To date, however, there is no plausible explanation for the proce

To date, however, there is no plausible explanation for the process chain MK0683 ic50 involved in the anthropogenic change of cloud microphysics caused by aerosols. Further, it can be concluded that the detection of changes in essential variables, i.e. cloud albedo, cloud brightness temperature and amount of precipitation, is most pronounced in the source regions of air pollutants. This might indicate the regional character of the processes rather than an influence on a global scale. However, the hydrological cycle and the radiation balance

in Europe were involved. In addition to the knowledge described above, there are significant observations which need further attention in future studies. These results are important constraints for evaluating the nature of cloud-mediated processes and to further quantify the magnitude of human impact on climate in Europe. The first result is related to time series of global irradiance in Estonia (Eerme et al. 2010). Measurement data show a conspicuous low relation of global irradiance for overcast and clear conditions (G/Gclear) during the summers of the late 1970s and the 1980s. Furthermore, the characteristics for normalised surface global solar radiation seem to be conspicuously connected to the results for Germany by Liepert & Kukla (1997), which are mentioned above. In principle, cloud MAPK Inhibitor Library mw properties could have changed as a consequence of anthropogenic

aerosols. However, if this is indeed the case, the origin of the impact has to be identified. Also requiring further investigation is whether both time series could 3-mercaptopyruvate sulfurtransferase be explained in a similar way by aerosol cloud-mediated processes in Europe. Another result which needs to be taken into account when assessing the influence of European aerosol system on clouds is the behaviour

of organic vapour emissions in Europe. Paasonen et al. (2013) found that rising biogenic organic vapour emissions in response to warming will enhance condensation on particles and their growth to the size of cloud condensation nuclei. The authors specify the strongest negative feedbacks at the most northern and remote sites. Moreover, phytoplankton related emissions, such as dimethyl sulphide (DMS) and volatile organic gases via their transformation into aerosol particles need to be considered for the Baltic Region. The subsequent formation of cloud condensation nuclei (CCN) (O’Dowd & de Leeuw 2007) can decrease the shortwave radiation flux at the surface and increase the reflected shortwave radiation flux at the top of the atmosphere as well as decrease the amount of precipitation (Krüger & Graßl 2011). Thus, biologically produced gases dissolved in oceanic waters and their subsequent emission into the marine troposphere play an important role and are likely to be involved in regional feedback processes in the climate system. Therefore, we need to investigate the influence of changing anthropogenic and natural emissions (e.g.

The animals were then food deprived for 56 h (IACUC approved), a

The animals were then food deprived for 56 h (IACUC approved), a time length previously shown to maximize food hoarding [4] and [18]. Before access to food was returned at light offset, half of the animals received an injection of PYY(3-36) (0.1 nmol in 200 nl), the active form of the peptide for satiation [52], into the Epigenetic pathway inhibitors Arc and the other half received the saline vehicle. Wheel revolutions,

food foraging, food intake, and food hoarding were measured at 1, 2, 4, 24 h and each day post-injection until all animals returned to pre-injection levels. After the animals returned to behavioral baseline, brain tissue was collected to verify cannula location (Fig. 1; 69 hits and 11 misses or removed their cannula; final group sizes: PYY(3-36): BW: n = 12, FW: n = 11, and 10REV: n = 13 and vehicle: BW: n = 9, FW: n = 11, and 10REV: n = 13). Raw data from Experiment 1 were transformed for each individual into percent change from vehicle before

statistical analyses using the formula: [((X-Vehicle)/Vehicle) × 100], where “X” equals the value measured in response to the dose of BIIE0246 CHIR-99021 chemical structure and “Vehicle” equals the value measured for that individual after vehicle injection. No statistical comparisons were made among the time intervals because the intervals were of unequal duration. No statistical comparisons are reported across test days in this counterbalanced-within subject design, as repeated measures two-way ANOVA (foraging treatment × Arc-injection) showed no effect of injection order. The data were analyzed using a two-way ANOVA (foraging treatment × Arc-injection; 3 × 4). For Experiment PJ34 HCl 2, data were not transformed into percent change from

