Caveolin-1-derived peptide attenuates cigarette smoke-induced airway and alveolar epithelial injury.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease with no effective treatment that can reduce mortality or slow the disease progression. COPD is the third leading cause of global death and is characterized by airflow limitations due to chronic bronchitis and alveolar damage/emphysema. Chronic cigarette smoke (CS) exposure damages airway and alveolar epithelium and remains a major risk factor for the pathogenesis of COPD. We found that the expression of caveolin-1, a tumor suppressor protein; p53; and plasminogen activator inhibitor-1 (PAI-1), one of the downstream targets of p53, was markedly increased in airway epithelial cells (AECs) as well as in type II alveolar epithelial (AT2) cells from the lungs of patients with COPD or wild-type mice with CS-induced lung injury (CS-LI). Moreover, p53- and PAI-1-deficient mice resisted CS-LI. Furthermore, treatment of AECs, AT2 cells, or lung tissue slices from patients with COPD or mice with CS-LI with a seven amino acid caveolin-1 scaffolding domain peptide (CSP7) reduced mucus hypersecretion in AECs and improved AT2 cell viability. Notably, induction of PAI-1 expression via increased caveolin-1 and p53 contributed to mucous cell metaplasia and mucus hypersecretion in AECs, and reduced AT2 viability, due to increased senescence and apoptosis, which was abrogated by CSP7. In addition, treatment of wild-type mice having CS-LI with CSP7 by intraperitoneal injection or nebulization via airways attenuated mucus hypersecretion, alveolar injury, and significantly improved lung function. This study validates the potential therapeutic role of CSP7 for treating CS-LI and COPD. NEW & NOTEWORTHY Chronic cigarette smoke (CS) exposure remains a major risk factor for the pathogenesis of COPD, a debilitating disease with no effective treatment. Increased caveolin-1 mediated induction of p53 and downstream plasminogen activator inhibitor-1 (PAI-1) expression contributes to CS-induced airway mucus hypersecretion and alveolar wall damage. This is reversed by caveolin-1 scaffolding domain peptide (CSP7) in preclinical models, suggesting the therapeutic potential of CSP7 for treating CS-induced lung injury (CS-LI) and COPD.

Genome-wide identification of MATE, functional analysis and molecular dynamics of DcMATE21 involved in anthocyanin accumulation in Daucus carota.

Anthocyanins are a subclass of flavonoids that are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Multidrug and toxic compound extrusion transporters (MATE) is a family of membrane transporters that transport ions and secondary metabolites, such as anthocyanins, in plants. Although various studies on MATE transporters have been carried out on different plant species, this is the first comprehensive report to mine the Daucus carota genome to identify the MATE gene family. Our study identified 45 DcMATEs through genome-wide analysis and detected five segmental and six tandem duplications from the genome. The chromosome distribution, phylogenetic analysis, and cis-regulatory elements revealed the structural diversity and numerous functions associated with the DcMATEs. In addition, we analyzed RNA-seq data obtained from the European Nucleotide Archive to screen for the expression of DcMATEs involved in anthocyanin biosynthesis. Among the identified DcMATEs, DcMATE21 correlated with anthocyanin content in the different D. carota varieties. In addition, the expression of DcMATE21 and anthocyanin biosynthesis genes was correlated under abscisic acid, methyl jasmonate, sodium nitroprusside, salicylic acid, and phenylalanine treatments, which were substantiated by anthocyanin accumulation in the in vitro cultures. Further molecular membrane dynamics of DcMATE21 with anthocyanin (cyanidin-3-glucoside) identified the binding pocket, showing extensive H-bond interactions with 10 crucial amino acids present in the transmembrane helix of 7, 8, and 10 of DcMATE21. The current investigation, using RNA-seq, in vitro cultures, and molecular dynamics studies revealed the involvement of DcMATE21 in anthocyanin accumulation in vitro cultures of D. carota.

miR-7-5p Antagomir Protects Against Inflammation-Mediated Apoptosis and Lung Injury via Targeting Raf-1 In Vitro and In Vivo.

