Lipid Signaling Requires ROS Production to Elicit Actin Cytoskeleton Remodeling during Plant Innate Immunity

In terrestrial plants a basal innate immune system, pattern-triggered immunity (PTI), has evolved to limit infection by diverse microbes. The remodeling of actin cytoskeletal arrays is now recognized as a key hallmark event during the rapid host cellular responses to pathogen attack. Several actin binding proteins have been demonstrated to fine tune the dynamics of actin filaments during this process. However, the upstream signals that stimulate actin remodeling during PTI signaling remain poorly characterized. Two second messengers, reactive oxygen species (ROS) and phosphatidic acid (PA), are elevated following pathogen perception or microbe-associated molecular pattern (MAMP) treatment, and the timing of signaling fluxes roughly correlates with actin cytoskeletal rearrangements. Here, we combined genetic analysis, chemical complementation experiments, and quantitative live-cell imaging experiments to test the role of these second messengers in actin remodeling and to order the signaling events during plant immunity. We demonstrated that PHOSPHOLIPASE Dβ (PLDβ) isoforms are necessary to elicit actin accumulation in response to flg22-associated PTI. Further, bacterial growth experiments and MAMP-induced apoplastic ROS production measurements revealed that PLDβ-generated PA acts upstream of ROS signaling to trigger actin remodeling through inhibition of CAPPING PROTEIN (CP) activity. Collectively, our results provide compelling evidence that PLDβ/PA functions upstream of RBOHD-mediated ROS production to elicit actin rearrangements during the innate immune response in Arabidopsis.     

Anti-Inflammatory Effect of Allicin Associated with Fibrosis in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling. Recent evidence supports that inflammation plays a key role in triggering and maintaining pulmonary vascular remodeling. Recent studies have shown that garlic extract has protective effects in PAH, but the precise role of allicin, a compound derived from garlic, is unknown. Thus, we used allicin to evaluate its effects on inflammation and fibrosis in PAH. Male Wistar rats were divided into three groups: control (CON), monocrotaline (60 mg/kg) (MCT), and MCT plus allicin (16 mg/kg/oral gavage) (MCT + A). Right ventricle (RV) hypertrophy and pulmonary arterial medial wall thickness were determined. IL-1β, IL-6, TNF-α, NFκB p65, Iκβ, TGF-β, and α-SMA were determined by Western blot analysis. In addition, TNF-α and TGF-β were determined by immunohistochemistry, and miR-21-5p and mRNA expressions of Cd68, Bmpr2, and Smad5 were determined by RT-qPCR. Results: Allicin prevented increases in vessel wall thickness due to TNF-α, IL-6, IL-1β, and Cd68 in the lung. In addition, TGF-β, α-SMA, and fibrosis were lower in the MCT + A group compared with the MCT group. In the RV, allicin prevented increases in TNF-α, IL-6, and TGF-β. These observations suggest that, through the modulation of proinflammatory and profibrotic markers in the lung and heart, allicin delays the progression of PAH.     

MAP3Kε1/2 Interact with MOB1A/1B and Play Important Roles in Control of Pollen Germination through Crosstalk with JA Signaling in Arabidopsis

Restriction of pollen germination before the pollen grain is pollinated to stigma is essential for successful fertilization in angiosperms. However, the mechanisms underlying the process remain poorly understood. Here, we report functional characterization of the MAPKKK kinases, MAP3Kε1 and MAP3Kε2, involve in control of pollen germination in Arabidopsis. The two genes were expressed in different tissues with higher expression levels in the tricellular pollen grains. The map3kε1 map3kε2 double mutation caused abnormal callose accumulation, increasing level of JA and precocious pollen germination, resulting in significantly reduced seed set. Furthermore, the map3kε1 map3kε2 double mutations obviously upregulated the expression levels of genes in JA biosynthesis and signaling. The MAP3Kε1/2 interacted with MOB1A/1B which shared homology with the core components of Hippo singling pathway in yeast. The Arabidopsis mob1a mob1b mutant also exhibited a similar phenotype of precocious pollen germination to that in map3kε1 map3kε2 mutants. Taken together, these results suggested that the MAP3Kεs interacted with MOB1s and played important role in restriction of the precocious pollen germination, possibly through crosstalk with JA signaling and influencing callose accumulation in Arabidopsis.     

