in goats. These differentially expressed proteins and phosphorylated proteins may constitute the proteomic backgrounds between the two different stages.

Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport and cause congenital hypophosphatemia.

The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg495His or Arg495Cys variants within the internal PDZ motif. The wild-type internal 494TRL496 PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a 494AAA496 substitution blocked hormone-inhibitable phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg495His or Arg495Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast with WT NPT2A, NPT2A Arg495His, or Arg495Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg495 changes the electrostatics, preventing phosphorylation of the upstream Thr494, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxy-terminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

Non-hippo kinases: indispensable roles in YAP/TAZ signaling and implications in cancer therapy.

The transcriptional co-activators Yes-associated protein (YAP) and PDZ-binding domain (TAZ) are the known downstream effectors of the Hippo kinase cascade. YAP/TAZ have been shown to play important roles in cellular growth and differentiation, tissue development and carcinogenesis. Recent studies have found that, in addition to the Hippo kinase cascade, multiple non-Hippo kinases also regulate the YAP/TAZ cellular signaling and produce important effects on cellular functions, particularly on tumorigenesis and progression. In this article, we will review the multifaceted regulation of the YAP/TAZ signaling by the non-Hippo kinases and discuss the potential application of the non-Hippo kinase-regulated YAP/TAZ signaling for cancer therapy.

Quantitative phosphoproteome analysis reveals differential whey phosphoproteins of bovine milk during lactation.

Whey proteins in bovine milk, as the most widely used nutritional components for infant formulae, have been paid more attention. However, the phosphorylation of proteins in bovine whey during lactation has not been thoroughly researched. In this study, a total of 185 phosphorylation sites on 72 phosphoproteins were identified in bovine whey during lactation. 45 differentially expressed whey phosphoproteins (DEWPPs) in colostrum and mature milk were focused on by bioinformatics approaches. Gene Ontology annotation indicated that blood coagulation, extractive space, and protein binding played a key role in bovine milk. The critical pathway of DEWPPs was related to the immune system according to KEGG analysis. Our study investigated the biological functions of whey proteins from a phosphorylation perspective for the first time. The results elucidate and increase our knowledge of differentially phosphorylation sites and phosphoproteins in bovine whey during lactation. Additionally, the data might offer fresh insight into the development of whey protein nutrition.

Regulatory role of phosphoproteins in the development of bovine small intestine during early life.

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study.

Transmembrane protein KIRREL1 regulates Hippo signaling via a feedback loop and represents a therapeutic target in YAP/TAZ-active cancers.

The Hippo tumor-suppressor pathway is frequently dysregulated in human cancers and represents a therapeutic target. However, strategies targeting the mammalian Hippo pathway are limited because of the lack of a well-established cell-surface regulator. Here, we show that transmembrane protein KIRREL1, by interacting with both SAV1 and LATS1/2, promotes LATS1/2 activation by MST1/2 (Hippo kinases), and LATS1/2 activation, in turn, inhibits activity of YAP/TAZ oncoproteins. Conversely, YAP/TAZ directly induce the expression of KIRREL1 in a TEAD1-4-dependent manner. Indeed, KIRREL1 expression positively correlates with canonical YAP/TAZ target gene expression in clinical tumor specimens and predicts poor prognosis. Moreover, transgenic expression of KIRREL1 effectively blocks tumorigenesis in a mouse intrahepatic cholangiocarcinoma model, indicating a tumor-suppressor role of KIRREL1. Hence, KIRREL1 constitutes a negative feedback mechanism regulating the Hippo pathway and serves as a cell-surface marker and potential drug target in cancers with YAP/TAZ dependency.

Dephosphorylation of Y228 and Y217 and phosphorylation of Y335 in p120 catenin activate convergent extension during zebrafish gastrulation.

