Next, we addressed the molecular role of Prdm8 within this repressor complex. Some members of the Prdm family have been shown to function as sequence specific transcription factors, while others are known to function as cofactors to mediate transcriptional repression (Davis et al., 2006, Duan et al., 2005, Gyory et al., 2004, Hayashi

et al., 2005 and Kim et al., 2003). Given the phenotypic similarity between Bhlhb5 and Prdm8 mutant mice, we first considered the hypothesis that a Bhlhb5 dimer is recruited to specific DNA elements through its consensus binding motif and then recruits Prdm8 to mediate transcriptional repression. If so, we reasoned that Bhlhb5 would bind normally to its DNA targets Adriamycin nmr SRT1720 nmr in the absence of Prdm8, but that Prdm8 would not associate with these sites in the absence of Bhlhb5. To address this hypothesis, we performed ChIP-qPCR from the dorsal telencephalon of wild-type or mutant mice and analyzed the binding of Bhlhb5 and Prdm8 at the RP58 promoter. As we had shown above ( Figures 5F and 5I), we again found that both Bhlhb5 and Prdm8 display robust binding to the proximal promoter of RP58 in wild-type mice ( Figure 6Bi). Furthermore, Bhlhb5 shows similar binding to

the RP58 promoter when ChIP-qPCR was performed in Prdm8 mutant mice, indicating that the binding of Bhlhb5 at this promoter is not dependent on Prdm8 ( Figure 6Bii). In sharp contrast, however, we found that Prdm8 was not bound to the RP58 promoter in Bhlhb5 mutant mice ( Figure 6Biii). Note that the observed absence of Prdm8 binding at this site is not due to a general absence of Prdm8 protein in Bhlhb5−/− mice (e.g., see Figure 1C). Thus, the inability of Prdm8 to bind to the RP58 promoter in the absence of Bhlhb5 suggests that Prdm8 requires Bhlhb5 for targeting to this genetic locus. Furthermore, the dependence of Prdm8 on Bhlhb5 for sequence-specific targeting to DNA appears to be a general phenomenon since we observed similar results when we tested several

other genomic loci including the Bhlhb5 promoter ( Figure S8A) and the Mephenoxalone Bhlhb5 binding site in the first Cdh11 intron ( Figure S8B). Based on these findings, we suggest a model in which Bhlhb5 functions by binding to specific DNA elements possibly as a homodimer and then recruiting Prdm8 to mediate the repression of target genes (Figure 8A). When the Bhlhb5 alone is present, it can bind to target genes, but it cannot repress them. Likewise, when Prdm8 alone is present, target genes are also not repressed, in this case because Prdm8 does not bind to DNA in the absence of Bhlhb5. Thus, both factors are required to mediate transcriptional repression of a specific set of target genes so that, when either Bhlhb5 or Prdm8 is knocked out, common genes are upregulated and highly similar phenotypes result.

, 2008a and Triplett et al., 2009). However, this topographic axon targeting precedes the major periods of synapse formation, functional maturation, and input refinement that coincide with the onset of environmental drive (Lu and Constantine-Paton, 2004). This “consolidation” phase of refinement

in the sSC is likely to involve both synaptic elaboration and elimination as individual retinal and cortical axons sort their terminals on postsynaptic cells. Little is known about this process or its cellular mechanisms, which allow precise refinement of converging projections. Alectinib purchase Simple Hebbian mechanisms are predicted to suppress the later-arriving cortical inputs unless their activity is closely synchronized with that of earlier synapses (Constantine-Paton et al., 1990). This has led us to the hypothesis that late arriving, broadly mapped,

inputs such as those from VC have specific adaptations to enable successful wiring. Here we control EO to precisely define the onset of pattern vision, and combine this with in vivo anterograde labeling of retinal http://www.selleckchem.com/products/BMS-754807.html and cortical afferents to sSC and anatomical reconstruction of cells expressing a genetically encoded eGFP in a population of collicular neurons located at the interface of the two projections. We follow changes in these neurons and the cortical input in age-matched animals with opened or closed eyelids using quantitative structural and whole-cell patch clamp analyses. These approaches identify structural and functional changes at synapses over the EO interval of identified sSC neurons, and highlight those changes specifically controlled by early visual experience. In vivo multiunit recording of spontaneous and visually evoked activity in sSC and VC layer 5 of intact awake pups are used to reveal the relative latencies of vision-driven activity in cortex and sSC. These data provide evidence for a spike-timing dependent mechanism