vehicle, because animals only were food deprived once and therefore could not serve as their own control, and the absolute values were analyzed using a two-way ANOVA (foraging treatment × Arc-injection; 3 × 2) within each individual time point for the same reason as above. All statistical analyses were performed using NCSS (version 2007, Kaysville, UT). Exact probabilities and test values were omitted for simplicity and clarity of presentation. Differences were considered statistically significant if P < 0.05. Tukey-Kramer Multiple Comparison Tests were used for post hoc tests when appropriate. Misplaced cannulae were not included in the final statistical comparisons. Wheel running. At each time interval, Arc injection of BIIE0246 did not significantly stimulate wheel running activity compared to vehicle injection at any of the three doses tested (0.1, 1.0 and 5.0 nmol; Fig. 2A). The lack of wheel running increase in the FW group, where food delivery was not contingent upon wheel running, suggests that there was not non-specific stimulation of locomotor activity. Food foraging.

All participants were native Japanese speakers with higher than c

All participants were native Japanese speakers with higher than college level education. The study protocol was approved by the ethics committee of Osaka City University and was conducted in accordance with the principles of the Declaration of Helsinki, with written informed consent obtained from all participants prior to enrollment in the study. This study comprised three experimental sessions (Story A session, Story B session, and Story C session) (Fig. 6A). After enrollment, participants

were randomly assigned to three groups in a single-blinded, three-crossover fashion to consecutively SP600125 concentration undergo these three experimental sessions. They were requested to carefully listen to and understand three spoken Japanese stories (Story A, Story B, and Story C) with their eyes closed. The stories were constructed of recorded narratives in

which 2–4 syllables of the latter portion of spoken keywords, which seemed to contribute to the understanding of the stories, were replaced by 300-ms white-noise stimuli with an inter-stimulus interval of 1.6–20.3 s (Fig. 6B). Two of the three stories (Story A and Story B) were played forward and one story (Story C) was played in reverse (Story C was the reverse version of Story A). All spoken words consisted of the same digitally recorded female voice. Sound pressure, frequency range, and duration of the spoken words and white noise were adjusted using MycoClean Mycoplasma Removal Kit Adobe Premier Elements buy Everolimus (Adobe Systems, Tokyo, Japan) and presented via an MEG-compatible sound system (Model ER-2; Etymotic Research, Elk Grove Village, IL) using Windows Media Player 9 (Microsoft Japan, Tokyo, Japan) implemented on a personal computer (Precision PWS390; Dell Computer, TX). The Story A session involved 50 white-noise stimuli with a total duration of 311 s and the Story B session involved 68 white-noise stimuli with a total duration of 412 s, and the Story A session and the Story B session constituted a forward condition. The

Story C session consisted of 101 white-noise stimuli with a total duration of 311 s, and the Story C session constituted a reverse condition. We recorded MEG during these three experimental sessions, and white noise was used as a stimulus. The reverse condition was performed as a control, and we compared MEG responses to white-noise stimuli during the forward condition with those during the reverse condition using time–frequency analyses, in order to investigate neural activations related to phonemic restoration. Immediately after the end of the Story A and Story B sessions, the participants were asked 8 questions about the contents of each story to assess the objective story-comprehension level. Each question comprised 4 choices with one correct answer.