Adjuvant Immune Enhancement of Subunit Vaccine Encoding pSCPI of Streptococcus iniae in Channel Catfish (Ictalurus punctatus)

Channel catfish (Ictalurus punctatus) is an important agricultural fish that has been plagued by Streptococcus iniae (S. iniae) infections in recent years, some of them severe. C5a peptidase is an important virulent factor of S. iniae. In this study, the subunit vaccine containing the truncated part of C5a peptidase (pSCPI) was mixed with aluminum hydroxide gel (AH), propolis adjuvant (PA), and Freund’s Incomplete Adjuvant (FIA). The immunogenicity of the pSCPI was detected by Western-blot in vitro. The relative percent survival (RPS), lysozyme activity, antibody titers, and the expression of the related immune genes were monitored in vivo to evaluate the immune effects of the three different adjuvants. The results showed that pSCPI exerted moderate immune protection (RPS = 46.43%), whereas each of the three adjuvants improved the immune protection of pSCPI. The immunoprotection of pSCPI + AH, pSCPI + PA, and pSCPI + FIA was characterized by RPS values of 67.86%, 75.00% and, 85.71%, respectively. Further, each of the three different adjuvanted pSCPIs stimulated higher levels of lysozyme activity and antibody titers than the unadjuvanted pSCPI and/or PBS buffer. In addition, pSCPI + FIA and pSCPI + PA induced expression of the related immune genes under investigation, which was substantially higher than the levels stimulated by PBS. pSCPI + AH significantly stimulated the induction of MHC II β, CD4-L2, and IFN-γ, while it induced slightly higher production of TNF-α and even led to a decrease in the levels of IL-1β, MHC I α, and CD8 α. Therefore, we conclude that compared with the other two adjuvants, FIA combined with pSCPI is a more promising candidate adjuvant against S. iniae in channel catfish.     

miR-29b Regulates TGF-β1-Induced Epithelial–Mesenchymal Transition by Inhibiting Heat Shock Protein 47 Expression in Airway Epithelial Cells

Tissue remodeling contributes to ongoing inflammation and refractoriness of chronic rhinosinusitis (CRS). During this process, epithelial-mesenchymal transition (EMT) plays an important role in dysregulated remodeling and both microRNA (miR)-29b and heat shock protein 47 (HSP47) may be engaged in the pathophysiology of CRS. This study aimed to determine the role of miR-29b and HSP47 in modulating transforming growth factor (TGF)-β1-induced EMT and migration in airway epithelial cells. Expression levels of miR-29b, HSP47, E-cadherin, α-smooth muscle actin (α-SMA), vimentin and fibronectin were assessed through real-time PCR, Western blotting, and immunofluorescence staining. Small interfering RNA (siRNA) targeted against miR-29b and HSP47 were transfected to regulate the expression of EMT-related markers. Cell migration was evaluated with wound scratch and transwell migration assay. miR-29b mimic significantly inhibited the expression of HSP47 and TGF-β1-induced EMT-related markers in A549 cells. However, the miR-29b inhibitor more greatly induced the expression of them. HSP47 knockout suppressed TGF-β1-induced EMT marker levels. Functional studies indicated that TGF-β1-induced EMT was regulated by miR-29b and HSP47 in A549 cells. These findings were further verified in primary nasal epithelial cells. miR-29b modulated TGF-β1-induced EMT-related markers and migration via HSP47 expression modulation in A549 and primary nasal epithelial cells. These results suggested the importance of miR-29b and HSP47 in pathologic tissue remodeling progression in CRS.     