Exacerbated inflammation and apoptosis are considered upstream events associated with acute lung injury (ALI). microRNAs are critical regulators of genes responsible for inflammation and apoptosis and are considered potential therapeutic targets for ameliorating ALI. This study was undertaken to uncover the role of miR-7-5p in LPS-induced lung injury. A LPS-induced inflammation model was established using BEAS-2B cells and C57BL/6 mice. Bioinformatics analysis and the luciferase reporter assay confirmed that Raf-1 is a target of miR-7-5p and that its expression was inversely correlated with expression of proinflammatory markers and miR-7-5p, whereas miR-7-5p inhibition in vitro led to subsequent restoration of Raf-1 expression and prevention of apoptosis. Intranasal (i.n.) administration of antagomir using the C57BL/6 mouse model further confirmed that miR-7-5p inhibition suppresses LPS-induced inflammation and apoptosis via modulating the miR-7-5p/Raf-1 axis. Our findings indicate that blocking miR-7-5p expression by antagomir protects mice from LPS-induced lung injury by suppressing inflammation and activation of mitochondria-mediated survival signalling. In conclusion, our findings demonstrate a previously unknown pathophysiological role of miR-7-5p in the progression of ALI, and targeted i.n. administration of miR-7-5p antagomir could aid in the development of potential therapeutic strategies against lung injury.

Distinct GmASMTs are involved in regulating transcription factors and signalling cross-talk across embryo development, biotic, and abiotic stress in soybean.

N-Acetylserotonin O-methyltransferase (ASMT) is the final enzyme involved in melatonin biosynthesis. Identifying the expression of ASMT will reveal the regulatory role in the development and stress conditions in soybean. To identify and characterize ASMT in soybean (GmASMT), we employed genome-wide analysis, gene structure, cis-acting elements, gene expression, co-expression network analysis, and enzyme assay. We found seven pairs of segmental and tandem duplication pairs among the 44 identified GmASMTs by genome-wide analysis. Notably, co-expression network analysis reported that distinct GmASMTs are involved in various stress response. For example, GmASMT3, GmASMT44, GmASMT17, and GmASMT7 are involved in embryo development, heat, drought, aphid, and soybean cyst nematode infections, respectively. These distinct networks of GmASMTs were associated with transcription factors (NAC, MYB, WRKY, and ERF), stress signalling, isoflavone and secondary metabolites, calcium, and calmodulin proteins involved in stress regulation. Further, GmASMTs demonstrated auxin-like activities by regulating the genes involved in auxin transporter (WAT1 and NRT1/PTR) and auxin-responsive protein during developmental and biotic stress. The current study identified the key regulatory role of GmASMTs during development and stress. Hence GmASMT could be the primary target in genetic engineering for crop improvement under changing environmental conditions.

Folate derivatives, 5-methyltetrahydrofolate and 10-formyltetrahydrofolate, protect BEAS-2B cells from high glucose-induced oxidative stress and inflammation.

