Type III Secretion System Genes of Dickeya dadantii 3937 Are Induced by Plant Phenolic Acids

Background: Dickeya dadantii is a broad-host range phytopathogen. D. dadantii 3937 (Ech3937) possesses a type III secretion system (T3SS), a major virulence factor secretion system in many Gram-negative pathogens of plants and animals. In Ech3937, the T3SS is regulated by two major regulatory pathways, HrpX/HrpY-HrpS-HrpL and GacS/GacA-rsmB-RsmA pathways. Although the plant apoplast environment, low pH, low temperature, and absence of complex nitrogen sources in media have been associated with the induction of T3SS genes of phytobacteria, no specific inducer has yet been identified. Methodology/Principal Findings: In this work, we identified two novel plant phenolic compounds, o-coumaric acid (OCA) and t-cinnamic acid (TCA), that induced the expression of T3SS genes dspE (a T3SS effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) in vitro. Assays by qRT-PCR showed higher amounts of mRNA of hrpL (a T3SS alternative sigma factor) and rsmB (an untranslated regulatory RNA), but not hrpS (a s 54-enhancer binding protein) of Ech3937 when these two plant compounds were supplemented into minimal medium (MM). However, promoter activity assays using flow cytometry showed similar promoter activities of hrpN in rsmB mutant Ech148 grown in MM and MM supplemented with these phenolic compounds. Compared with MM alone, only slightly higher promoter activities of hrpL were observed in bacterial cells grown in MM supplemented with OCA/TCA. Conclusion/Significance: The induction of T3SS expression by OCA and TCA is moderated through the rsmB-Rsm

The precancer risk of betel quid chewing, tobacco use and alcohol consumption in oral leukoplakia and oral submucous fibrosis in southern Taiwan

In areas where the practise of betel quid chewing is widespread and the chewers also often smoke and drink alcohol, the relation between oral precancerous lesion and condition to the three habits is probably complex. To explore such association and their attributable effect on oral leukoplakia (OL) and oral submucous fibrosis (OSF), a gender–age-matched case–control study was conducted at Kaohsiung, southern Taiwan. This study included 219 patients with newly diagnosed and histologically confirmed OL or OSF, and 876 randomly selected community controls. All information was collected by a structured questionnaire through in-person interviews. A preponderance of younger patients had OSF, while a predominance of older patients had OL. Betel quid chewing was strongly associated with both these oral diseases, the attributable fraction of OL being 73.2% and of OSF 85.4%. While the heterogeneity in risk for areca nut chewing across the two diseases was not apparent, betel quid chewing patients with OSF experienced a higher risk at each exposure level of chewing duration, quantity and cumulative measure than those who had OL. Alcohol intake did not appear to be a risk factor. However, cigarette smoking had a significant contribution to the risk of OL, and modified the effect of chewing based on an additive interaction model. For the two oral premalignant diseases combined, 86.5% was attributable to chewing and smoking. Our results suggested that, although betel quid chewing was a major cause for both OL and OSF, its effect might be difference between the two diseases. Cigarette smoking has a modifying effect in the development of oral leukoplakia

Genome-Wide Identification of HrpL-Regulated Genes in the Necrotrophic Phytopathogen Dickeya dadantii 3937

BACKGROUND: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. METHODOLOGY/PRINCIPAL FINDINGS: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens

Inference of Co-Evolving Site Pairs: an Excellent Predictor of Contact Residue Pairs in Protein 3D structures

Residue-residue interactions that fold a protein into a unique three-dimensional structure and make it play a specific function impose structural and functional constraints on each residue site. Selective constraints on residue sites are recorded in amino acid orders in homologous sequences and also in the evolutionary trace of amino acid substitutions. A challenge is to extract direct dependences between residue sites by removing indirect dependences through other residues within a protein or even through other molecules. Recent attempts of disentangling direct from indirect dependences of amino acid types between residue positions in multiple sequence alignments have revealed that the strength of inferred residue pair couplings is an excellent predictor of residue-residue proximity in folded structures. Here, we report an alternative attempt of inferring co-evolving site pairs from concurrent and compensatory substitutions between sites in each branch of a phylogenetic tree. First, branch lengths of a phylogenetic tree inferred by the neighbor-joining method are optimized as well as other parameters by maximizing a likelihood of the tree in a mechanistic codon substitution model. Mean changes of quantities, which are characteristic of concurrent and compensatory substitutions, accompanied by substitutions at each site in each branch of the tree are estimated with the likelihood of each substitution. Partial correlation coefficients of the characteristic changes along branches between sites are calculated and used to rank co-evolving site pairs. Accuracy of contact prediction based on the present co-evolution score is comparable to that achieved by a maximum entropy model of protein sequences for 15 protein families taken from the Pfam release 26.0. Besides, this excellent accuracy indicates that compensatory substitutions are significant in protein evolution.Comment: 17 pages, 4 figures, and 4 tables with supplementary information of 5 figure

BPR1K653, a Novel Aurora Kinase Inhibitor, Exhibits Potent Anti-Proliferative Activity in MDR1 (P-gp170)-Mediated Multidrug-Resistant Cancer Cells

Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells.BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats.BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments

Performance evaluation on an air-cooled heat exchanger for alumina nanofluid under laminar flow