BACKGROUND: The cadherin-associated protein p120 catenin regulates convergent extension through interactions with cadherin proteins, Cdc42, and Rac1, as we previously showed in zebrafish (Danio rerio). Phosphorylation of p120 catenin changes the nature of its activity in vitro but is virtually unexplored in embryos. We used our previously developed antisense RNA splice-site morpholino targeted to endogenous p120 catenin-δ1 to cause defects in axis elongation probing the functions of three p120 catenin tyrosine-phosphorylation sites in gastrulating zebrafish embryos. RESULTS: The morpholino-induced defects were rescued by co-injections with mouse p120 catenin-δ1-3A mRNAs mutated at residues Y228 and Y217 to a non-phosphorylatable phenylalanine (F) or mutated at residue Y335 to a phosphomimetic glutamic acid (E). Co-injection of the complementary mutations Y228E, Y217E, or Y335F mRNAs partially rescued embryos whereas dual mutation to Y228E-Y217E blocked rescue. Immunopurification showed Y228F mutant proteins preferentially interacted with Rac1, potentially promoting cell migration. In contrast, the phosphomimetic Y228E preferentially interacted with E-cadherin increasing adhesion. Both Y228F and Y335F strongly bind VAV2. CONCLUSIONS: p120 catenin serves dual roles during gastrulation of zebrafish. Phosphorylation and dephosphorylation of tyrosine residues Y217, Y228, and Y335 precisely balance cell adhesion and cell migration to facilitate somite compaction and axis elongation.

Phyllanthus taxodiifolius Beille Disrupted N-cadherin, Vimentin, Paxillin and Actin Stress Fibers in Glioblastoma.

OBJECTIVE: Glioblastoma is the most aggressive and lethal brain tumor in adults with highly invasive properties. In this present study, we explored the effects of Phyllanthus taxodiifolius Beille extract on molecules known to be hallmarks of aggressive glioblastoma including N-cadherin and vimentin, mesenchymal markers, as well as paxillin, a major adaptor protein that regulates the linking of focal adhesions to the actin cytoskeleton. METHODS: P. taxodiifolius were air-dried, powdered and percolated with methanol, filtered, concentrated and lyophilized to yield a crude methanol extract. C6 glioblastoma cell line was used in this study. The expression of N-cadherin and vimentin, as well as the activation of paxillin was determined using Western blot analysis. The effect of the extract on focal adhesions and actin cytoskeleton were investigated using immunofluorescence staining and confocal imaging. RESULTS: In the presence of 40 µg/ml Phyllanthus taxodiifolius Beille extract, the expression of N-cadherin and vimentin were significantly decreased (p<0.001 and p<0.05, respectively). Activation of paxillin was also diminished as indicated by a reduction of phosphorylated-paxillin (p<0.01). Consequently, actin stress fibers in glioblastoma cells were abolished as evidenced by the decrease in focal adhesion (p<0.001) and stress fibers numbers (p<0.001). CONCLUSION: Our study demonstrates for the first time that P. taxodiifolius interferes with multiple key molecules related to pathological hallmarks of glioblastoma. These molecules are involved with cell contacts, focal adhesions, and the formation and stabilization of actin stress fibers, which are required for glioblastoma metastatic behavior. These results provide further evidence supporting the potential of P. taxodiifolius and its bioactive compounds as anti-cancer agents.

Proteomic and Phosphoproteomic Analyses Reveal a Complex Network Regulating Pollen Abortion and Potential Candidate Proteins in TCMS Wheat.

Thermosensitive sterile lines are natural materials for exploring the effects of anther development on male fertility. To study the possible molecular mechanisms regulating protein activity during the induction of male sterility, proteomic and phosphoproteomic analyses with tandem mass tags (TMTs) were used to study the binucleate anther of the thermosensitive sterile wheat line YS3038. A total of 9072 proteins, including 5019 phosphoproteins, were identified. Enrichment analyses of differentially abundant proteins (DAPs) and phosphoproteins (DAPPs) in metabolic pathways showed that both were mainly related to energy metabolism. Soluble sugar and ATP content were significantly decreased, free fatty acid content was significantly increased, and ROS was abnormally accumulated in male sterile YS3038-A. In addition, 233 kinase-substrate pairs involved in potential phosphorylation control networks were predicted to regulate fertility. Candidate proteins were identified, and a quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to validate the TMT results. TaPDCD5 is likely to be involved in fertility conversion of YS3038 by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS). Our data provide new insights into the mechanism of TCMS, which has value for identifying potential candidate proteins associated with the formation or abortion of pollen and promotion of wheat heterosis utilization.