Folate (vitamin B9) and its biologically active derivatives are well-known antioxidant molecules protecting cells from oxidative degradation. The presence of high glucose, often found in diabetic patients, causes oxidative stress resulting in cellular stress and inflammatory injury. Cells in organs such as the lung are highly prone to inflammation, and various protective mechanisms exist to prevent the progressive disorders arising from inflammation. In the present study, the synthetic form of folate, i.e. folic acid, and active forms of folate, i.e. 5-methyltetrahydrofolate and 10-formyltetrahydrofolate, were evaluated for their antioxidant and antiinflammatory potential against high glucose (50 mM)-mediated oxidative stress and inflammation in BEAS-2B cells, an immortalised bronchial epithelial cell line. High glucose treatment showed a 67% reduction in the viability of BEAS-2B cells, which was restored to the viability levels seen in control cultures by the addition of active folate derivatives to the culture media. The DCFH-DA fluorometric assay was performed for oxidative stress detection. The high glucose-treated cells showed a significantly higher fluorescence intensity (1.81- and 3.8-fold for microplate assay and microscopic observation, respectively), which was normalised to control levels on supplementation with active folate derivatives. The proinflammatory NF-κB p50 protein expression in the active folate derivative-supplemented high glucose-treated cells was significantly lower compared to the folic acid treatment. In support of these findings, in silico microarray GENVESTIGATOR database analysis showed that in bronchiolar small airway epithelial cells exposed to inflammatory condition, folate utilization pathway genes are largely downregulated. However, the folate-binding protein gene, which encodes to the folate receptor 1 (FOLR1), is significantly upregulated, suggesting a high demand for folate by these cells  in inflammatory situations. Supplementation of the active folate derivatives 5-methyltetrahydrofolate and 10-formyltetrahydrofolate resulted in significantly higher protection over the folic acid from high glucose-induced oxidative stress and inflammation. Therefore, the biologically active folate derivatives could be a suitable alternative over the folic acid for alleviating inflammatory injury-causing oxidative stress.

Exogenous Serotonin and Melatonin Regulate Dietary Isoflavones Profoundly through Ethylene Biosynthesis in Soybean [Glycine max (L.) Merr.].

Serotonin and melatonin are important signaling and stress mitigating molecules. However, their role and molecular mechanism in the accumulation of isoflavones are not clearly defined. To elucidate their functions, serotonin and melatonin were applied to in vitro cultures of soybean at different concentrations and analyzed to assess the accumulation of isoflavone content followed by transcript levels of biosynthesis genes at different time intervals. Increased total phenolics, total flavonoids, and different forms of isoflavone content were observed in the treatments. Expression levels of critical genes in isoflavone, ethylene, jasmonic acid, abscisic acid, and melatonin biosynthesis and related transcription factor were quantified. A correlation was observed between the expression of ethylene biosynthesis genes (S-adenosylmethionine synthase and 1-aminocyclopropane-1-carboxylate oxidase) and isoflavone biosynthesis genes (chalcone synthase, chalcone reductase, and isoflavone synthase). We hypothesize that, under serotonin and melatonin treatments, ethylene biosynthesis may play a role in the increase/decrease in isoflavone content in soybean culture.

Suppression of N-glycan processing enzymes by deoxynojirimycin in tomato (Solanum lycopersicum) fruit.

The present study investigated the potential of a small molecule inhibitor, 1-deoxynojirimycin (DNJ), to extend the shelf life of tomatoes. The optimum concentration of DNJ and the proper ripening stage for treatment were standardized using response surface methodology, following a central composite design. The concentration of DNJ used for the analysis was 0.15 mM, and 0.30 mM and the ripening stages of the tomato fruit analysed were immature green, mature green, breaker, ripen and over-ripen. Analysis of the influence of the DNJ treatment of the fruit using quadratic multiple regression models considering the factors colour, texture, and free sugars revealed significant responses. A DNJ concentration of 0.30 mM and fruit-ripening stage of mature green was found to be optimal for the treatment. DNJ-treatment maintained fruit firmness throughout ripening with a significant reduction in reducing sugar formation. Enzyme activity of the N-glycan processing enzymes involved in cell wall softening, α-mannosidase and ß-d-N-acetylhexosaminidase revealed a significant reduction in their activity by 2 and 3.5-fold, respectively. Down-regulation of expression of important ripening-related and softening process-associated genes, aminocyclopropane carboxylic synthase-4, aminocyclopropane carboxylic oxidase, polygalacturonase and pectin methylesterases at 4, 5, 6 and 5-fold, respectively, was also observed. The present results showed that the treatment of mature green tomato fruit with DNJ at a concentration of 0.30 mM can delay the ripening of the tomato fruit by inhibiting cell wall and N-glycan processing enzymes.