This study analyzes the characteristics of alumina (Al2O3)/water nanofluid to determine the feasibility of its application in an air-cooled heat exchanger for heat dissipation for PEMFC or electronic chip cooling. The experimental sample was Al2O3/water nanofluid produced by the direct synthesis method at three different concentrations (0.5, 1.0, and 1.5 wt.%). The experiments in this study measured the thermal conductivity and viscosity of nanofluid with weight fractions and sample temperatures (20-60°C), and then used the nanofluid in an actual air-cooled heat exchanger to assess its heat exchange capacity and pressure drop under laminar flow. Experimental results show that the nanofluid has a higher heat exchange capacity than water, and a higher concentration of nanoparticles provides an even better ratio of the heat exchange. The maximum enhanced ratio of heat exchange and pressure drop for all the experimental parameters in this study was about 39% and 5.6%, respectively. In addition to nanoparticle concentration, the temperature and mass flow rates of the working fluid can affect the enhanced ratio of heat exchange and pressure drop of nanofluid. The cross-section aspect ratio of tube in the heat exchanger is another important factor to be taken into consideration

Rescue of Photoreceptor Degeneration by Curcumin in Transgenic Rats with P23H Rhodopsin Mutation

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects

The dopamine D1 receptor is expressed and induces CREB phosphorylation and MUC5AC expression in human airway epithelium

Background Dopamine receptors comprise two subgroups, Gs protein-coupled “D1-like” receptors (D1, D5) and Gi-coupled “D2-like” receptors (D2, D3, D4). In airways, both dopamine D1 and D2 receptors are expressed on airway smooth muscle and regulate airway smooth muscle force. However, functional expression of the dopamine D1 receptor has never been identified on airway epithelium. Activation of Gs-coupled receptors stimulate adenylyl cyclase leading to cyclic AMP (cAMP) production, which is known to induce mucus overproduction through the cAMP response element binding protein (CREB) in airway epithelial cells. We questioned whether the dopamine D1 receptor is expressed on airway epithelium, and whether it promotes CREB phosphorylation and MUC5AC expression. Methods We evaluated the protein expression of the dopamine D1 receptor on native human airway epithelium and three sources of cultured human airway epithelial cells including primary cultured airway epithelial cells, the bronchial epithelial cell line (16HBE14o-), and the pulmonary mucoepidermoid carcinoma cell line (NCI-H292) using immunohistochemistry and immunoblotting. To characterize the stimulation of cAMP through the dopamine D1 receptor, 16HBE14o- cells and NCI-H292 cells were treated with dopamine or the dopamine D1 receptor agonists (SKF38393 or A68930) before cAMP measurements. The phosphorylation of CREB by A68930 in both 16HBE14o- and NCI-H292 cells was measured by immunoblot. The effect of dopamine or A68930 on the expression of MUC5AC mRNA and protein in NCI-H292 cells was evaluated by real-time PCR and immunofluorescence staining, respectively. Results The dopamine D1 receptor protein was detected in native human airway epithelium and three sources of cultured human airway epithelial cells. Dopamine or the dopamine D1-like receptor agonists stimulated cAMP production in 16HBE14o- cells and NCI-H292 cells, which was reversed by the selective dopamine D1-like receptor antagonists (SCH23390 or SCH39166). A68930 significantly increased phosphorylation of CREB in both 16HBE14o- and NCI-H292 cells, which was attenuated by the inhibitors of PKA (H89) and MEK (U0126). Expression of MUC5AC mRNA and protein were also increased by either dopamine or A68930 in NCI-H292 cells. Conclusions These results suggest that the activation of the dopamine D1 receptor on human airway epithelium could induce mucus overproduction, which could worsen airway obstructive symptoms

Novel Quinazolinone MJ-29 Triggers Endoplasmic Reticulum Stress and Intrinsic Apoptosis in Murine Leukemia WEHI-3 Cells and Inhibits Leukemic Mice

The present study was to explore the biological responses of the newly compound, MJ-29 in murine myelomonocytic leukemia WEHI-3 cells in vitro and in vivo fates. We focused on the in vitro effects of MJ-29 on ER stress and mitochondria-dependent apoptotic death in WEHI-3 cells, and to hypothesize that MJ-29 might fully impair the orthotopic leukemic mice. Our results indicated that a concentration-dependent decrease of cell viability was shown in MJ-29-treated cells. DNA content was examined utilizing flow cytometry, whereas apoptotic populations were determined using annexin V/PI, DAPI staining and TUNEL assay. Increasing vital factors of mitochondrial dysfunction by MJ-29 were further investigated. Thus, MJ-29-provaked apoptosis of WEHI-3 cells is mediated through the intrinsic pathway. Importantly, intracellular Ca2+ release and ER stress-associated signaling also contributed to MJ-29-triggered cell apoptosis. We found that MJ-29 stimulated the protein levels of calpain 1, CHOP and p-eIF2α pathways in WEHI-3 cells. In in vivo experiments, intraperitoneal administration of MJ-29 significantly improved the total survival rate, enhanced body weight and attenuated enlarged spleen and liver tissues in leukemic mice. The infiltration of immature myeloblastic cells into splenic red pulp was reduced in MJ-29-treated leukemic mice. Moreover, MJ-29 increased the differentiations of T and B cells but decreased that of macrophages and monocytes. Additionally, MJ-29-stimulated immune responses might be involved in anti-leukemic activity in vivo. Based on these observations, MJ-29 suppresses WEHI-3 cells in vitro and in vivo, and it is proposed that this potent and selective agent could be a new chemotherapeutic candidate for anti-leukemia in the future