For morphological study of cell death, cells were stained with 50 μg/mL of acridine orange and 50 μg/mL of ethidium bromide and then observed and photographed under a fluorescent microscope. Flow cytometry analysis

after Anexin V and PI staining Apoptosis was detected by flow cytometry using Annexin V-FITC Apoptosis Detection Kit (Nanjing KeyGen Biotech, Nanjing, China). Briefly, cells were double stained with annexin V-FITC and propidium PX-478 datasheet iodide (PI) following manufacturer’s instruction. Early apoptosis is defined by Annexin V+/PI- staining (Q4) and late apoptosis is defined by Annexin V+/PI+ staining (Q2) as determined by FACScan (Beckman coulter cell, Brea, CA, USA). Immunoblot analysis Cells were treated as indicated in each figure legend and then cell extracts were prepared by lysing cells in M2 buffer [20 mmol/L Tris-HCl (pH 7.6), 0.5% NP40, 250 mmol/L NaCl, 3 mmol/L EDTA, 3 mmol/L EGTA, 2 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride, 20 mmol/L β-glycerophosphate, 1 mmol/L sodium vanadate, and 1 μg/mL leupeptin]. Cell extracts were subjected to SDS-PAGE and analyzed by Western blot using various antibodies.

The proteins GSK3326595 order were observed by enhanced chemiluminescence (Millipore, Billerica, MA, USA) using BIO-RAD Image station. Each https://www.selleckchem.com/products/VX-809.html experiment was repeated at least three times and representative results are shown in each figure. Detection of ROS Cells cultured in 12-well plates were treated with saikosaponin or cisplatin alone or both as indicated in each figure legend. Cells were then stained for 30 minutes with 5 μM of H2O2-sensitive fluorescent dye CM-H2DCFDA or 5 μM of.O2 –sensitive dye dihydroethidium (DHE), washed 3 times with PBS, and subsequently assayed by FACScan (Beckman coulter cell, Brea, CA, USA) as reported previously [21]. Statistical analysis All numerical data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was analyzed

by paired Student’s t test using SPSS statistics software package and P < 0.05 was used for significance. Results Saikosaponin-a and -d sensitize cancer cells to cisplatin induced cytotoxicity Both SSa and SSd have been reported to induce proliferation inhibition and cell death in various cancer cells (5-9). However, 5-Fluoracil purchase the effect of combination of these saikosaponins with chemotherapeutic drugs has never been investigated. We addressed this question by treating a cervical cancer cell line HeLa with SSa and cisplatin alone or both. Cell death was detected and quantified by an LDH release assay. While treatment with SSa alone caused marginal cell death (~10% cell death at 10 μM), it significantly sensitized cancer cells to cisplatin-induced cell death in a dose-dependent manner (~50% cell death at 10 μM concentration of SSa) (Figure 1A). A similar dose-dependent potentiation of cytotoxicity was observed with increasing cisplatin concentrations and a fixed SSa concentration (10 μM, Figure 1B).

calamagrostidis (4B) 5′ Stromata hairy when young, red to dark reddish brown; ostiolar dots absent or indistinct; conidia green H. junci (1 T) 6 Stromata upright, height usually exceeding the width, with a sterile stipe (formerly Podostroma, buy U0126 Podocrea) 7 6′ Stromata different 10 7 On wood and bark, stromata clavate or irregular, fertile part yellow; slow-growing; anamorph on CMD trichoderma-like, green-conidial when fresh H. alutacea (2P) 7′ On the ground on forest litter; anamorphs on CMD

verticillium-like or reduced, white-conidial; predominantly in North Europe 8 8 Stromata large, to more than 10 cm long; fertile part reddish brown to brownish orange, pigment inhomogeneously distributed; distal ascospore cell Tariquidar in vitro 3.0–5.5 × 3.0–4.2 μm; conidia large, 5–21 × 3–9 μm, typically produced on solitary phialides H. nybergiana (2P) 8′ Stromata smaller, typically <5 cm long, fertile part paler, yellowish; distal ascospore cell 2.7–4.0 × 2.3–3.5 μm; anamorph verticillium-like 9 9 Colour not changing upon drying,