Polyamine Induction in Postharvest Banana Fruits in Response to NO Donor SNP Occurs via l-Arginine Mediated Pathway and Not via Competitive Diversion of S-Adenosyl-l-Methionine.

Nitric oxide (NO) is known to antagonize ethylene by various mechanisms; one of such mechanisms is reducing ethylene levels by competitive action on S-adenosyl-L-methionine (SAM)-a common precursor for both ethylene and polyamines (PAs) biosynthesis. In order to investigate whether this mechanism of SAM pool diversion by NO occur towards PAs biosynthesis in banana, we studied the effect of NO on alterations in the levels of PAs, which in turn modulate ethylene levels during ripening. In response to NO donor sodium nitroprusside (SNP) treatment, all three major PAs viz. putrescine, spermidine and spermine were induced in control as well as ethylene pre-treated banana fruits. However, the gene expression studies in two popular banana varieties of diverse genomes, Nanjanagudu rasabale (NR; AAB genome) and Cavendish (CAV; AAA genome) revealed the downregulation of SAM decarboxylase, an intermediate gene involved in ethylene and PA pathway after the fifth day of NO donor SNP treatment, suggesting that ethylene and PA pathways do not compete for SAM. Interestingly, arginine decarboxylase belonging to arginine-mediated route of PA biosynthesis was upregulated several folds in response to the SNP treatment. These observations revealed that NO induces PAs via l-arginine-mediated route and not via diversion of SAM pool.

Effects of methyl jasmonate and carotenogenic inhibitors on gene expression and carotenoid accumulation in coriander (Coriandrum sativum L.) foliage.

Coriander (Coriandrum sativum L.), a commonly used annual herb that accumulates carotenoids upon methyl jasmonate (MeJA) treatment, provides an excellent model to investigate carotenogenesis and gene regulation. To explore key mechanisms involved in enhancing carotenoids, transcriptional expression profile of ten carotenogenic genes in the presence of MeJA and various gene specific inhibitors were investigated. Foliar application of MeJA (10 µM) increased expression levels of CsPDS (phytoene desaturase), CsZDS (ς-carotene desaturase), CsCHYE (carotene ε - hydroxylase) and CsLCYE (lycopene ß-cyclase) genes, and their transcript levels were strongly associated with carotenoid content, where, three days after treatment, 3.9 & 6.1 fold increase was observed for ß-carotene and lutein respectively. The regulatory effect of key genes, CsPDS, CsZDS, CsLCYE and LCYB were further confirmed by using gene-specific inhibitors fosmidomycin, norflurazon and amitrol. Norflurazon- the phytoene desaturation inhibitor leads to a decrease in ß-carotene and lutein content correlated with CsPDS, CsZDS gene induction. Our results clearly demonstrate that MeJA induced-signalling network evokes carotenogenic genes, leading to the accumulation of carotenoids. This knowledge may help to develop precise strategies for remodelling carotenoid pathway so that desired levels of a particular carotenoid in leafy vegetables is achievable.

Novel Folate Binding Protein in Arabidopsis Expressed during Salicylic Acid-Induced Folate Accumulation.

Increasing the quantity of natural folates in plant foods is recently gaining significant interest, owing to their acute deficiencies in various populations. This study observed that foliar salicylic acid treatment enhanced the accumulation of folates in Arabidopsis, which correlated with the increase in a folate binding protein (FBP) and the expression of mRNA of a putative folate binding protein At5G27830. A protein band corresponding to ∼43 kDa was observed after resolving the affinity-purified protein on SDS-PAGE, and the partial amino acid sequence indicated that the protein is indeed At5G27830. Docking studies performed with At5G27830 confirmed specific binding of folic acid to predicted site. Heterologous expression of At5G27830 in the yeast resulted in significant uptake and accumulation of folic acid in cells. This novel study of a plant FBP will be useful for folate metabolic engineering of a wide range of crops.

Evaluation of folate-binding proteins and stability of folates in plant foliages.