24 Ischemia was evoked by inflation of the arm cuff to 200 mm Hg

24 Ischemia was evoked by inflation of the arm cuff to 200 mm Hg during 5 minutes. Blood pressure was measured on the left arm by the auscultatory method, using a calibrated mercury sphygmomanometer with an appropriate cuff size, once at the beginning of preischemia FBF measurement and once at the beginning of postischemia FBF measurement. FBF was calculated by a semiautomatic method, which has shown high intra- and interevaluator reproducibility (intraclass correlation coefficients between 0.98

and 0.99).25 Mean blood pressure (MBP) was used to calculate forearm vascular conductance (FVC; FBF/MBP). Thereafter, the area under the FVC curve was calculated pre- and postischemia. The percent increase in area under the FVC during postischemia, above the correspondent area under the FVC during preischemia, was considered as the Epacadostat in vitro study’s vascular reactivity measure and used as the main end point INNO-406 cell line for statistical analyses. The sample size was estimated on the basis of

pilot data and results from previous studies.12 and 13 For a 2-way analysis of variance (ANOVA) (2 groups and 4 repeated measures), a total sample size of 120 subjects would be necessary to detect a difference of 35% between groups’ vascular reactivity (group main effect), considering a standard deviation within groups of 90%, P value of 0.05, and power of 0.80. Shapiro–Wilk’s test was used to verify variables’ distribution, Pregnenolone Levene’s

test was used to verify homoscedasticity, and Mauchly’s test was used to verify sphericity. Some variables were not normally distributed (ie, age, BMI, triglycerides, HDL, glycemia, VO2peak, SBP, and vascular reactivity), and thus were transformed into natural logarithms for inferential analyses. After logarithm transformation, there was no violation of the homoscedasticity assumption in any analyses. Nonetheless, vascular reactivity results deviated from the sphericity assumption, which required a correction that is described next. Three genetic models (dominant, recessive, and additive) were assessed to verify which model was better to fit the vascular reactivity data on partial correlations adjusted by all sample characteristics. In these partial correlations, eNOS gene polymorphisms were analyzed as dummy variables as follows: dominant model (heterozygous + polymorphic homozygous = 0 vs wild homozygous = 1), recessive model (polymorphic homozygous = 0 vs wild homozygous + heterozygous = 1), and additive model (polymorphic homozygous = 0 vs heterozygous = 1 vs wild homozygous = 2). Then, subjects’ characteristics according to genotypes and haplotypes were compared using independent Student t test or chi-square test.

Resulting data points were fitted to a dose–response curve The d

Resulting data points were fitted to a dose–response curve. The dose of 45 nmol/50 nL was used in the following protocols and 50 nL of ACSF was microinjected as vehicle control. Number of rats used, n = 12. These doses were established from data in the literature ( Pizzirusso et al., 1998). The third group of animals was used to evaluate the involvement of muscarinic receptors in the cardiovascular response to the injection of Ach into the vlPAG. Different doses of the nonselective muscarinic receptor antagonist atropine (1,

3 or 9 nmol/50 nL) were microinjected into the vlPAG 10 min before microinjection of Ach. Each animal received only one dose of atropine. Number of rats used, n = 16. These doses were established from data in the literature ( Crippa et al., 1999). In the last part of the study, we determined whether the cardiovascular response was due to a central effect of Ach. Animals received this website intravenously the same dose of atropine as injected into the vlPAG (9 nmol) 10 min prior to the injection of 45 nmol of Ach into that area. Number of rats used, n = 6. At the end of the experiments, 50 nL of 1% Evan’s blue dye was injected into the vlPAG or the dPAG as a marker of the injection site. Animals were submitted to intracardiac perfusion with 0.9% NaCl followed by 10% formalin. Brains were removed SGI-1776 research buy and post-fixed for 48 h at 4 °C and serial 40 μm-thick sections were cut using a cryostat (CM1900,

