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Dendrograms on the left are derived from Figure 3a (branch lengths do not represent inferred distances). Detected orthologs are only present in the genomes in bold. Arrows in black represent genes in an OG of the highlighted pattern and grey arrows represent other genes nearby in

the genome. Blue lines linking genes indicate inferred orthology. Gene numbers correspond to the last part of the original gene names. Numbers in colours other than black indicate genes with products putatively secreted (red) or with transmembrane domains (green). The clusters are (a) one including a wrongly annotated check details pathogenicity-related gene (yapH) and a phage gene (Φ-hk97); and (b) one possibly related to the type IV secretion system. The second cluster (Figure 5b) is present in XamC and Xfa0 but not in Xfa1, despite the high genome-wide similarity presented between Xfa1 and Xfa0 (Figure 2a). The classification of putative homologs of the genes in this cluster (see methods) revealed that it is mainly composed of sequences similar to proteins in Escherichia coli, Siphoviridae, Savolitinib Stenotrophomonas sp. SKA14, Salmonella enterica and

Pseudomonas aeruginosa (Additional file 5). Moreover, members of the Siphoviridae viral family are known to be Pseudomonas and Xanthomonas phages, suggesting the presence of virus-mediated LGT. We cannot attribute the pattern to the mixture of chromosomal and plasmidic DNA in draft genomes (XamC and Xfa0), because none of the sequences presented Celecoxib similarity with genes in Xanthomonas plasmids. Note that the gene at the locus XAUC_17260_1

(Xfa0:1726 in Figure 5b) was originally annotated as yapH, but its product is a large protein of 1231 aa in Xfa0 and 1482 aa in XamC, putatively xenologous with a component of a phage tail (group COG4733 in the COG database). Two genes in the cluster (XamCg00977 and XamCg00978) presented a G+C content more than one standard deviation below the mean of the coding sequences in the XamC genome (i.e., 64.82 ± 3.31%), and a low CAI with respect to the whole predicted coding sequences (0.516 and 0.486, respectively). The other seven genes in the cluster presented average features, which would have precluded their identification as units potentially under LGT. Discussion The results of the genome-based phylogenetic reconstruction suggest that certain changes should be considered in the nomenclature of the Xanthomonas genus. For instance, X. fuscans was recently proposed as a new species [27], but here we show that it should be considered as a later heterotypic synonym of X. citri, as previously suggested [18, 31]. Other clades in the standing bacterial nomenclature [63] within the Xanthonomonas genus were consistent with the phylogenetic reconstruction. Nevertheless, we observed a paralogy in the genus Xanthomonas when Xylella fastidiosa was included with X. albilineans outside the Xanthomonas group. Our results suggest that X.

DCs were then collected and suspended in cold staining buffer (PBS containing 1% FCS, 0.1 mL) in microcentrifuge tubes. Afterwards, 20 μL of FITC-labeled anti-CD83, CD86, and HLA-DR monoclone antibodies (BD Pharmingen, San Jose, CA, USA) were added and Belinostat incubated with DCs for 30 min at 4°C. The DCs were washed again with cold staining buffer for three times, and the cell surface markers were analyzed by flow cytometry. Cellular viability study The influence of GO on DC viability was checked with

a standard MTS cell viability assay according to the manufacturer’s direction. Briefly, DCs were treated with GO (0.1 μg/mL) or D-Hank’s solution in 24-well plates for 2 h at 37°C in 5% CO2, washed thoroughly, and then added into 96-well plates with a density of 1 × 104/well. After 1, 4, and 24 h of incubation, the viability of DCs was evaluated with the MTS cell viability Semaxanib price assay (n = 6). Statistical analysis Statistical difference was determined by Student’s t test, and a value of p < 0.05 was considered statistically significant. Results GO was prepared from natural graphite by a modified Hummer's method [24]. In order to get exfoliated single-layer nanosized GO, the GO solution was further processed and cracked by ultrasonication. The GO nanosheets were next collected via centrifugation at 50,000 g and dispersed in water as the stock solution. Atomic force microscopy (AFM) characterization (Figure 1A)