Potential role of green tea amino acid l-theanine in the activation of innate immune response by enhancing expression of cytochrome b558 responsible for the reactive oxygen species-generating ability of leukocytes.

l-Theanine (N-ethyl- l-glutamine) is an analog of l-glutamine and l-glutamic acid, accounts for up to 50% of all free amino acids in green tea, and elicits an umami taste. As l-theanine also shows various physiological activities including immune response-modifying activities, it is expected to be an excellent health-promoting phytochemical agent. To know the influences of l-theanine on the human innate immune response, we investigated the effect of l-theanine on the superoxide anion (O2 - )-generating system of leukocytes using U937 cells. The O2 - -generating system in leukocytes consists of membrane cytochrome b558 protein (a complex of p22-phox and gp91-phox proteins) and cytosolic p40-phox, p47-phox, and p67-phox proteins. Addition of 500 µM l-theanine caused remarkable enhancement of the all-trans retinoic acid (ATRA)-induced O2 - -generating activity (to ~470% of ATRA-treated cells), but not l-glutamine and l-glutamic acid. Semiquantitative RT-PCR showed that the transcription level of gp91-phox is significantly increased in ATRA and l-theanine-co-treated cells. Chromatin immunoprecipitation revealed that l-theanine enhances acetylations of Lys-9 and Lys-14 residues of histone H3 within the chromatin surrounding the promoter region of the gp91-phox gene. Immunoblotting demonstrated that membrane cytochrome b558 proteins remarkably accumulate in ATRA + l-theanine-treated cells. These results suggested that l-theanine brings about a remarkable accumulation of cytochrome b558 protein via upregulating the transcription of the gp91-phox gene during leukocyte differentiation, resulting in marked augmentation of the O2 - -generating ability, which is one of the most important functions of leukocytes responsible for the innate immune system.

Transcriptional repression of estrogen receptor alpha by YAP reveals the Hippo pathway as therapeutic target for ER+ breast cancer.

Extensive knowledge has been gained on the transcription network controlled by ERα, however, the mechanism underlying ESR1 (encoding ERα) expression is less understood. We recently discovered that the Hippo pathway is required for the proper expression of ESR1. YAP/TAZ are transcription coactivators that are phosphorylated and inhibited by the Hippo pathway kinase LATS. Here we delineated the molecular mechanisms underlying ESR1 transcription repression by the Hippo pathway. Mechanistically, YAP binds to TEAD to increase local chromatin accessibility to stimulate transcription of nearby genes. Among the YAP target genes, Vestigial-Like Protein 3 (VGLL3) competes with YAP/TAZ for binding to TEAD transcription factor and recruits the NCOR2/SMRT repressor to the super-enhancer of ESR1 gene, leading to epigenetic alteration and transcriptional silencing. We developed a potent LATS inhibitor VT02956. Targeting the Hippo pathway by VT02956 represses ESR1 expression and inhibits the growth of ER+ breast cancer cells as well as patient-derived tumour organoids. Moreover, histone deacetylase inhibitors, such as Entinostat, induce VGLL3 expression to inhibit ER+ breast cancer cells. Our study suggests LATS as unexpected cancer therapeutic targets, especially for endocrine-resistant breast cancers.

Novel protein kinase C phosphorylated kinase inhibitor-matrine suppresses replication of hepatitis B virus via modulating the mitogen-activated protein kinase signal.

HBV (hepatitis B virus) infection still threatens human health. Therefore, it is essential to find new effective anti-HBV compounds. Here, we identified matrine as a novel inhibitor of PKC (protein kinase C) phosphorylated kinase by screening a natural compound library. After HepG2.215 cells were treated with matrine, we carried out a phosphorylated proteomics sequence study and analyzed the prediction of related kinase expression level. In the case of HBV infection, it was found that PKC kinase mediates the activation of mitogen-activated protein kinase (MAPK) signaling pathway known as son of sevenless (SOS) activation. It was also found that PKC kinase inhibits the expression of C-X-C Motif Chemokine Ligand 8 (CXCL8) by inhibiting the activity of activating transcription factor 2/ cAMP response element binding protein (ATF2/CREB), and this effect is independent of its activated MAPK signaling pathway. Finally, Western blot was used to detect the expression of MAPK, ATF2, CREB3 phosphorylation and nonphosphorylation in matrine-treated cells and PKC-treated cells. PKC phosphorylated kinase inhibitor-matrine suppresses the replication of HBV via modulating the MAPK/ATF2 signal. Matrine is a good clinical drug to enhance the autoimmunity in the adjuvant treatment of chronic HBV infection.