Leica, Germany). Brain sections were stained with 1% neutral red for light microscopy analysis. The actual location of microinjection sites in the area was determined after the analysis of serial sections and represented according to the rat brain atlas of Paxinos and Watson (1997). Nonlinear regression analysis

was used to compare MAP and HR results from different Ach doses microinjected into the vlPAG or the dPAG. Baseline MAP and HR values were compared using the paired Student’s t test (before treatment vs. after treatment). Percentages of response inhibition by vlPAG pretreatment with muscarinic antagonists were analysed utilizing nonlinear regression Clomifene analysis. We used Prism software (GraphPad, USA) to perform statistical analysis. *P < 0.05 was assumed to be statistically significant. The authors would like to thank Ms. Ivanilda A.C. Fortunato, Idália I.B. Aguiar and Simone S. Guilhaume for technical support. Cristiane Busnardo (Fapesp proc. 2009/05308-8) is a post-doctoral fellow in the Department of Pharmacology of the School of Medicine of Ribeirão Preto-USP. Milena Vieira Deolindo (Fapesp proc. 07/50166-1) is a PhD student enrolled in the Graduation Program on Pharmacology of the School of Medicine of Ribeirão Preto-USP. "
“Neuroticism, a stable temperament that arises early in life, is one of the best-established vulnerability factors for depression (Kotov, Gamez, Schmidt, & Watson, 2010). High levels of neuroticism are associated with an increased overall risk of depression (e.g.

1 nM–300 μM) were determined

The role of NO in the relax

1 nM–300 μM) were determined.

The role of NO in the relaxation induced by ACh was analyzed by incubating the selleck inhibitor vessels with NG-nitro-l-arginine methyl ester (L-NAME, 100 μM, nonspecific NOS inhibitor) for 30 min before phenylephrine or KCl administration. The contribution of K+ channels to ACh-induced relaxation was assessed in aortas previously incubated for 30 min with the K+ channel blockers tetraethylammonium (TEA, 2 mM, nonselective blocker of K+ channels), 4-aminopyridine (4-AP, 5 mM, Kv blocker), iberiotoxin (IbTX, 30 nM, selective BKCa blocker), apamin (0.5 μM, selective blocker of small-conductance Ca2+-activated K+ channels — SKCa) and charybdotoxin (ChTX, 0.1 μM, blocker of KCa and Kv). In some experiments, the concentration–response curves to sodium nitroprusside (SNP, 0.01 nM–0.3 μM) were performed Selleckchem BMS 354825 in segments contracted with phenylephrine (1 μM). The role of the Kv and BKCa channels in the SNP-induced

relaxation was analyzed by incubating the vessels with 4-AP and IbTX, respectively, for 30 min before phenylephrine administration. The influence of the endothelium on the response to SNP in untreated and lead-treated rats was investigated after its mechanical removal, which was performed by rubbing the lumen with a needle. The absence of endothelium was confirmed by the inability of 10 μM acetylcholine (ACh) to produce relaxation. The functional activity of the Na±/K+-ATPase was measured in segments from untreated and lead-treated rats using K+-induced relaxation, as described by Weeb and Bohr (1978) and modified by Rossoni et al. (2002). After a 30-min equilibration period in normal Krebs, the preparations were incubated for 30 min in K+-free Krebs. The vessels were subsequently pre-contracted with

phenylephrine, and once a plateau was attained, the KCl concentration was increased stepwise (1, 2, 5 and 10 mM) with each step lasting for 2.5 min. After a washout period, the preparations were incubated next with 100 μM ouabain (OUA) for 30 min to inhibit sodium pump activity, and the K+-induced relaxation curve was repeated. To study the involvement of NO, inducible NO synthase (iNOS) and K+ channels in OUA-sensitive Na+K+-ATPase functional activity, the rings were incubated with L-NAME (100 μM), aminoguanidine (50 μM) and TEA (2 mM), respectively. Moreover, the influence of the endothelium was investigated, repeating the same protocols after its mechanical removal. The oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate O2− production in situ, as previously described by Wiggers et al. (2008). Hydroethidine freely permeates cells and is oxidized in the presence of O2− to ethidium bromide, which is trapped by intercalation with DNA. Ethidium bromide is excited at 546 nm and has an emission spectrum of 610 nm.