Lecithin Inclusion by α-Cyclodextrin Activates SREBP2 Signaling in the Gut and Ameliorates Postprandial Hyperglycemia

Background: α-cyclodextrin (α-CD) is one of the dietary fibers that may have a beneficial effect on cholesterol and/or glucose metabolism, but its efficacy and mode of action remain unclear. Methods: In the present study, we examined the anti-hyperglycemic effect of α-CD after oral loading of glucose and liquid meal in mice. Results: Administration of 2 g/kg α-CD suppressed hyperglycemia after glucose loading, which was associated with increased glucagon-like peptide 1 (GLP-1) secretion and enhanced hepatic glucose sequestration. By contrast, 1 g/kg α-CD similarly suppressed hyperglycemia, but without increasing secretions of GLP-1 and insulin. Furthermore, oral α-CD administration disrupts lipid micelle formation through its inclusion of lecithin in the gut luminal fluid. Importantly, prior inclusion of α-CD with lecithin in vitro nullified the anti-hyperglycemic effect of α-CD in vivo, which was associated with increased intestinal mRNA expressions of SREBP2-target genes (Ldlr, Hmgcr, Pcsk9, and Srebp2). Conclusions: α-CD elicits its anti-hyperglycemic effect after glucose loading by inducing lecithin inclusion in the gut lumen and activating SREBP2, which is known to induce cholecystokinin secretion to suppress hepatic glucose production via a gut/brain/liver axis.     

Methyl Brevifolincarboxylate Attenuates Free Fatty Acid-Induced Lipid Metabolism and Inflammation in Hepatocytes through AMPK/NF-κB Signaling Pathway

The prevalence of non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver diseases worldwide. This study examined the potential protective effects of a naturally occurring polyphenolic compound, methyl brevifolincarboxylate (MBC) on fatty liver injury in vitro. The results showed that MBC at its non-cytotoxic concentrations, reduced lipid droplet accumulation and triglyceride (TG) levels in the oleic acid (OA)-treated human hepatocarcinoma cell line, SK-HEP-1 and murine primary hepatocytes. In OA-treated SK-HEP-1 cells and primary murine hepatocytes, MBC attenuated the mRNA expression levels of the de novo lipogenesis molecules, acetyl-coenzyme A carboxylase (Acc1), fatty acid synthase (Fasn) and sterol regulatory element binding protein 1c (Srebp1c). MBC promoted the lipid oxidation factor peroxisome proliferator activated receptor-α (Pparα), and its target genes, carnitine palmitoyl transferase 1 (Cpt1) and acyl-coenzyme A oxidase 1 (Acox1) in both the SK-HEP-1 cells and primary murine hepatocytes. The mRNA results were further supported by the attenuated protein expression of lipogenesis and lipid oxidation molecules in OA-treated SK-HEP-1 cells. The MBC increased the expression of AMP activated protein kinase (AMPK) phosphorylation. On the other hand, MBC treatment dampened the inflammatory mediator’s, tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), IL-8, and IL-1β secretion, and nuclear factor (NF)-κB expression (mRNA and protein) through reduced reactive oxygen species production in OA-treated SK-HEP-1 cells. Taken together, our results demonstrated that MBC possessed potential protective effects against NAFLD in vitro by amelioration of lipid metabolism and inflammatory markers through the AMPK/NF-κB signaling pathway.     

Molecular Identification of the G-Protein Genes and Their Expression Profiles in Response to Nitrogen Deprivation in Brassica napus