that underlies the successful stabilization of cortical synapses on sSC neurons with EO, Rolziracetam and the net synaptic loss observed when the eyes remain closed. In this study, we focus on a distinct population of sSC neurons with vertically distributed and predominantly dorsal dendrites (dorsally oriented vertical [DOV] neurons) lying within both retinal and cortical terminal zones. These cells were labeled early in eGFP mice and are also identifiable with IR-DIC optics using laminar position, somatic shape, and dendritic orientation (Tokunaga and Otani, 1976). Based on earlier work (Lu and Constantine-Paton, 2004, Philpot et al., 2001 and Yoshii et al., 2003), and the finding that normal levels of PSD-95 are required to produce NMDA receptor-dependent long-term potentiation and depression (Béïque and Andrade, 2003 and Migaud et al., 1998), we hypothesized that PSD-95 is crucial to rapid, EO-induced synaptic remodeling through its stabilization of synapses sensitive to the new stimuli. PSD-95 is highly expressed in DOV neurons and sSC synapses (see Figure S1 available online).

, 2001). The remaining error rate could then be accounted for by the stochastic nature Crenolanib and

inherent noise of guidance cue binding as considered in the stochastic version of our model, and discussed in more detail in Mortimer et al. (2009). A particularly intriguing aspect of the model is that it provides a mechanism for integrating information from multiple attractive and repulsive cues. It is known that receptors for guidance cues can interact to determine growth cone responses (Stein and Tessier-Lavigne, 2001). However, alternatively (or in addition) guidance cues could interact via their effect on the calcium signaling pathway we have modeled. For instance, the application of repulsive guidance cues, MK-2206 solubility dmso which individually produce only small calcium influxes, could together produce large influxes, potentially

cancelling the repulsion, or even switching it to attraction. This possibility remains to be explored. The mathematical model of the signaling pathway shown in Figure 1A is adapted from that of Graupner and Brunel (2007), originally proposed for the switch between LTP and LTD. We extended the model to two compartments in order to provide a “distribution” of inputs and outputs over the growth cone. This allows the determination of whether each combination of calcium, cAMP, and spatially nonuniform calcium influx results in attraction or repulsion. For details see Supplemental Experimental Procedures. All experimental procedures involving animals were approved by the Animal

Ethics Committee of the University of Queensland. SCGs were isolated by microdissection from postnatal day 1–3 Wistar rat pups as per Higgins et al. (1991). The SCGs were then cut into thirds, Diflunisal incubated in 0.25% trypsin (GIBCO, Melbourne, Australia) at 37°C for 15 min and then triturated through flamed-polished Pasteur pipettes for 10 min to dissociate individual cells. The cells were plated in Opti-MEM solution (GIBCO) containing 10 μg/ml natural mouse laminin (Invitrogen, Melbourne, Australia) and 0.5 nM NGF (2.5S mouse NGF; Biosensis, Thebarton, Australia) and incubated overnight at 37°C on 35 mm Petri dishes. Growth cone turning assays were carried out at 37°C on a heated microscope stage (Fryer Co., Huntley, IL). Growth cones with a straight trailing axon of more than 20 μm were selected for the assay. Steep gradients of 10%–15% change in concentration across 10 μm were generated using the pulsatile ejection method previously reported by Lohof et al. (1992) (see also Pujic et al., 2008). Forty kilodaltons dextran labeled with fluorescent tetramethylrhodamine (Molecular Probes Inc., Melbourne, Australia) was added to the pipette solution to monitor the chemical gradient produced. KT5720 (Alexis Biochemicals, San Diego, CA) or Sp-cAMPs (BioLog, Bremen, Germany) were added to the prewarmed assay medium when appropriate.