fertile part sharply delimited from the stipe; conidia ellipsoidal, 2.8–6.2 × 2.0–3.0 μm H. leucopus (2P) 9′ Colour changing to ochre upon drying, perithecia decurrent on the stipe; conidia subglobose to ellipsoidal, 2.5–4.5 × 2.0–3.7 μm H. seppoi (2P) 10 Stromata hypomyces-like, perithecia seated on or in a subiculum; AZD8931 purchase anamorphs white-conidial 11 10′ Perithecia embedded in a fleshy, at least partially pseudoparenchymatous stroma 16 11 Ascospore cells conical, 4–6 × 2–3 μm, with minute acute appendages; anamorph verticillium-like Arachnocrea stipata 11′ Ascospores rounded 12 12 On aphyllophoralean fungi; anamorphs gliocladium-like 13 12′ On wood and bark, overgrowing fungi or bryophytes; PTK6 anamorphs verticillium-like 14 13 On Skeletocutis spp. and other polypores; perithecia yellowish, amber to olive; subiculum white, KOH- Protocrea farinosa 13′

On Oligoporus and Tyromyces spp., perithecia orange, subiculum white or orange, KOH+ purple Protocrea pallida 14 Perithecia ochre, orange or brown, subiculum white or brownish, KOH-; perithecia small, up to 200 μm diam; distal ascospore cell 2.3–3.7 × 2.0–3.2 μm H. delicatula (3E) 14′ Subiculum with different colours, more compact, KOH+; distal ascospore cell 3.0–5.5 × 2.5–4.0 μm 15 15 Subiculum red in fertile areas, purple in KOH H. parmastoi (3E) 15′ Subiculum olive-brown to yellow-brown, turning brown to grey in KOH H. alcalifuscescens (3E) 16 Stromata effuse to subpulvinate at maturity, extending to >1 cm; margin often attached on the substrate at least when young; surface not conspicuously hairy or velutinous except in H.

RNA obtained was treated with 0.6 U of RQ1 DNase (Promega) for Selleckchem Bafilomycin A1 30 min at 37°C, followed by phenol extraction and ethanol precipitation, in order to eliminate contaminating genomic DNA. The RNA integrity was assessed by agarose/formaldehyde gel electrophoresis and quantified in a Nanodrop 2000

device (Thermo Scientific). The reactions were performed using primers RND3 and RND4 (located within the coding region of CCNA_02805 and CCNA_02806, respectively). cDNA was synthesized from 0.25 μg of RNA using Super Script™ First Strand Synthesis System (Life Technologies) in a 20 μl final volume, following the manufacturer’s instructions. PCR amplification was performed using 1.2 μg of cDNA as template, 10 pmol each primer, 5% DMSO in a final volume of 25 μl using Taq DNA polymerase (Fermentas). The PCR conditions were: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 1 min, with a final cycle at 72°C

for 5 minutes. A negative control reaction was performed as described above, without the addition of reverse transcriptase. The PCR products were analyzed on 1% agarose gel electrophoresis. Construction of the czrA and nczA mutant and complemented strains In-frame deletions were constructed by allelic exchange using the pNPTS138 suicide vector and C. crescentus NA1000 strain. Two genomic regions upstream and downstream of the gene to be deleted were amplified by PCR using pfx Platinum DNA polymerase (Life Technologies) and primers RND7/RND8 (785 bp, HindIII/EcoRI) and RND9/RND10 (752 bp, EcoRI/MluI) to czrA gene and CDK inhibitor review primers RND11/RND12 (870 bp, HindIII/BamHI) and RND13/RND14 (654 bp, BamHI/MluI) to nczA gene. A terminal adenine was added with Taq DNA Polymerase (Life Technologies) and subsequently the fragments were cloned into vector pGEM-T Easy (Promega). The fragments were cloned in tandem into the pNPTS138 vector, the plasmids were transferred to C. crescentus strain NA1000 by Axenfeld syndrome Fosbretabulin clinical trial conjugation with E. coli S17-1, and recombinant

colonies were selected in PYE-kanamycin plates. A colony containing the integrated plasmid was inoculated in PYE medium without antibiotics for 48 hours, and loss of the plasmid was selected in PYE media containing 3% sucrose. The deletions were confirmed by PCR. Double mutant ΔczrAΔnczA was obtained by introducing the pNPTS138 vector containing the 5′ and 3′-flanking regions of czrA into the ΔnczA strain. PCR products using primers RND15/RND16 (3243 bp) and RND17/RND18 (3132 bp), containing the coding regions of czc1 and czc2 genes respectively, were used to generate complemented strains. Each fragment was cloned into the suicide vector pNPT228XNE, and the plasmid was inserted into the mutant strains by conjugation with E. coli S17-1. The insertion of the recombinant vector occurs at the xylose utilization locus, and expression of the cloned genes is induced with 0.2% xylose. Growth assays in the presence of metals Initial cultures at OD600 = 0.