The present study reports the presence of folate binding proteins (FBPs) in the plants, coriander and Arabidopsis, and their contributions toward folate enhancement. After observing that salicylic acid (SA) enhanced the accumulation of folates in coriander, a study was conducted in Arabidopsis, where twofold increase in folates occurred in foliage upon SA treatment. For obtaining insights into genes involved in SA-induced folate accumulation, microarray data of responsive genes in Arabidopsis were screened. Based on the expression profiles, 19 genes were further analysed by qPCR. The results revealed that folate biosynthetic genes were largely down-regulated, whereas a gene of a putative folate-binding protein (FPB) was up-regulated, which correlated with a significant increase of FPBs in foliage. This new information on a plant FBP appears useful for metabolic engineering of a wide range of crops to enhance the content and stability of the folates during post-harvest storage.

Epigallocatechin gallate protects BEAS-2B cells from lipopolysaccharide-induced apoptosis through upregulation of gastrin-releasing peptide.

Gastrin-releasing peptide (GRP) plays a major role in the development and maintenance of lung epithelial cells by promoting cell division, whereas its suppression causes growth arrest and apoptosis. The present study shows that human bronchial epithelial BEAS-2B cells challenged with lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, downregulated GRP expression and induced apoptosis via upregulation of p53 and active caspase-3, signifying the importance of GRP in lung epithelial cell survival. However, in the presence of epigallocatechin-3-gallate (EGCG), a polyphenol in green tea, BEAS-2B cells resisted LPS-induced apoptosis and restored the expression of GRP and its downstream effectors such as epidermal growth factor receptor and NF-κB, as analysed by immunoblotting and qPCR. Based on our findings, we objectify that cytoprotective functions of EGCG, via upregulation of GRP in cells challenged with LPS, are novel and can be further explored in a therapeutic point of view for diseases such as septic shock.

Regulation of p53-mediated changes in the uPA-fibrinolytic system and in lung injury by loss of surfactant protein C expression in alveolar epithelial cells.

Pulmonary surfactant protein C (SP-C) expression by type II alveolar epithelial cells (AECs) is markedly reduced in diverse types of lung injuries and is often associated with AEC apoptosis. It is unclear whether loss of SP-C contributes to the increased p53 and urokinase-type plasminogen activator (uPA) system cross-talk and apoptosis of AECs. Therefore, we inhibited SP-C expression in human and murine AECs using lentivirus vector expressing shRNA and tested p53 and downstream changes in the uPA-fibrinolytic system. Inhibition of SP-C expression in AECs induced p53 and activated caspase-3, indicating AEC apoptosis. We also found that bleomycin or cigarette smoke exposure failed to inhibit SP-C expression or apoptosis in AECs in p53- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice. Depletion of SP-C expression by lentiviral SP-C shRNA in PAI-1-deficient mice failed to induce p53 or apoptosis in AECs, whereas it increased both AEC p53 and apoptosis in wild-type and uPA-deficient mice. SP-C inhibition in AECs also increased in CXCL1 and CXCL2 and their receptor CXCR2 as well as ICAM-1 expression, which is indicative of a proinflammatory response. Overexpression of p53-binding 3'-UTR sequences in AECs inhibited PAI-1 induction while maintaining uPA and uPAR protein and mRNA expression. Furthermore, caveolin-1 expression and phosphorylation were increased in AECs, indicating an intricate link between caveolin-1 and Src kinase-mediated cell signaling and AEC apoptosis due to loss of SP-C expression through p53 and uPA system-mediated cross-talk. The role of uPA, PAI-1, and p53 in the regulation of AEC apoptosis after injury was also determined in knockout mice.