All other unlabeled chemicals and reagents were analytical graded. A. bisporus (AB) were commercially purchased from Cuddalore in vegetable markets, Tamil Nadu. A voucher specimen (No. 217) was deposited in Department of Botany, Annamalai University. Powder of AB (50 g) were extracted by stirring with 500 ml of ethanol (30 °C) at 150 rpm for 24 h

and filtered through Whatman No. 4 filter paper. The residues of ethanol extract was then rotary evaporated at 40 °C to dryness, re-dissolved in ethanol to a inhibitors concentration of 10 mg/ml and stored at 4 °C for further use. The terpenoids content of the A. bisporus extracts were determined by the method of Puncal D Test. The flavonoid content of the sample were detected with few ml of ammonia shows the presence of fluorescence OTX015 ic50 this website at 366 nm indicates the presence of flavonoids. The steroids content of the sample were detected by added a few ml of concentrated sulfuric acid solution to the extract. Formation of green color indicates the presence of steroids. The Carbohydrates and Sugars content of the sample were detected by added a few ml of concentrated sulfuric acid solution to the extract and heated formation of charring indicates the presence of carbohydrates. The alkaloids content of the sample were detected by the method of Dragandorff’s test. The

proteins content of the sample were detected by the method of Ninhydrin test. The Tannins content of the sample were detected by 1 ml of

Aluminum chloride. The total phenolic concentration in ABE and ABCNPs was expressed as gallic acid equivalents and was measured according to the method described by Bandoniene et al8 with slight modifications. The Total flavonoid contents (TFC) of the A. bisporus were extracted with 5% NaNO2, 10% AlCl3 and 1 M NaOH were measured at 510 nm with a known quercetin concentration as a standard. The results were expressed as milligrams of quercetin equivalents (CE) per gram of sample. AB loaded chitosan nanoparticles were synthesized by ionic gelation method using tripolyphosphate as a gelating agent. A known amount of chitosan was dissolved in 1% (v/v) acetic acid and allowed to stir for 1 h 3 mg/ml AB ethanol GPX6 extract have prepared already was then added to the freshly prepared chitosan dispersion. The pH of the medium was maintained at 5.0 using 1 M NaOH and then further stirred for 1 h. Finally, 1 mg/ml of TPP was added to the chitosan- AB ethanol extract under mild magnetic stirring. The resulting mixture was allowed to stir for 2 h to form AB encapsulated chitosan nanoparticles. The AB loaded chitosan nanoparticles were collected after the centrifugation of 10,000 rpm for 45 min with 4 °C.9 The powdered samples were collected with the help of lyophilizer and stored at 4 °C for further use. The ABE and ABCNPs were used for analyzing their DPPH radical scavenging activities where determined by the method of Chen.

This solution was used as standard solution. The magnesium was estimated by titrimetric method using standard EDTA with Erio-chrome black-T indicator at pH10 using ammonia as a buffer. Vitamin B was determined spectrocolorimetrically

with the reagent ferric sulfate and KCNS. Vitamin A was estimated spectrocolorimetrically using acidic antimony chloride reagent by the standard graph method. The total flavonoid and phenolic contents were quantified by spectrophotometeric method using Folin’s Ciocalteaus reagent. The other secondary metabolites such as alkaloids, tannins, lignins, glycosides, serpentines, terpenoids and saponins quantified by HPLC method and C18 general purpose column. The inhibitors mobile phase consisted of solvent A (Methanol) and solvent B (0.5% (v/v) orthophosphoric acid in water). The data were interpreted by the Millenium Chromatography Manager V4.0 Software.4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 Fresh Panobinostat concentration leaves were collected,

shade dried and powdered mechanically. About 100 g of the powder were extracted with 1000 mL of 70% ethanol by hot percolation method using soxhlet extractor for 4 h. The extract obtained was evaporated at 45 °C to get a semi solid mass. The yield of ethanolic extract was found to be 40%. This extract was used Anticancer Compound Library supplier for further studies.14, 15, 16, 17 and 18 To determine the DPPH assay of sample by Gyamfi et al., method, free radical scavenging potential of P. wightianus leaf extracts was tested against a methanolic solution of DPPH (α, α-diphenyl-β-picryl hydrazyl). When antioxidants react with DPPH, the DPPH was converted only to α, α-diphenyl-β-picryl hydrazine with a discoloration. The degree of discoloration indicates the scavenging potentials of the antioxidant extract. The change

in the absorbance produced at 517 nm has been used as a measure of antioxidant activity. The change in absorbance of the samples was measured. Free radical scavenging activity was expressed as the inhibition percentage calculated using the formula. Percentageofanti-radicalactivity=[A−B/A]×100where, ‘A’ is absorbance of control & ‘B’ is absorbance of sample. To determine the reducing power assay of sample by Yildrim et al., 1 mL of leaf extract was mixed with phosphate buffer (2.5 mL 0.2 M, pH 6.6) and potassium ferricyanide (2.5 mL). The mixture was incubated at 50 °C for 20 min. A portion (2.5 mL) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and ferricchloride (0.5 mL, 0.1%) and absorbance measured at 700 nm. Increased absorbance of the reaction mixture indicates stronger reducing power. The activity was compared with ascorbic acid standard. Percentagescavengingactivity=Acontrol−AtestAcontrol×100where Acontrol is the absorbance of the control. Atest is the absorbance in the presence of the sample.