aureus strains isolated from hospitalized patients (4 MRSA and 4 MSSA) examined with respect of their ability to survive after PDI treatment, showed different pattern of response. Based on statistical analysis we divided those strains into two groups: sensitive and resistant to PDI. In the group of resistant strains (2002, 4246, MAPK inhibitor 1397, 7259) the drop in the survival rate did not exceed 1.5 log10 units. In the second group of strains, called sensitive, (472, 80/0, 2288, 5491) the drop in survival rate was at least 1.5 log10 units reduction in viable counts. In our previous reports we already showed a strain-dependent response to PDI targeted

S. aureus cells, where the observed efficacy of photokilling reached even 5 log10 units reduction. The differences between our previous studies and the one presented

here might have probably resulted from a different photosensitizer used – PpIX vs. protoporphyrin IX diarginate (PpIXArg2) [24, 25]. Other groups also observed the phenomenon of PDI-strain dependence, however, the mechanism underlying the diverse response to PDI was not explored [43, 44]. Our data shows that at lower concentration of a photosensitizer (10 μM) a substantial drop in bacterial survival occurred, whereas at higher selleck chemicals llc concentrations (25-50 μM), no further decrease in survival was noticed. We associate this phenomenon with poor solubility of PpIX in water solutions but the solubility itself does not justify the observed variability in killing curves. Similar results were obtained by the group of Wilson (2008). In the study they used another anionic photosensitizer, indocyanine green (ICG) against S. aureus and observed that the concentration of 25 μg/ml resulted in 6 log10 units reduction in viable counts, but higher ICG concentrations (50 heptaminol and 100 μg/ml), resulted in lesser, about 4 and 5 log10 units reduction in survival counts, respectively [45]. Possible explanation of this phenomenon may be the self shielding effect of the non-bound PS in solution at higher concentrations. Effective

photodynamic therapy is a result of a combination of several factors. Beside the biophysical properties of a sensitizer itself, also total light delivered, time of incubation with a photosensitizer, presence of additional proteins are crucial. In our work we did not focused on BMN 673 research buy examining the dependence of killing rate vs. light dose. We performed all photodynamic inactivation studies on one light dose (12 J/cm2) chosen as optimal based on our previously published data concerning S. aureus photoinactivation as well as phototoxicity assays performed on dermal human fibroblasts [46, 47]. In our previous attempts to explore the differences of porphyrin-based photokilling towards S. aureus cells, we found biofilm production ability to correlate with higher resistance to PDI treatment. However, it was also noted that among S. aureus isolates with elevated resistance to PDI, biofilm non-producing strains were also observed.

*Significant (P < 0.05) difference between post and pre supplementation. Cardiopulmonary variables There was no significant change in O2 or CO2 during constant-load exercise, and no differences were

found between groups before or after Selleckchem Pitavastatin supplementation (Table 2). RER, on the other hand, was significantly overall higher post compared to pre supplementation in the Cr/Gly/Glu group (P = 0.01) but not in the Cr/Gly/Glu group (Table 2). A significant 3- or 2-way interaction for heart rate (HR) was not found, thus the main effects were interpreted. During exercise, HR increased significantly over time (P = 0.01). Overall, HR was significantly lower post supplementation (P = 0.39) (Figure 3). In pre supplementation trials HR during exercise was not significantly different between the 2 groups. Table Ruboxistaurin purchase 2 Cardiopulmonary responses throughout exercise Variable   Time (min)     Trial 10 20 30 40 O2 (ml/kg/min) Cr/Gly/Glu Pre 42.9 ± 6.1 43.1 ± 7.4 44.2 ± 6.2 44.6 ± 7.3     Post 42.2 ± 6.7 42.1 ± 6.6 40.8 ± 6.4 42.3 ± 6.2   Cr/Gly/Glu/Ala Pre 40.9 ± 4.8 41.9 ± 5.1 42.7 ± 4.8 42.3 ± 5.2     Post 41.8 ± 3.4 41.5 ± 2.9 41.8 ± 4.1 42.3 ± 3.7 CO2 (ml/kg/min) Cr/Gly/Glu Pre 41.5 ± 6.1 41.0 ± 7.4