provided morphological information of the GO nanosheets. The height profile showed that the thickness of GO nanosheets was around 1.1 nm (Figure 1A), indicating single-layer

nanosheets. Moreover, the lateral size of GO nanosheets was about 60 to 360 nm, with an average dimension of 140 nm. The GO was negatively charged with an average zeta potential of -28 mV (Figure 1B). The GO solutions were used without further treatments in the following experiments. Figure 1 Characterization of GO nanosheets and their antigen loading capability. (A) AFM topographic image of nanosized GO sheets deposited on mica (top) and the height profile along the black line (bottom). Scale bar is 500 nm. (B) Distributions of size and zeta potential of GO. (C) Loading rates of Ag on GO at various peptide/GO feed ratios. Prostatic acid phosphatase To induce a specific anti-glioma immune response, DCs must be exposed to glioma antigens. The antigen used in the study was a peptide (ELTLGEFLKL, termed Ag) from the protein survivin, which is widely expressed in malignant gliomas [20–22]. Survivin is a member of the inhibitor of apoptosis (IAP) protein family, which can regulate two important cellular processes: it inhibits apoptosis and promotes cell proliferation. Hence, survivin expression is generally associated with poor prognosis [30, 31]. The peptide ELTLGEFLKL can bind to HLA-A*0201, the most common human leukocyte antigen (HLA) serotype, and stimulate DCs to generate CD8+ immune responses against glioma cells [20–22, 26].

Analysis of amplified 16S rRNA gene sequences was done in comparison with the RDP II database (match length >1200 nucleotides). The percentages of the phylogenetically classified sequences are plotted on y-axis. The detailed affiliation of different phylotypes with their closest neighbour in database is presented in Additional file 4: Table S1. The majority of phylotypes that belong to Alphaproteobacteria were from AS clone library. These OTUs were related (85-99%) to Rhizobiales, Sphingomonadales and Rhodospirillales while six OTUs from SS1 & SS2 libraries showed affiliation (89-99%)

to Rhodobacterales, Rhizobiales and Rhodospirillales. A cluster of 25 sequences from AS clone library (7 OTUs), which contributes 58.7% of the total AS Betaproteobacterial population were related (87-99%) to Limnobacter thiooxidans from family Burkholderiaceae, formed one of its largest cluster. The only SS1 OTU HSS79 showed 97% similarity BVD-523 cost to uncultured Betaproteobacteria whereas no OTU was observed in SS2 clone library. The 22 OTUs (4 from www.selleckchem.com/products/ABT-888.html AS and 18 from SS1 & SS2 clone libraries) were related to different species of uncultured Gammaproteobacteria. Most of the SS1 & SS2 clone sequences were related to cultured bacteria like Salinisphaeraceae bacterium, Methylohalomonas lacus, sulphur-oxidizing bacterium and Marinobacter

species. The presence of sulphur-oxidizing and Marinobacter bacteria PFKL in saline soils may suggest the presence of sulphur in these saline environments. These saline soils

indeed contain sulphur (Table 1). Deltaproteobacterial OTUs from SS1 & SS2 clone libraries formed a tight cluster with deep sea bacterium, uncultured Deltaproteobacteria and Marinobacterium. OTUs belonging to photoautotrophic Cyanobacteria and chemoautotrophic nitrifying Nitrospira were found only in AS clone library. Two phylotypes BSS159 and BSS49 were related (91%) to Cyanobacteria and uncultured Nitrospira, respectively and more may be present as rarefaction curves did not reached saturation, although started to level off. The photoautotrophic Chloroflexi related sequences were mostly from SS1 & SS2 clone libraries within the families Caldilineaceae, Sphaerobacteraceae and Anaerolineaceae. One OTU RS187 had 88% homology with Sphaerobacter thermophilus, no other OTUs were more than 91% similar to that of any described organism (Additional file 4: Table S1). There were only two OTUs from AS clone library which showed affiliation (>92%) to uncultured Chloroflexi. van der Meer et al. (2005) [27] suggested that Cyanobacteria and Chloroflexi utilize different spectra of light, and CO2 from the atmosphere for photosynthesis. Firmicutes related sequences were found mostly in AS and SS2 clone library. One phylotype RS190 was affiliated with Bacillus polygoni (95%) a moderately halophilic, non-motile, obligate alkaliphile isolated from indigo balls.

