Additional file 1: Figure S1. of AMP-Conjugated Quantum Dots: Low Immunotoxicity Both In Vitro and In Vivo

Intracellular Cd2+ concentrations in macrophage J774A.1 cells incubated with quantum dots (50 nM) for 12 h were measured. Data are presented as means ± s.d. (n = 6). Figure S2. Colloidal stability of AMP-QDs in physiological media. AMP-QDs were incubated for 24 h in 0.01 M PBS (a) or cell culture medium containing 10 % foetal bovine serum, (b) and their size distributions were measured by a dynamic light scattering (DLS) with Zetasizer NanoZS Instrument. The hydrodynamic size of AMP-QDs in 0.01 M PBS (a) and cell culture medium (b) were determined to be 8.49 nm and 8.56 nm, respectively

Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments

<p>Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. <i>Halorosellinia</i> sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3 min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15 min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of <i>Halorosellinia</i> sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.</p

A σ^E-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in <i>Vibrio alginolyticus</i>

<div><p>In vibrios, the expression of virulence factors is often controlled by LuxR, the master quorum-sensing regulator. Here, we investigate the interplay between LuxR and σ^E, an alternative sigma factor, during the control of virulence-related gene expression and adaptations to temperature elevations in the zoonotic pathogen <i>Vibrio alginolyticus</i>. An <i>rpoE</i> null <i>V</i>. <i>alginolyticus</i> mutant was unable to adapt to various stresses and was survival-deficient in fish. In wild type <i>V</i>. <i>alginolyticus</i>, the expression of LuxR-regulated virulence factors increased as the temperature was increased from 22°C to 37°C, but mutants lacking σ^E did not respond to temperature, indicating that σ^E is critical for the temperature-dependent upregulation of virulence genes. Further analyses revealed that σ^E binds directly to -10 and -35 elements in the <i>luxR</i> promoter that drive its transcription. ChIP assays showed that σ^E binds to the promoter regions of <i>luxR</i>, <i>rpoH</i> and <i>rpoE</i> at high temperatures (e.g., 30°C and 37°C). However, at higher temperatures (42°C) that induce thermal stress, σ^E binding to the <i>luxR</i> promoter decreased, while its binding to the <i>rpoH</i> and <i>rpoE</i> promoters was unchanged. Thus, the temperature-dependent binding of σ^E to distinct promoters appears to underlie a σ^E-controlled switch between the expression of virulence genes and adaptation to thermal stress. This study illustrates how a conserved temperature response mechanism integrates into quorum-sensing circuits to regulate both virulence and stress adaptation.</p></div

RpoE binds directly to the <i>luxR</i> promoter region.

<p>(<b>A</b>) EMSAs were performed with purified RpoE, and the <i>luxR</i> promoter region was analyzed. The amount of RpoE protein (nM) that was used is indicated, and 20 ng of each Cy5-labelled probe was added to the EMSA reactions. The shifts were verified to be specific in experiments in which we added a 5- to 50-fold excess of unlabeled specific DNA and non-specific competitor DNA (poly(dI:dC)). (<b>B</b>) Plot showing the affinity of RpoE to the <i>luxR</i> promoter. The densitometric intensities of bound DNA fragments were plotted against RpoE concentrations. The arrow indicates the concentration of RpoE that caused half-maximal binding (<i>K</i>_d). (<b>C</b>)Footprinting analysis of RpoE binding to a binding site in the <i>luxR</i> promoter. Electropherograms of a DNase I digest of the P_<i>luxR</i> promoter probe (400 ng) after incubation with 0 or 400 nM RpoE. The respective nucleotide sequences that were protected by His-RpoE are indicated below, and the specific -10 and -35 regions are underlined. (<b>D-E</b>) <i>In vitro</i> transcription was performed using a P_<i>rpoE</i> template, (<b>D</b>) a P_<i>luxR</i> template (<b>E</b>), NTP, and RNAP core enzyme as well as RpoE. Various concentrations of purified LuxR were added into the reaction mixture to determine its effect on <i>luxR</i> transcription (<b>E</b>). The transcripts were purified, reverse-transcribed (RT, +) and detected using PCR. As a control, the same purified transcripts were also treated using the same process but without reverse transcriptase (RT, -).</p

RpoE binds to target DNA in response to temperature changes <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>A</b>) ChIP assays were used to analyze RpoE binding to the <i>luxR</i>, <i>rpoE</i> and <i>rpoH</i> promoter <i>in vivo</i>. Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using PCR with the primers P_<i>luxR</i>chip-F/R, P_<i>rpoE</i>-chipF/R, P_<i>rpoH</i>-chipF/R and control-F/R (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005645#ppat.1005645.s010" target="_blank">S2 Table</a>). (<b>B</b>) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene <i>gyrB</i>. Results were calculated using the ΔΔ<i>C</i>_T method. * <i>P</i> <0.05, ** <i>P</i> <0.01, <i>t-</i>test. (<b>C</b>) Plot showing the affinity of RpoE binding to the promoters of <i>luxR</i>, <i>rpoE</i>, and <i>rpoH</i> at different temperatures as determined using EMSA (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005645#ppat.1005645.s004" target="_blank">S4 Fig</a>). The intensities of the bound DNA fragments were determined using a densitometer and plotted against the RpoE concentrations. Triplicate assays were performed, and a representative plot is shown. (<b>D</b>) The promoter strength of <i>luxR</i>, <i>rpoE</i>, and <i>rpoH</i> in the presence of similar levels of RpoE at different temperatures in <i>E</i>. <i>coli</i> DH5α cells. The three indicated promoters were fused to different fluorescence reporters and cloned into the same cloning plasmid, pMD19T. The fluorescence reads for each of the promoters after incubation in the presence of arabinose inducing pBAD-driven <i>rpoE-flag</i> expression were subtracted from the reads obtained with no arabinose and normalized to both the corresponding reads at 22°C and the densitometry values of RpoE expression. The results are presented as the mean ± S.D. (<i>n</i> = 3).</p

RpoE controls Asp expression via the QS regulator LuxR.