41.7 ± 4.9 41.8 ± 7.6 find more     Post 41.4 ± 4.7 42.0 ± 4.8 42.0 ± 4.6 42.1 ± 5.1   Cr/Gly/Glu/Ala Pre 42.3 ± 7.2 41.2 ± 7.3 39.9 ± 6.7 41.2 ± 6.6     Post 41.2 ± 3.1 41.0 ± 3.5 41.2 ± 3.5 41.3 ± 3.9 RER Cr/Gly/Glu Pre 0.94 ± 0.0 0.94 ± 0.0 0.94 ± 0.1 0.93 ± 0.0     Post* 0.98 ± 0.0 0.97 ± 0.0 0.97 ± 0.0 0.97 ± 0.0   Cr/Gly/Glu/Ala Pre 0.98 ± 0.0 0.98 ± 0.0 0.96 ± 0.0

0.97 ± 0.0     Post 0.97 ± 0.0 0.97 ± 0.0 0.97 ± 0.0 0.96 ± 0.0 Oxygen consumption (O2) and carbon dioxide production (CO2), and respiratory exchange ratio (RER) in Cr/Gly/Glu and Cr/Gly/Glu/Ala groups during exercise before and after supplementation. Data presented as Mean ± SD. Figure 3 Heart rate (HR) during exercise before (grey triangles) and after (black circles) supplementation in the Cr/Gly/Glu/Ala and Cr/Gly/Glu groups. Data presented as Mean ± SD. *(P = 0.01) for significant difference between after and before supplementation. Core temperature (tcore) responses Pre supplementation Exoribonuclease Tcore was similar in the 2 groups of participants (P > 0.05). A significant 3- or 2-way interaction was absent for Tcore; hence the interpretation of the main effects. Throughout the exercise period, Tcore increased significantly (P = 0.01; Figure 4). Overall, Tcore was significantly lower during exercise conducted after supplementation (P = 0.01). Figure 4 Core temperature (Tcore) during exercise before (grey triangles) and after (black circles) supplementation in the Cr/Gly/Glu/Ala and Cr/Gly/Glu groups.

Side branches sometimes rebranching to form

complex, dense, non-transparent structures. Phialides solitary or in whorls of 3–5(–7). Sparse conidial development also on long aerial hyphae. Phialides (5.5–)7–12(–17) × (2.7–)3.2–4.0(–4.7) μm, l/w = (1.4–)1.9–3.4(–5.0), (1.5–)2.0–3.0(–4.0) μm wide at the base (n = 60), terminal phialides often longer than the flanking ones in the fascicle, lageniform to narrowly subcylindrical, sometimes sinuous, less commonly ampulliform or sometimes ventricose, inequilateral and with a long neck, widest point at various positions. Conidia find more (3.0–)3.5–6.5(–10.5) × (2.2–)2.5–3.3(–4.2), l/w = (1.1–)1.2–2.2(–3.4) (n = 75), hyaline, yellowish in mass, oval to oblong, often attenuated toward

one end, smooth, with guttules often in a group at each end. At 15°C development slower; at 30°C faster, with more abundant yellowish conidiation submerged in the agar, morphologically indistinguishable from granules on the surface of the APR-246 agar. Coconut-like odour also formed at all other temperatures. Most abundant chlamydospores and yellow crystals formed at 30 and 35°C. At 35°C growth continuing for >1 week, with only few hyphae on the agar surface and scanty effuse, simple conidiation without any granulation after 4–5 days. On PDA 9–11 mm at 15°C, 28–29 mm at 25°C, 27–31 mm at 30°C, 3–6 mm at 35°C; mycelium covering the plate after 7–8 days at 25°C; growth slower than on CMD, with hyphae more thickly and densely arranged than on CMD. Colony thick, dense, not or indistinctly zonate, with a thin, finely granular centre of extremely densely interwoven to condensed hyphae and an ill-defined, diffuse margin with surface hyphae forming strands. Surface whitish, turning yellow or greenish, downy to floccose by a reticulum of aerial hyphae forming thick strands and numerous narrow ID-8 branches without