A critical review of in vitro research methodologies used to study mineralization in human dental pulp cell cultures.

BACKGROUND: The pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains. OBJECTIVES: The purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes. METHOD: The literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review. RESULTS: Most published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta-glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21-days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein-1 (DMP -1), the most frequent. DISCUSSION: While this review highlights variability among experimental approaches, it does however identify a consensus experimental approach. CONCLUSION: Standardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

Curcumin by activation of adenosine A2A receptor stimulates protein kinase a and potentiates inhibitory effect of cangrelor on platelets.

Curcumin is a natural polyphenol derived from the turmeric plant (Curcuma longa) which exhibits numerous beneficial effects on different cell types. Inhibition of platelet activation by curcumin is well known, however molecular mechanisms of its action on platelets are not fully defined. In this study, we used laser diffraction method for analysis of platelet aggregation and Western blot for analysis of intracellular signaling mechanisms of curcumin effects on platelets. We identified two new molecular mechanisms involved in the inhibitory effects of curcumin on platelet activation. Firstly, curcumin by activation of adenosine A2A receptor stimulated protein kinase A activation and phosphorylation of Vasodilator-stimulated phosphoprotein. Secondly, we demonstrated that curcumin even at low doses, which did not inhibit platelet aggregation, potentiated inhibitory effect of ADP receptor P2Y12 antagonist cangrelor which partly could be explained by activation of adenosine A2A receptor.

Rutaecarpine, an Alkaloid from Evodia rutaecarpa, Can Prevent Platelet Activation in Humans and Reduce Microvascular Thrombosis in Mice: Crucial Role of the PI3K/Akt/GSK3ß Signal Axis through a Cyclic Nucleotides/VASP-Independent Mechanism.

The role of activated platelets in acute and chronic cardiovascular diseases (CVDs) is well established. Therefore, antiplatelet drugs significantly reduce the risk of severe CVDs. Evodia rutaecarpa (Wu-Chu-Yu) is a well-known Chinese medicine, and rutaecarpine (Rut) is a main bioactive component with substantial beneficial properties including vasodilation. To address a research gap, we investigated the inhibitory mechanisms of Rut in washed human platelets and experimental mice. At low concentrations (1-5 µM), Rut strongly inhibited collagen-induced platelet aggregation, whereas it exerted only a slight or no effect on platelets stimulated with other agonists (e.g., thrombin). Rut markedly inhibited P-selectin expression; adenosine triphosphate release; [Ca2+]i mobilization; hydroxyl radical formation; and phospholipase C (PLC)γ2/protein kinase C (PKC), mitogen-activated protein kinase, and phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3ß (GSK3ß) phosphorylation stimulated by collagen. SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor) did not reverse Rut-mediated antiplatelet aggregation. Rut was not directly responding to vasodilator-stimulated phosphoprotein phosphorylation. Rut significantly increased the occlusion time of fluorescence irradiated thrombotic platelet plug formation. The findings demonstrated that Rut exerts a strong effect against platelet activation through the PLCγ2/PKC and PI3K/Akt/GSK3ß pathways. Thus, Rut can be a potential therapeutic agent for thromboembolic disorders.

In Vitro Antiplatelet Activity of Mulberroside C through the Up-Regulation of Cyclic Nucleotide Signaling Pathways and Down-Regulation of Phosphoproteins.

Physiological agonists trigger signaling cascades, called "inside-out signaling", and activated platelets facilitate adhesion, shape change, granule release, and structural change of glycoprotein IIb/IIIa (αIIb/ß3). Activated αIIb/ß3 interacts with fibrinogen and begins second signaling cascades called "outside-in signaling". These two signaling pathways can lead to hemostasis or thrombosis. Thrombosis can occur in arterial and venous blood vessels and is a major medical problem. Platelet-mediated thrombosis is a major cause of cardiovascular disease (CVD). Therefore, controlling platelet activity is important for platelet-mediated thrombosis and cardiovascular diseases. In this study, focus on Morus Alba Linn, a popular medicinal plant, to inhibit the function of platelets and found the containing component mulberroside C. We examine the effect of mulberroside C on the regulation of phosphoproteins, platelet-activating factors, and binding molecules. Agonist-induced human platelet aggregation is dose-dependently inhibited by mulberroside C without cytotoxicity, and it decreased Ca2+ mobilization and p-selectin expression through the upregulation of inositol 1, 4, 5-triphosphate receptor I (Ser1756), and downregulation of extracellular signal-regulated kinase (ERK). In addition, mulberroside C inhibited thromboxane A2 production, fibrinogen binding, and clot retraction. Our results show antiplatelet effects and antithrombus formation of mulberroside C in human platelets. Thus, we confirm that mulberroside C could be a potential phytochemical for the prevention of thrombosis-mediated CVDs.