Thetford, Emilys Wood, near Brandon, MTB 35-31/2, 52°28′08″ N, 00°38′20″ E, elev. 20 m, on partly decorticated branch of Fagus sylvatica 3 cm thick, mainly on wood, and a white Corticiaceae, soc. Hypocrea minutispora and Trichoderma stilbohypoxyli, holomorph, 13 Sep. 2004, H. Voglmayr & W. Jaklitsch, RGFP966 order W.J. 2713 (WU 29300, culture C.P.K. 2357). Same area, on partly decorticated branches of Fagus sylvatica 3–4 cm thick, on bark and wood, soc. Hypocrea minutispora, holomorph, 13 Sep. 2004, H. Voglmayr & W. Jaklitsch,

W.J. 2714 (combined with WU 29300, culture C.P.K. 1901). Notes: Hypocrea neorufoides is closely related to H. neorufa. The teleomorphs of these species are indistinguishable. H. neorufoides is widespread in Europe and more common than H. neorufa, particularly in southern England and eastern Austria. Morphologically these species establish an intermediate position between Trichoderma sect. Trichoderma and the pachybasium core group,

deviating from other species of the first section in more distinct surface cells and in a yellow perithecial wall, and in thick, i.e. pachybasium-like conidiophores. Contrary to H. neorufa the conidiation in T. neorufoides develops continuously from effuse and verticillium-like to a pachybasium-like shrub conidiation without statistically significant differences in the sizes of phialides and conidia. Nevertheless, both measurements are given in order to highlight the differences to H. neorufa. Additional Enzalutamide differences from H. neorufa are a lower growth optimum, particularly on SNA and PDA, a different macroscopic growth pattern on PDA, larger and more variable conidia and slightly longer phialides. The pigmentation of the reverse on PDA is distinctly less pronounced Cobimetinib than in H. neorufa. The shrub conidiation of H. neorufoides on CMD often disappears after several transfers and only simple effuse conidiation remains. Hypocrea ochroleuca Berk. & Ravenel, Grevillea 4: 14 (1875). Fig. 12 Fig. 12 Teleomorph

of Hypocrea ochroleuca. a, b. Fresh stromata. c, d, f, g. Dry stromata (f. vertical section showing layered subperithecial tissue). e, h. Stromata in 3% KOH after rehydration. i. Stroma surface in face view. j. Perithecium in section. k. Cortical and subcortical tissue in section. l Subperithecial tissue in section. m. Stroma base in section. n. Hairs on the stroma surface. o Ascospores. p, q Asci with ascospores (q. in cotton blue/lactic acid). a–f, h–q. WU 29310. g. holotype K 56075. Scale bars: a = 1.5 mm. b = 2.5 mm. c = 1 mm. d, e, g, h = 0.5 mm. f = 150 μm. i, o = 5 μm. j, k, m = 20 μm. l, n, p, q = 10 μm Anamorph: Trichoderma sp. Fig. 13 Fig. 13 Cultures and anamorph of Hypocrea ochroleuca (CBS 119502). a–c. Cultures after 7 days (a. on CMD; b. on PDA; c. on SNA). d. Conidiation shrubs (CMD, 4 days). e–g. Conidiophores on growth plates (4 days; e. CMD; f, g. SNA). h–l. Conidiophores (CMD, 4–7 days). m, n. Phialides (CMD, 6 days). o. Conidia in chains and clumps (SNA, 22 days). p–r.