any noticeable orientation. Autolytic activity and coilings conspicuous at 25 and 30°C. Conidiation finely granular, colourless to white, on numerous single phialides or short verticillium-like, seated on surface and aerial hyphae, effuse, spreading across the entire colony. Reverse and to some extent also surface turning light yellow from the centre, 3A3, 3B5–6, 4B4–5. Odour indistinct to slightly mushroomy. At 35°C growth slow, forming small sterile, white, hairy colonies. On SNA 11–12 mm at 15°C, 33–35 mm at 25°C, 42–44 mm at 30°C, 9–15 mm at 35°C; mycelium covering the plate after 5–6 days at 25°C. Colony thin, hyaline, growth predominantly submerged in the agar, hyphae GSK2126458 datasheet loosely arranged and sometimes forming several separated strands rather than a continuous colony. Aerial hyphae scant, more common and longer at the whitish and downy distal margin. Autolytic activity and coilings conspicuous at 25 and 30°C. Surface hyphae soon degenerating.

For example, when N(t) is equal to 20 × K N , the growth rate is theoretically ~95% of μ max. Such a 5% decrease is typically undetectable by optical density measurements [36]. Therefore, in theory, as long as the initial cell density is X 0 << Y × 20 × K N , variations in the inoculum density have negligible impact on growth curve reproducibility. This therefore sets an upper limit to the inoculum density. Besides the lower and upper limits of inoculum density, another important condition for the growth curve synchronization is that the lag phase must be independent BIIB057 price of inoculum concentration. We can confirm if this is true by testing

whether the time shift (τ) between growth curves starting from cell densities X 1 and X2 (where X 2 > X 1) obeys the following relationship Below, we show how we tested this condition empirically for all growth curves aligned by calculating the linear regression between τ and ln (X 2/X 1). Application to virulence

see more factor secretion by Pseudomonas aeruginosa We used high-resolution OD600 curves of wild-type P. aeruginosa PA14 to demonstrate the growth curve synchronization method. The wild-type strain will be referred to as WT (see Table 1 for list of strains used). Figure 1 shows 8 growth curves obtained by serial dilution before (Figure 1A) and after alignment (Figure 1B). Although visual inspection shows the alignment was successful, we evaluated the quality of the alignment by plotting the time delays (τ) as a function of the log of the dilutions (Figure 2). For this case, we obtained Selleck AZD9291 R2 = 0.996, CYTH4 which confirmed the alignment is appropriate and confirms that the lag phase is independent of inoculum density, which is a central requirement of our method. Figure

1C shows GFP expression measured for the same samples. GFP expression is under the control of the rhlAB-promoter, making GFP an indication of the expression of rhamnolipid synthesis genes. Figure 1D shows the alignment of GFP expression obtained using the time delays calculated from the original synchronization based on OD600. This alignment shows that gene expression monitored by a reporter protein can be synchronized using the same time-shift, without the need for a separate calculation, again supporting our theoretical model. Figure 1 Alignment of growth curves and GFP expression in WT strain. A) Median growth curves constructed from 8 replicates of cultures inoculated between 0.0025 OD600 (dark blue) and 2 × 10-5 OD600 (dark red). B) Growth curve alignment for the median growth curves. C) Median GFP expression curves, constructed from the same samples as the growth curves. D) GFP curves aligned using the time-shift calculated from the OD600 alignment. Figure 2 Determining the reproducibility of the lag phase in WT cells. If the mathematical assumption τ = (1/μ max) ln (X2/X1) is correct, then τ as a function of ln (X2/X1) should yield a straight line with a slope of 1/μ max.