<p>(<b>A</b>) <i>luxR</i> transcriptional assays were performed in wt and Δ<i>luxO</i> strains that were grown at different temperatures. The wt and Δ<i>luxO</i> strains that carried the P_<i>luxR</i>-<i>lacZ</i> reporter plasmid were cultured in LBS medium and assayed for <i>β</i>-galactosidase activity. The results are presented as the mean ± S.D. (<i>n</i> = 3). (<b>B</b>) Western blot assays show LuxR expression in wt, Δ<i>rpoE</i> and <i>rpoE</i>⁺ strains grown at different temperatures. Bacteria were cultured in LBS medium for 9 h and then harvested. Proteins from equal cell volumes were resolved using 12% SDS-PAGE. (<b>C</b>) Extracellular Asp activity in the <i>V</i>. <i>alginolyticus</i> strains. The bacteria were centrifuged after they were cultured in LBS medium for 9 h, and the supernatants were harvested to measure protease activity. The results are presented as the mean ± S.D. (<i>n</i> = 3). **, <i>P</i><0.01, and ***, <i>P</i><0.001, based on ANOVA comparisons. (<b>D</b>) Western blot assays were performed to analyze Asp and LuxR expression in wt and <i>rpoE-</i> and <i>luxO</i>-related mutants. pBAD33::<i>rpoE</i> was introduced into the Δ<i>rpoE</i>Δ<i>luxO</i> strain to analyze Asp and LuxR expression in the presence (+) and absence (-) of L-arabinose (Ara).</p

Effects of vaccination with the live attenuated <i>V. anguillarum</i> on Wnt signaling pathway.

<p>The genes related to Wnt signaling pathway were modulated after vaccination with the live attenuated <i>V. anguillarum</i>. Red: up-regulated, Green: down-regulated, Black: not found to be modulated. WNT: wingless-type MMTV integration site family, DKK1: Dickkopf homologue 1, LRP5/6: LDL-receptor-related protein 5/6, CK1: casein kinase, GSK3β: glycogen synthase kinase 3β, AXIN: axis inhibition protein, DVL: mammalian homologue of <i>Drosophila</i> dishevelled, APC : adenomatous polyposis coli, PKA: cAMP-dependent protein kinase catalytic subunit alpha, PS-1: presenilin 1, PYGO: legless CREB binding protein, LGS: pygopus CREB binding protein, TCF: T-cell factor, GRG:Groucho, CTBP: C-terminal binding protein, HDAC: histone deacetylases, STBM: VANGL planar cell polarity protein 2, DAAM: dishevelled associated activator of morphogenesis, RHOA: ras homolog family member A, RAC: ras-related C3 botulinum toxin substrate, ROCK: Rho-associated coiled-coil containing protein kinase, JNK: Jun N-terminal kinase, JUN: jun proto-oncogene, PLC: phospholipase C, PKC: protein kinase C, CAMKII: calcium calmodulin mediated kinase II, NFAT: nuclear factor of activated T cells.</p

Validation of relative expression between microarray data and RT-qPCR results at 28 days post vaccination.

<p>iNOS2a: inducible nitric oxide synthase 2, itga3b: integrin alpha 3b, STAT5: Signal Transducer and Activator of Transcription 5, IL7R: interleukin 7 receptor, IL22: interleukine 22, Bcl6ab: B-cell CLL/lymphoma 6a, Cdc42l: cell division cycle 42 like, Mycb: myelocytomatosis oncogene b, ABCB8: ATP-binding cassette sub-family B member 8, Cts1a: cathepsin L 1 a.</p

List of differentially expressed genes related to adaptive immunity.

<p>List of differentially expressed genes related to adaptive immunity.</p

RpoE is involved in the temperature-dependent <i>in vivo</i> virulence of <i>V</i>. <i>alginolyticus</i>.

<p>(<b>A-D</b>) Virulence was analyzed in the <i>rpoE</i> mutant in zebrafish. A series of dilutions of the wt and <i>rpoE</i> mutant strains were intramuscularly (i.m.) inoculated into fish that were acclimated at 22°C or 30°C for 4 weeks. A total of 30 fish were used for each of the dilutions. The infected fish were cultivated at 22°C or 30°C and monitored for 7 days. <i>P</i> values were calculated using Kaplan-Meier survival analysis with a log rank test. (<b>E</b>) CI assays for the Δ<i>rpoE</i> strain against the wt (<i>lacZ</i>⁺), which was a wt strain carrying <i>lacZ</i> following the <i>glmS</i> locus. The CI values of the 1:1 Δ<i>rpoE</i> vs. wt (<i>lacZ</i>⁺) inoculums were first tested in LBS medium at 30°C for 24 h. Then, 1:1 [the indicated strains vs. wt (<i>lacZ</i>⁺)] inoculums were i.m. administered into zebrafish and cultivated at 30°C for 24 h before the fish were anesthetized and their cells were numerated on IPTG plates. ***, <i>P</i><0.001, based on ANOVA followed by Bonferroni’s multiple-comparison post-test to compare the data to the values for the corresponding wt/wt (<i>lacZ</i>⁺) samples.</p