However, clinically GC resistance occurs in 10-30% of untreated ALL patients and is more frequently seen in T-lineage ALL (T-ALL) than B-precursor ALL and GC resistance always leads to the failure of chemotherapy [4]. T-ALL is a highly malignant tumor representing 10%-15% of pediatric and 25% of adult ALL in humans and it is clinically regarded as a high-risk disease with a relapse rate of about 30% [5, 6]. T-ALL has a less favorable prognosis than B-cell ALL. The mechanisms that underlie the development

of GC resistance are poorly understood and likely vary with disease type, treatment regimen, and the genetic background of the patient [7]. However, an increasing number of reports indicate that activation of mammalian target of rapamycin Selleckchem Staurosporine (mTOR) signaling pathway may contribute to GC resistance in hematological malignancies [8–11]. A recent study, using a database of drug-associated gene expression profiles to screen for molecules whose profile overlapped with a gene expression signature Doxorubicin solubility dmso of GC sensitivity/resistance in ALL cells, demonstrated that the mTOR inhibitor rapamycin profile matched the signature of GC sensitivity [12]. We recently demonstrated that nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogene originated from t(2;5)(p23;q35) in a subset of non-Hodgkin’s lymphoma transformed lymphoid

cells to become resistant to GC or Dex treatment by activating mTOR signaling pathway and rapamycin could re-sensitize the transformed lymphocytes to Dex treatment [13]. Rapamycin, the best studied mTOR inhibitor, was originally isolated from the soil bacterium Lck Streptomyces hygroscopicus in the mid-1970 s [14]. Although

it was initially developed as a fungicide and immunosuppressant, antitumor activity of rapamycin has been described in vitro and in vivo [15–18]. mTOR is a serine-threonine protein kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family. Inhibition of mTOR kinase leads to dephosphorylation of its two major downstream signaling components, p70 S6 kinase (p70S6K) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), which in turn inhibits the translation of specific mRNAs involved in cell cycle and proliferation and leads to G1 growth arrest [19, 20]. A major regulator of the mTOR pathway is the PI3K/AKT kinase cascade and activation of PI3K/AKT/mTOR has been found in lymphoid malignancies [21]. Most studies have shown that rapamycin acts as a cytostatic agent by arresting cells in the G1 phase [15–20]. Although cell cycle arrest can temporarily halt tumor progression, the affected clones could re-grow since the tumor cells have not been killed. Cell cycle inhibitor seems to work best in combination with chemotherapy. However, combination of cell cycle inhibitor with cytotoxic agents might be agonistic or antagonistic [22, 23].

C. García-Estrada is supported by the Torres Quevedo Program

(PTQ04-3-0411) cofinanced by the ADE Inversiones y Servicios of Castilla y León (04B/07/LE/0003). I. Vaca received a fellowship of the Diputación de León. The expert help of Carlos Barreiro and Patricia Martín (Instituto de Biotecnología, INBIOTEC) with the mass spectrometry and DNA sequencing analyses, respectively, is acknowledged. Authors wish to thank B. Martín, STA-9090 mouse J. Merino, A. Casenave and B. Aguado (Instituto de Biotecnología, INBIOTEC) for their excellent technical assistance. References 1. Martín JF, Liras P: Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu Rev

Microbiol 1989, 43:173–206.CrossRefPubMed 2. Álvarez E, Cantoral JM, Barredo JL, Díez B, Martín JF: Purification to homogeneity and characterization of the acyl-CoA: Lenvatinib ic50 6-APA acyltransferase of Penicillium chrysogenum. Antimicrob Agents Chemother 1987, 31:1675–1682.PubMed 3. Martín JF, Ingolia TD, Queener SW: Molecular genetics of penicillin and cephalosporin antibiotic biosynthesis. Molecular Terminal deoxynucleotidyl transferase Industrial Mycology (Edited by: Leong SA, Berka R). New York: Marcel Dekker 1990, 149–195. 4. Lamas-Maceiras M, Vaca I, Rodríguez E,