References Baker ML, Jiang W, Rixon FJ et al (2005) Common ancestry of herpes viruses and tailed DNA bacteriophages. J Virol 79:14967–14970CrossRefPubMed PI3K inhibitor Bamford DH (2003) Do viruses form lineages across different domains of life? Res Microbiol 154:231–236CrossRefPubMed Bamford DH, Grimes JM, Stuart DI (2006) What does structure tell us about virus evolution? Curr Opin Struct Biol 15:655–663CrossRef Bandea C (1983) 7-Cl-O-Nec1 in vivo J Theor Biol 105:591–602CrossRefPubMed Bell PJ (2001) Viral eukaryogenesis: was the ancestor of the nucleus a complex DNA virus? J Mol Evol 53:251–256CrossRefPubMed

Bizet A, Karlsson EA, Ekefjärd K, Prevost MC, Forterre P, Tenaillon O, Bernander R, Prangishvili D (2009) A Unique Virus Release Mechanism in Archea. Proc Natl Acad Sci, in press Bragg JG, Chisholm SW (2008) Modeling the fitness consequences of a cyanophage-encoded photosynthesis gene. PLoS ONE 3(10):e3550CrossRefPubMed Brosius

J (2003) The contribution of RNAs and retroposition to evolutionary novelties. Genetica 118:99–116CrossRefPubMed Cavicchioli R (2007) Archaea: molecular and cellular biology. ASM Claverie JM (2006) Viruses take center stage in cellular evolution. Genome Biol 7:110CrossRefPubMed De Parseval N, Heidmann T (2005) Human endogenous retroviruses: from infectious DZNeP elements to human genes. Cytogenet Genome Res 110:318–332CrossRefPubMed Edwards RA, Rohwer F (2005) Opinion: viral metagenomics. Nat Rev Microbiol 3:504–510CrossRefPubMed Engels F (1883) Dialectics of nature, 1–410. Wellred, London, 2006 Filée J, Forterre P (2005) Viral proteins functioning in organelles: a cryptic origin? Trends Microbiol 13:510–513CrossRefPubMed Filée J,

Forterre P, Sen-Li T et al (2002) Evolution of DNA polymerase families: evidences for multiple gene exchange between cellular and viral proteins. Niclosamide J Mol Evol 54:763–773CrossRefPubMed Filée J, Forterre P, Laurent J (2003) The role played by viruses in the evolution of their hosts: a view based on informational protein phylogenies. Res Microbiol 154:237–43CrossRefPubMed Forterre P (1992) New hypotheses about the origins of viruses, prokaryotes and eukaryotes. In: Thanh Vân JK, Mounolou JC, Schneider J, Mc Kay C (eds) Frontiers of life. Edition Frontières, Gif-sur-Yvette, pp 221–234 Forterre P (1999) Displacement of cellular proteins by functional analogues from plasmids or viruses could explain puzzling phylogenies of many DNA informational proteins. Mol Microbiol 33:457–465CrossRefPubMed Forterre P (2002) The origin of DNA genomes and DNA replication. Curr Opin Microbiol 5:525–532CrossRefPubMed Forterre P (2005) The two ages of the RNA world, and the transition to the DNA world: a story of viruses and cells. Biochimie 87:793–803CrossRefPubMed Forterre P (2006a) Three RNA cells for ribosomal lineages and three DNA viruses to replicate their genomes: a hypothesis for the origin of cellular domain.

1% (wt/vol) glycine solution (1:100), pooled and stored at −20°C. Circular dichroism spectroscopy Purified recombinant proteins were dialyzed against sodium phosphate buffer (pH 7.4). Circular dichroism (CD) spectroscopy measurements were performed at 20°C using a Jasco J-810 spectropolarimeter (Japan Spectroscopic, Tokyo) equipped with a Peltier unit for temperature control. Far-UV CD spectra were measured using a 1 mm – path – length

cell at 0.5 nm intervals. selleck chemicals The spectra were presented as an average of five scans recorded from 185 to 260 nm. The molar ellipticity (Φ) is expressed in deg.cm.dmol1. Antiserum Five female BALB/c mice (4–6 weeks old) were immunized subcutaneously with 10 μg of the recombinant proteins. The recombinant proteins were adsorbed in 10% (vol/vol) of Alhydrogel (2% Al(OH)3, Brenntag Biosector, Denmark), used as adjuvant. Two subsequent booster injections see more were given at two – week intervals with the same preparation of 10 μg

of the proteins. Negative – control mice were injected with PBS. One week after each immunization, the mice were bled from the retro – orbital plexus and the pooled sera were analyzed by enzyme -linked immunosorbent assay (ELISA) for determination of antibody titers. All animal studies were approved by the Ethics Committee of the Instituto Butantan, São Paulo, SP, Brazil. The Committee in Animal Research in Instituto Butantan adopts the guidelines of the Brazilian College of Animal Experimentation. Immunoblotting O-methylated flavonoid assay The purified recombinant proteins were loaded into 12% SDS – PAGE and transferred to nitrocellulose membranes (Hybond ECL; GE Healthcare) in semi – dry equipment. Membranes were blocked with 5% non-fat dried milk and 2.5% BSA in PBS containing 0.05% Tween 20 (PBS – T) and then incubated with anti – rLIC11834