p53 and miR-34a Feedback Promotes Lung Epithelial Injury and Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. The pathogenesis of interstitial lung diseases, including its most common form, IPF, remains poorly understood. Alveolar epithelial cell (AEC) apoptosis, proliferation, and accumulation of myofibroblasts and extracellular matrix deposition results in progressive loss of lung function in IPF. We found induction of tumor suppressor protein, p53, and apoptosis with suppression of urokinase-type plasminogen activator (uPA) and the uPA receptor in AECs from the lungs of IPF patients, and in mice with bleomycin, cigarette smoke, silica, or sepsis-induced lung injury. Treatment with the caveolin-1 scaffolding domain peptide (CSP) reversed these effects. Consistent with induction of p53, AECs from IPF lungs or mice with diverse types of lung injuries showed increased p53 acetylation and miR-34a expression with reduction in Sirt1. This was significantly reduced after treatment of wild-type mice with CSP, and uPA-deficient mice were unresponsive. Bleomycin failed to induce miR-34a in p53- or plasminogen activator inhibitor-1 (PAI-1)-deficient mice. CSP-mediated inhibition of miR-34a restored Sirt1, suppressed p53 acetylation and apoptosis in injured AECs, and prevented pulmonary fibrosis (PF). AEC-specific suppression of miR-34a inhibited bleomycin-induced p53, PAI-1, and apoptosis and prevented PF, whereas overexpression of precursor-miR-34a increased p53, PAI-1, and apoptosis in AECs of mice unexposed to bleomycin. Our study validates p53-miR-34a feedback as a potential therapeutic target in PF.

Salicylic acid-induced elicitation of folates in coriander (Coriandrum sativum L.) improves bioaccessibility and reduces pro-oxidant status.

Foliage of Coriandrum sativum is a rich source of natural folates amenable for enhancement through salicylic acid-mediated elicitation, thereby holding a great promise for natural-mode alleviation of this vitamin (B(9)) deficiency. In the present study we report salicylic acid-mediated differential elicitation of different forms of folates - 5-methyltetrahydrofolate, 5-formyltetrahydrofolate and 10-formyltetrahydrofolate - their stabilities during microwave-drying and bioaccessibilities from fresh and dried foliage. The first two compounds nearly doubled and the third increased sixfold post-elicitation, with all three showing concomitant increase in bioaccessibilities. Although a slight decrease in bioaccessibility was observed in dried foliage, over twofold increase of each form of folate upon elicitation would deliver much higher levels of natural folates from this traditional culinary foliage, which is widely used in many cuisines. Elicitor-mediated folate enhancement also imparted reduction of oxidative status and the enhancement of antioxidant enzyme activities in coriander foliage.

Enhancement of folate content and its stability using food grade elicitors in coriander (Coriandrum sativum L.).

Folate (vitamin B9) content was evaluated in 10 varieties of coriander with the aim of enhancing its concentration and stability, because of three reasons: 1) coriander is among a few widely used greens in the world and suits many cuisines, 2) folate deficiency is prevalent in developing countries causing anaemia, infant mortality and neural tube closure defects, and 3) natural folate is preferred due to doubts about health risks associated with the synthetic form. In C. sativum, the highest folate content of 1,577 µg/100 g DW was found in var. GS4 Multicut foliage of mature plants (marketable stage) with an insignificantly higher content (1,599.74 µg/100 g DW) at flowering, which is a stage not preferred in markets. In callus cultures treated with plant growth regulators (GRs) (6-benzylaminopurine, kinetin and abscisic acid) substantial increase in folate occurred after 6 h, whereas elicitors (methyl jasmonate and salicylic acid) caused rapid 2-fold increase of folate, particularly in response to salicylic acid. Based on these observations, foliar applications were done for in vivo plants, where salicylic acid (250 µM, 24 h) also enhanced folate level by 2-folds (3,112.33 µg/100 g DW), although the content varied with diurnal rhythms. Stability of folates in treated coriander foliage was 10 % higher than in untreated foliage when stored at 25 °C and 4 °C. This study has established for the first time that coriander foliage is rich in folates, which can be doubled by elicitation and impart 10 % more stability than control during processing and storage.