HD2-type histone deacetylases: unique regulators of plant development and stress responses.

Plants have developed sophisticated and complex epigenetic regulation-based mechanisms to maintain stable growth and development under diverse environmental conditions. Histone deacetylases (HDACs) are important epigenetic regulators in eukaryotes that are involved in the deacetylation of lysine residues of histone H3 and H4 proteins. Plants have developed a unique HDAC family, HD2, in addition to the RPD3 and Sir2 families, which are also present in other eukaryotes. HD2s are well conserved plant-specific HDACs, which were first identified as nucleolar phosphoproteins in maize. The HD2 family plays important roles not only in fundamental developmental processes, including seed germination, root and leaf development, floral transition, and seed development but also in regulating plant responses to biotic and abiotic stresses. Some of the HD2 members coordinate with each other to function. The HD2 family proteins also show functional association with RPD3-type HDACs and other transcription factors as a part of repression complexes in gene regulatory networks involved in environmental stress responses. This review aims to analyse and summarise recent research progress in the HD2 family, and to describe their role in plant growth and development and in response to different environmental stresses.

Phosphoproteomic Analysis of Subcutaneous and Omental Adipose Tissue Reveals Increased Lipid Turnover in Dairy Cows Supplemented with Conjugated Linoleic Acid.

Phosphoproteomics is a cutting-edge technique that can be utilized to explore adipose tissue (AT) metabolism by quantifying the repertoire of phospho-peptides (PP) in AT. Dairy cows were supplemented with conjugated linoleic acid (CLA, n = 5) or a control diet (CON, n = 5) from 63 d prepartum to 63 d postpartum; cows were slaughtered at 63 d postpartum and AT was collected. We performed a quantitative phosphoproteomics analysis of subcutaneous (SC) and omental (OM) AT using nanoUPLC-MS/MS and examined the effects of CLA supplementation on the change in the phosphoproteome. A total of 5919 PP were detected in AT, and the abundance of 854 (14.4%) were differential between CON and CLA AT (p ≤ 0.05 and fold change ± 1.5). The abundance of 470 PP (7.9%) differed between OM and SC AT, and the interaction treatment vs. AT depot was significant for 205 PP (3.5% of total PP). The integrated phosphoproteome demonstrated the up- and downregulation of PP from proteins related to lipolysis and lipogenesis, and phosphorylation events in multiple pathways, including the regulation of lipolysis in adipocytes, mTOR signaling, insulin signaling, AMPK signaling, and glycolysis. The differential regulation of phosphosite on a serine residue (S777) of fatty acid synthase (FASN) in AT of CLA-supplemented cows was related to lipogenesis and with more phosphorylation sites compared to acetyl-coenzyme A synthetase (ACSS2). Increased protein phosphorylation was seen in acetyl-CoA carboxylase 1 (ACACA;8 PP), FASN (9 PP), hormone sensitive lipase (LIPE;6 PP), perilipin (PLIN;3 PP), and diacylglycerol lipase alpha (DAGLA;1 PP) in CLA vs. CON AT. The relative gene expression in the SC and OM AT revealed an increase in LIPE and FASN in CLA compared to CON AT. In addition, the expression of DAGLA, which is a lipid metabolism enzyme related to the endocannabinoid system, was 1.6-fold higher in CLA vs. CON AT, and the expression of the cannabinoid receptor CNR1 was reduced in CLA vs. CON AT. Immunoblots of SC and OM AT showed an increased abundance of FASN and a lower abundance of CB1 in CLA vs. CON. This study presents a complete map of the SC and the OM AT phosphoproteome in dairy cows following CLA supplementation and discloses many unknown phosphorylation sites, suggestive of increased lipid turnover in AT, for further functional investigation.