(1:500), anti – rLIC12253 (1:500) mouse serum or anti – his antibody (1:1,000) (GE Healthcare) for 2 h at room temperature. After washing, the membranes were incubated with horseradish peroxidase (HRP) – conjugated anti – mouse IgG (1:5,000; Sigma) in PBS – T for 1 h. The protein’s reactivity was revealed by ECL reagent kit chemiluminescence substrate (GE Healthcare) with subsequent exposition to X – Ray film. ELISA for detection of human antibodies Human IgG antibodies against Lsa33 or Lsa25 were detected by ELISA as previously described [59]. In brief, serum samples of negative (24) and positive (33) MAT from confirmed – leptospirosis patients were diluted 1:400 and evaluated for total IgG using goat HRP – conjugated anti-human IgG antibodies (1:5,000, Sigma). Cutoff values were set at three standard deviations above the mean OD492nm of sera from 11 health individuals, unexposed to leptospirosis, from the city of São Paulo, CP673451 order Brazil and one pool of normal serum samples from USA (Sigma).

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in a continental island system: molecular phylogeography of the Aegean Nigella arvensis alliance (Ranunculaceae) inferred from chloroplast DNA. Mol Ecol 14:4065–4083CrossRefPubMed Brofas G, Karetsos G, Panitsa M et al (2001) The flora and vegetation of Gyali island, SE Aegean, Greece. Willdenowia 31:51–70 Burton RM (1991) A check-list and evaluation of the flora of Nisiros (Dodecanese, Greece). Willdenowia 20:15–38 Carlström A (1987) A survey of the flora and phytogeography of Rodhos, Simi, Tilos and the Marmaris Peninsula (SE Greece, SW Turkey). PhD thesis, University

of Lund, Sweden Christodoulakis D (1986) Flora and vegetation of Samos. PhD thesis, University of Patras, Greece. (In Greek with an English summary) Christodoulakis D (1996) The flora of Ikaria (Greece, E. Aegean Islands). Phyton 36:63–91 Christodoulakis Bioactive Compound Library concentration D (2000) The flora of Samiopoula (E Aegean Islands, Greece): a biological, chorological and ecological analysis. Bot Chron 13:287–301 Davis SD, Heywood VH, Hamilton AC (1994) Centers of plant diversity: a guide and strategy for their conservation. WWF and IUCN, Cambridge Georghiou K, Delipetrou P (2008) Database «Chloris»: endemic, rare, threatened and protected plants of Greece. Synonyms, distribution, conservation and protection status, biology, ecology, bibliography. University of Athens Gittenberger E (1991) What Glutamate dehydrogenase about non-adaptive radiation? Biol J Linn Soc 43:263–272CrossRef Greuter W (1970) Zur Paläogeographie

und Florengeschichte der südlichen Ägäis. Feddes Repert 81:233–242CrossRef Greuter W (1972) Betrachtungen zur Pflanzengeographie der Südägäis. In: Strid A (ed) Evolution in the Aegean. Opera Bot 30:49–64 Greuter W (1979) The origins and evolution of island floras as exemplified by the Aegean archipelago. In: Bramwell D (ed) Plants and islands. Academic Press, London, pp 87–106 Greuter W (1995) Origin and peculiarities of Meditteranean island floras. Ecol Mediterr 21(1–2):1–10 Greuter W (2001) Diversity of Mediterranean island floras. Bocconea 13:55–64 Greuter W, Pleger R, Raus T (1983) The vascular flora of the Karpathos island group (Dodecanesos, Greece). A preliminary checklist. Willdenowia 13:43–78 Groombridge B (1992) Global biodiversity: status of the Earth’s living resources. Chapman & Hall, London Höner D (1991) Mehrjährige Beobachtungen kleiner Vegetationsflächen im Raume von Karpathos (Nomos Dhodhekanisou, Griechenland). Diss Bot 173:1–185 Jahn R, Schönfelder P (1995) Exkursionsflora für Kreta.