Casqueiro J, Martín JF: Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N acyltransferase. Biochem J 2006, 395:147–155.CrossRefPubMed 5. Wang FQ, Liu J, Dai M, Ren ZH, Su CY, He JG: Molecular cloning and functional identification of a novel phenylacetyl-CoA ligase gene from Penicillium chrysogenum. Biochem Biophys Res Commun 2007, 360:453–458.CrossRefPubMed 6. Fierro F, Barredo JL, Díez B, Gutiérrez S, Fernández FJ, Martín JF: The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences. Proc Natl Acad Sci USA 1995, 92:6200–6204.CrossRefPubMed 7. Fierro F, García-Estrada C, Castillo NI, Rodríguez R, Velasco-Conde T, Martín JF: Transcriptional and bioinformatic analysis of the 56.8 kb DNA region amplified in tandem repeats containing the penicillin gene cluster in Penicillium chrysogenum. Fung Genet Biol 2006, 43:618–629.CrossRef 8.

However, its activity depends on environmental stimuli (e.g., cyclic AMP levels, temperature, heat shock, osmolarity, membrane

biosynthesis, and H-NS protein [8]), cell division, flagella formation, and motility [9–11]. A number of Gram-negative pathogenic bacteria have evolved a specialized type III protein secretion system to deliver effector virulence proteins into host cells [12, 13]. There learn more are two types of type III secretion systems: the translocation-associated type III secretion system (T3aSS) and the bacterial flagellum type III secretion system (T3bSS). The various bacterial type III secretion systems characterized thus far all have Sec independence, ATPase dependence, presence of a hollow filamentous organelle that extends from the outer membrane, a cell-envelope-spanning secretion channel, and nine conserved proteins [14]. The bacterial flagellum type III secretion system also serves as the bacterial flagellum (a biological nanomachine with an ion-powered rotary motor). For the flagellum, the T3bSS apparatus functions to secrete components including the rod, hook, and filament subunits for extracellular assembly. The core of the flagellum is hollow, and secreted subunits polymerize at the growing end of the flagellum. A cap at the tip of the flagellum ensures efficient polymerization Dabrafenib of secreted subunit proteins [15, 16]. This secretion

apparatus is just one mechanism utilized by Gram-negative plant and animal pathogens for the secretion and translocation of virulence determinants into susceptible eukaryotic cells [17]. In Salmonella typhimurium, the expression of class 1 genes (i.e., flhD and flhC) activates expression of Cyclin-dependent kinase 3 genes required for flagella assembly and regulates expression class 2 genes (e.g., fliAZY and flhBAE), which in turn regulates expression of class 3 genes encoding flagellar structural proteins (e.g., fliC, flgMN, and MotAB) [18].

In Xenorhabdus nematophila, it was shown that the EnvZ-OmpR-FlhDC-FliA regulatory network coordinately controls flagella synthesis as well as exoenzyme and antibiotic production [8]. In this paper, we describe the transcriptional regulation of fliC and flhA expression by flhD/C and also show that flhD/C has an effect on extracellular secretion of the Carocin S1 protein, but not on Carocin S1 gene expression. Our results indicate that the type III secretion system of Pectobacterium carotovorum subsp. carotovorum has a new secretory function. Methods Bacterial strains, plasmids, media, and growth conditions The strains and plasmids used are shown in Table 1. Pectobacterium carotovorum subsp. carotovorum strains were propagated at 28°C in 1.4% nutrient agar (NA) or with shaking in Luria-Bertani (LB) medium with NaCl (5 g/L). E. coli strains were propagated at 37°C in LB medium with shaking. Rifampicin, kanamycin, and ampicillin (all at 50 mg/L) were added to either medium when necessary.