, 2007) NGAL concentration was measured by Therapeutics Research

, 2007). NGAL concentration was measured by Therapeutics Research Centre, University of Queensland, Brisbane, Australia in October 2009. These assays were conducted using the Triage® NGAL Test, a point-of-care fluorescence immunoassay using the Triage Meter according to product guidelines. Median values and inter-quartile ranges were determined for each

renal biomarker and compared non-parametrically. The rate of change of creatinine and cystatin C concentrations in serial samples were determined and compared between survivors and deaths. Receiver-operating characteristic (ROC) curves were constructed to determine the best threshold (as determined by Youden’s index (Youden, 1950)) for the rate of change of creatinine (dCr/dt) and cystatin C (dCyC/dt) concentrations for predicting death, including likelihood ratios, sensitivities and specificities. Sensitivity is the proportion of Thiazovivin nmr all deaths that were predicted to die Palbociclib by the test (cut-off), specificity is the proportion of survivors predicted to survive by the test. All analyses were conducted

using GraphPad Prism version 4.03 for Windows, GraphPad Software, San Diego, USA, and P < 0.05 was considered statistically significant. Prediction of outcome on the basis of the admission paraquat concentration was determined according to Senarathna et al. (2009). Paraquat exposure was confirmed in 20 patients who were eligible for inclusion; the other 6 patients were excluded. 14 of the 16 patients who were discharged alive were followed up in the community and three of these patients subsequently died. Altogether, seven patients died at 18 h, 48 h, 65 h, 11 days, 12 days, 15 days and 20 days after exposure. On the basis of the admission paraquat concentration, all actual deaths were predicted to die according to the Proudfoot nomogram (Eddleston et al., 2003). A total of 86 blood samples from different Reverse transcriptase time points were assayed, although in some cases the volume was too

small for every test to be conducted. Serial concentrations of creatinine and cystatin C for individuals are shown in Fig. 1a and b, respectively. In the case of creatinine and cystatin C, increasing concentrations during the first 24–48 h were observed which were suitable for further analyses. Because biochemical data from patients who died were unavailable beyond 75 h post-ingestion, all subsequent analyses in surviving patients were limited to data obtained within the same period. The plasma concentration of NGAL was measured in 14 patients and serial changes are shown in Fig. 1c. No relationship was observed that could be used to separate survivors from the four deaths captured in this study (which occurred 48 h, 65 h, 11 days and 12 days post-ingestion). Of these deaths, NGAL was not elevated in one patient while in the other three patients the highest concentration was 331 ng/mL and most were less than 100 ng/mL.

, Ltd , Ningbo, China), 4 5 m × 1 5 m × 1 7 m (length × width × h

, Ltd., Ningbo, China), 4.5 m × 1.5 m × 1.7 m (length × width × height) in size. The air

temperature and relative humidity in the chamber were controlled using signaling pathway electric resistance heaters and a bubbling system. The air temperature in the chamber at nighttime (19:00 to 7:00) was maintained at 25–28 °C with a wind speed of 0.5 m s− 1. The relative humidity was maintained at 75%–85%, similar to that of the unwarmed control environment. The air temperatures in the rice canopy in the chamber and the ambient environment were monitored every 10 min at night with a Thermo Recorder (ZDR-41, Zeda Instrument Co., Ltd., Hangzhou, China). The differences in rice canopy air temperatures at nighttime were automatically adjusted to approximately 3.0–3.5 °C higher in the chamber than in the ambient control environment (Fig. 1). Germinated rice seeds were sown in plastic boxes on 13 May 2010. After one month of growth, rice seedlings were transplanted to Protein Tyrosine Kinase inhibitor the plastic pots. There were two holes seedlings for each pot and two seedlings for each hole. Fertilizer was applied as 0.75 g N, 0.38 g P2O5 and 0.38 g K2O per pot. All of the P2O5 and K2O and 50% of the N were applied