Heterotrimeric guanine nucleotide binding protein (G-protein) consisting of Gα, Gβ, and Gγ subunits is one of the key signal transducers in plants. Recent studies indicated that G-protein has been proposed as an important mediator of nitrogen responses in rice, wheat, and Arabidopsis. However, little is known about these G-proteins in Brassica napus (B. napus), except for three identified G-proteins, BnGA1, BnGB1, and BnGG2. Therefore, the aim of the present study is to characterize the members of the G-protein gene family in allotetraploid B. napus and to analyze their expression profiles in response to nitrogen deprivation. In total, 21 G-protein family members were identified in B. napus, encoding two Gα, six Gβ, and 13 Gγ. Sequence and phylogenetic analyses showed that although genome-wide triploid events increased the number of genes encoding Gα, Gβ, and Gγ subunits, the gene structure and protein properties of the genes encoding each G-protein subunit were extremely conserved. Collinearity analysis showed that most G-protein genes in B. napus had syntenic relationships with G-protein members of Arabidopsis, Brassica rape (B. rapa), and Brassica oleracea (B. oleracea). Expression profile analysis indicated that Gα and C-type Gγ genes (except BnGG10 and BnGG12 were highly expressed in flower and ovule) were barely expressed in most organs, whereas most Gβ and A-type Gγ genes tended to be highly expressed in most organs. G-protein genes also showed various expression patterns in response to nitrogen-deficient conditions. Under nitrogen deficiency, Gα and five C-type Gγ genes were upregulated initially in roots, while in leaves, Gα was downregulated initially and five C-type Gγ genes were highly expressed in different times. These results provide a complex genetic dissection of G-protein genes in B. napus, and insight into the biological functions of G-protein genes in response to nitrogen deficiency.     

Overexpression of Peroxisome-Localized GmABCA7 Promotes Seed Germination in Arabidopsis thaliana

Peroxisome is one of the important organelles for intracellular lipid metabolism in plant cells and β-oxidation of fatty acids in peroxisomes provides the energy for oil-containing seed germination. In this study, we identified an ATP-binding cassette (ABC) transporter gene, GmABCA7 from soybean, which is highly expressed in the different developmental stages of seeds. Transient expression of GmABCA7 in tobacco epidermal cells showed that GmABCA7 was specifically localized at the peroxisomes. Overexpression of GmABCA7 in Arabidopsis does not change seed phenotypes, or the overall levels of lipid, protein and sugar stored in the seeds; however, the transgenic seeds produced more gluconeogenic pathway precursors such as succinate and malate and germinated earlier compared to the wild type seeds. These results suggest that GmABCA7 may affect the β-oxidation of fatty acids and play an important role in seed germination.     

Suppression of PGC-1α Drives Metabolic Dysfunction in TGFβ2-Induced EMT of Retinal Pigment Epithelial Cells

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFβ2) suppressed PGC-1α expression. Correspondingly, TGFβ2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.     

Interactive Effects of Dietary Lipid and Phenotypic Feed Efficiency on the Expression of Nuclear and Mitochondrial Genes Involved in the Mitochondrial Electron Transport Chain in Rainbow Trout

A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on the expression of mitochondrial and nuclear genes involved in electron transport chain in all-female rainbow trout Oncorhynchus mykiss. Three practical diets with a fixed crude protein content of 40%, formulated to contain 10% (40/10), 20% (40/20) and 30% (40/30) dietary lipid, were fed to apparent satiety to triplicate groups of either low-feed efficient (F120; 217.66 ± 2.24 g initial average mass) or high-feed efficient (F136; 205.47 ± 1.27 g) full-sib families of fish, twice per day, for 90 days. At the end of the experiment, the results showed that there is an interactive effect of the dietary lipid levels and the phenotypic feed efficiency (growth rate and feed efficiency) on the expression of the mitochondrial genes nd1 (NADH dehydrogenase subunit 1), cytb (Cytochrome b), cox1 (Cytochrome c oxidase subunits 1), cox2 (Cytochrome c oxidase subunits 2) and atp6 (ATP synthase subunit 6) and nuclear genes ucp2α (uncoupling proteins 2 alpha), ucp2β (uncoupling proteins 2 beta), pparα (peroxisome proliferator-activated receptor alpha), pparβ (peroxisome proliferatoractivated receptor beta) and ppargc1α (proliferator-activated receptor gamma coactivator 1 alpha) in fish liver, intestine and muscle, except on ppargc1α in the muscle which was affected by the diet and the family separately. Also, the results revealed that the expression of mitochondrial genes is associated with that of nuclear genes involved in electron transport chain in fish liver, intestine and muscle. Furthermore, this work showed that the expression of mitochondrial genes parallels with the expression of genes encoding uncoupling proteins (UCP) in the liver and the intestine of rainbow trout. This study for the first time presents the molecular basis of the effects of dietary lipid level on mitochondrial and nuclear genes involved in mitochondrial electron transport chain in fish.     