as basal dressing. Half of the remaining N was applied as side dressing at the early tillering stage in the latter of June, and the rest of the N was applied at panicle initiation in the latter part of August. Water depth in all pots was maintained at about 5 cm above the soil surface during the entire rice growing cycle. All pots were kept under ambient conditions outside the chamber before rice anthesis. During the post-anthesis phase, half of the pots were

placed in the chamber for 12 h at night (from 19:00 to 7:00) and moved outside after 7:00 every day. The warmed and unwarmed pots were kept in the same ambient environment at the daytime from 7:00 to 19:00 every day during the post-anthesis phase. At the anthesis and maturity stages, plants from three pots of each treatment were sampled and divided into leaf, stem, and panicle. All plant samples were oven-dried at 80 °C for 24 h and weighed. Post-anthesis biomass accumulation was calculated as the difference in total aboveground dry matter between the anthesis stage and harvest. Nine pots from each treatment were harvested to determine grain yield and its components. At 0, 21 and Chorioepithelioma 35 days post-anthesis (DPA), fifteen flag leaves of main stems were selected for measurements of net photosynthesis rate (from 9:00 to 11:00) and night respiration rate (from 22:00 to 23:00) with a portable photosynthesis system (Li-6400; Li-Cor, Inc., Lincoln, NE, USA). Another fifteen flag leaves were sampled in each treatment at 7, 14, 21, 28 and 35 DPA for measurements of chlorophyll a and b contents by the method of aqueous acetone extraction [13]. At the anthesis stage, approximately 200 rice panicles were labeled for the determination of grain filling rate.

1-c) The F3 progenies derived from these five recombinants showe

1-c). The F3 progenies derived from these five recombinants showed the expected segregating or homozygous resistant responses after challenge with isolate 001-99-1, completely corresponding to their genotypes at the two marker loci ( Fig. 1-c). Thus Pi60(t) was delimited to a 274 kb region flanked by InDel markers K1-4 and E12. For fine mapping of the Pi61(t) locus, a total of 2102 99-26-2-susceptible F2 individuals were genotyped with 14 InDel and SSR markers, viz. G2, G7, RM101, E4, T7, M1, M2, M9, G8, 12-5, P1, RRS63, RM27990 and 12-6 ( Table 3). As a result, Pi61(t) was located to a 0.15 cM interval (200 kb) on the short arm of chromosome 12, flanked by

markers M2 (0.10 cM) and Vemurafenib cost S29 (0.05 cM) and co-segregating with marker M9 ( Fig. 2-b). For Pi60(t), the target 274 kb region (6,374,147–6,648,601 bp) was covered by four PAC/BAC clones, including 48 putative genes annotated in the Gramene and

TIGR databases ( Fig. 1-d); these included 8 intact NBS-LRR genes (Os11g11550, Os11g11580, Os11g11770, Os11g11790, Os11g11810, Os11g11940, Os11g11950 and Os11g11960), 12 expressed proteins, 16 hypothetical proteins and 12 retrotransposons. Sequence alignment of the NBS-LRR genes showed that 93-11 contained only six NBS-LRR genes, viz. BGIOSGA034264, BGIOSGA034263, BGIOSGA035032, BGIOSGA035036, BGIOSGA034259 and BGIOSGA034258, corresponding to Os11g11770, Os11g11790 (SasRGA4 allele of Pia), Os11g11810 (SasRGA5 allele of Pia), Os11g11940, Os11g11950 and Os11g11960 at identity levels of 79.1%, 89.5%, 45.7%, 96.4%, 84.5% and 89.2% in

protein sequence, respectively. For Pi61(t), the target 200 kb region (9,924,675–10,124,186) in the Nipponbare sequence was covered by Urocanase six PAC/BAC clones, including 44 putative genes annotated in the Gramene and TIGR database ( Fig. 2-c), viz. 5 tandem NBS-LRR type genes, Os12g17410, Os12g17420, Os12g17430, Os12g17480 and Os12g17490 in a 40 kb cluster, 21 retrotransposons, 1 transposon, 11 hypothetical proteins and 6 expressed proteins. However, only four NBS-LRR genes can be amplified in cv. 93-11 using the specific primers ( Table 4), viz. BGIOSGA018510, BGIOSGA018508, BGIOSGA018507 and BGIOSGA018506, corresponding to Os12g17410, Os12g17430, Os12g17480 and Os12g17490 at identity levels of 68.7%, 99.3% (2-amino acid differences), 99.7% (3-amino acid differences) and 99.7% (3-amino acid differences) in protein sequences, respectively. Two other major blast R genes, Pi30(t) and cloned Pia/PiCO39, were previously mapped in the vicinity of Pi60(t) (6,374,147–6,648,601 bp) on chromosome 11 [11], [37] and [38]. Pi30(t) was roughly located within an interval of 6.1 Mb (441,392–6,578,785), and presumed to be Pia [59]. Sequencing of the two Pia/PiCO39 alleles in 93-11 showed that the two alleles, viz.

Candida suspensions were spectrophotometrically standardised to a

Candida suspensions were spectrophotometrically standardised to a concentration of 1 × 106 cells/mL. The resulting suspensions were used for all the further procedures. Aliquots of 100 μL of Candida standardised suspension were individually transferred to separate wells of a 96-well microtitre plate. After inoculation, an equal volume of diluted Cur solutions (100 μL) was added to the appropriate wells to give final concentrations of 5, 10 and 20 μM.

After dark incubation of 1, 5, 10 and 20 min, the samples were irradiated on the LED device for 4 min, which corresponded to 5.28 J/cm2 (P+L+). 41 To determine whether LED light alone had any effect on cell viability, additional samples were made with no PS (P−L+). The effect of Cur alone was also determined by exposing the yeast suspensions to the PS in an identical manner to those described above, but with no Protease Inhibitor Library concentration light exposure (P+L−). The suspensions that were not exposed to LED light or Cur acted as overall control (P−L−). All experiments were performed five times on two independent occasions. The microtitre plate containing the no-light samples was kept in the dark for 24 min, corresponding to the pre-irradiation time plus light exposure time.

Ten-fold serial dilutions (10−1, 10−2 and 10−3) were generated from the fungal click here suspensions and plated on SDA in duplicate. The plates were then aerobically incubated at 37 °C for 48 h. After incubation, yeast colony counts of each plate were quantified and the colony forming unit per millilitre (CFU mL−1)

was determined. A loopful of recently cultivated yeast was subcultured in RPMI 1640 overnight in an orbital shaker (AP 56, Phoenix Ind Com Equipamentos Científicos Ltda, Araraquara, SP, Brazil) at 120 rpm and 37 °C. The cells grown were harvested by centrifugation at 4000 rpm for 7 min, and the supernatants were discarded. The pellet was washed twice in PBS, and finally resuspended in PBS. Candida suspensions were spectrophotometrically standardised to a concentration of 1 × 106 cells/mL. Aliquots of 100 μL of the resulting aminophylline standardised Candida cell suspensions were transferred to appropriate wells of a 96-well microtitre plate and incubated at 37 °C in an orbital shaker (75 rpm). After 90 min of the adhesion phase, the supernatants were removed from the plate wells and gently washed twice with 150 μL of PBS to remove the non-adherent cells. Next, 150 μL of freshly prepared RPMI 1640 were added to each well and the plates were incubated in an orbital shaker for 48 h at 37 °C in order to generate single-species biofilms. After incubation, the wells were carefully washed twice with PBS to remove non-adherent cells. Aliquots of 150 μL of Cur at 20, 30 and 40 μM were added to each appropriate well directly onto the biofilm. The experimental conditions were identical to those of the planktonic cultures: P+L+, P−L+, P+L− and P−L−. All experiments were performed five times on three independent occasions.