Identification of the High Molecular Weight Isoform of Phostensin

Phostensin is encoded by KIAA1949. 5′-RACEanalysis has been used to identify the translation start site of phostensin mRNA, indicating that it encodes 165 amino acids with an apparent molecular weight of 26 kDa on SDS-PAGE. This low-molecular-weight phostensin is present in human peripheral blood mononuclear cells and many leukemic cell lines. Phostensin is a protein phosphatase-1(PP1) binding protein. It also contains one actin-binding motif at its C-terminal region and binds to the pointed ends of actin filaments, modulating actin dynamics. In the current study, a high-molecular-weight phostensin is identified by using immunoprecipitationin combination with a proteomic approach. This new species of phostensin is also encoded by KIAA1949 and consists of 613 amino acids with an apparent molecular weight of 110 kDa on SDS-PAGE. The low-molecular-weight and high-molecular-weight phostensins were named as phostensin-α and phostensin-β, respectively. Although phostensin-α is the C-terminal region of phostensin-β, it is not degraded from phostensin-β. Phostensin-β is capable of associating with PP1 and actin filaments, and is present in many cell lines.     

Preparation and Characterization of a Chloroperoxidase-like Catalytic Antibody

The small molecule, meso-tetra(α,α,α,α-o-phenylacetamidophenyl) porphyrin (Mr1147.0) was used as complete antigen to elicit MAb through the immunization and cell fusion techniques. The MAb 1F2 obtained was demonstrated to be very pure by MALDI/TOFMS. The subtype of MAb 1F2 is IgG2a, which has a relative molecular weight of 156,678.8 Da.No significant change in the intensity of absorption peaks in UV and CD spectra was observed over a pH range between 6 and 12. The high stability of the abzyme and the tight binding between Fe porphyrin and antibody were also demonstrated. V_max, K_m, κcat, κcat/K_m for abzyme are 5.18 × 10⁻⁸ Ms⁻¹, 1.50 × 10⁻⁸ M, 0.518 s⁻¹, 3.45 × 10⁷ M⁻¹s⁻¹, respectively. The data obtained indicate that catalytic antibody has high catalytic activity. The chloroperoxidase activity of MAb 1F2-Fe porphyrin complex is stable from 10 °C to 60 °C.     

The Role of PTP1B O-GlcNAcylation in Hepatic Insulin Resistance

Protein tyrosine phosphatase 1B (PTP1B), which can directly dephosphorylate both the insulin receptor and insulin receptor substrate 1 (IRS-1), thereby terminating insulin signaling, reportedly plays an important role in insulin resistance. Accumulating evidence has demonstrated that O-GlcNAc modification regulates functions of several important components of insulin signal pathway. In this study, we identified that PTP1B is modified by O-GlcNAcylation at three O-GlcNAc sites (Ser104, Ser201, and Ser386). Palmitate acid (PA) impaired the insulin signaling, indicated by decreased phosphorylation of both serine/threonine-protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) following insulin administration, and upregulated PTP1B O-GlcNAcylation in HepG2 cells. Compared with the wild-type, intervention PTP1B O-GlcNAcylation by site-directed gene mutation inhibited PTP1B phosphatase activity, resulted in a higher level of phosphorylated Akt and GSK3β, recovered insulin sensitivity, and improved lipid deposition in HepG2 cells. Taken together, our research showed that O-GlcNAcylation of PTP1B can influence insulin signal transduction by modulating its own phosphatase activity, which participates in the process of hepatic insulin resistance.