8 ml/min was used. Detection was carried out at 220 nm. The injection volume was 20 μl; analysis was performed at ambient temperature. An accurately weighed quantity of miglitol (10 mg) was transferred to 10 ml volumetric flask and dissolved in water and diluted up to the mark with water to get a 1 mg/ml solution.

The series inhibitors standard solutions were prepared by dilution of aliquots of the standard stock solution with mobile phase to get concentration in the range of 10–50 μg/ml of miglitol. Twenty microliter of the each standard solution was injected to HPLC system. The peak areas were plotted against the corresponding concentrations to obtain the calibration graph. The system suitability is used to verify whether the resolution and reproducibility of the chromatographic system are adequate for analysis to be done. The tests Selleck JQ1 were performed by collecting data from five replicate injections of standard solutions. A 20 μl standard drug solution was injected separately and system suitability parameters Entinostat cell line were recorded. Twenty tablets were weighed and average weight was calculated. The tablets were triturated to a fine powder. An accurately weighed quantity of powder equivalent to 10 mg of miglitol

was transferred to 50 ml volumetric flask. About 20 ml of water was added and sonicated for 15 min; further volume was made up to the mark with same solvent. The resulting solution was filtered and filtrate was appropriately diluted with mobile phase to get approximate conc. of 25 μg/ml of miglitol. Twenty micro liters of the test and standard solutions were injected separately after the equilibration of mobile phase with stationary phase. The chromatograms were recorded upto 8 min and area of each peak was noted. The optimized RP-HPLC method was completely validated according to the procedure described in ICH guidelines and United State Pharmacopoeia for validation of analytical methods. The performance parameters evaluated for the method were linearity, precision, accuracy, limits of detection and quantitation

and ruggedness. Linearity was studied by diluting standard stock solution at five aminophylline different concentrations (n = 3) covering the range of 10–50 μg/ml for miglitol, respectively. A graph was plotted for the concentration of the corresponding drug versus peak area. The correlation coefficient (r2) for each drug was calculated. Repeatability study was carried out by analyzing sample solution six times, at 100% of test concentration within the same day using proposed method. Similarly, the intra and inter day precision was evaluated by analyzing tablet sample on the same day and on different days at different time interval, respectively. The contents of drugs and the % relative standard deviation (% R.S.D.) value were calculated.

190,000 animal bites were reported to the National Center for Disease Prevention and Control (NCDPC) in 2008, 50% of the bite victims were children. One highlight of the Manila meeting was the enthusiastic acknowledgment of the commitment made by the Philippines government to supporting AP24534 molecular weight rabies control efforts. Dr Yolanda Oliveros, Director IV, NCDPC, Department of Health (DOH), stressed that the country had strengthened its National Rabies Prevention and Control Program by enacting the “Anti-Rabies Act” of 2007, which

supports the rabies program, with the aim of eliminating rabies throughout the Philippines by 2020. She also mentioned that several pilot projects had already been initiated. Three ongoing pilot projects were reviewed during the AREB meeting; two of them in Visayas, one in the province of Camarines Sur. The rabies-free Visayas project was launched recently. Visayas is one of the three island groups in the Philippines (the other two being Luzon and Mindanao). Almost one-third of the total cases of human rabies in the Philippines occur in this region, which has a population in excess of 17 million (19% of the Philippine population). The project, coordinated by WHO and funded by the Bill & Melinda Gates Foundation, is conducted through the collaborative

efforts of the Department of Health, the Department Selleckchem AG 14699 of Agriculture, and local governmental units. It aims to prevent human rabies through the control and eventual elimination of canine rabies. The main strategy of the project is based on community participation and relies on increasing dog vaccination coverage while concomitantly optimizing management of humans exposed to rabies. The project also includes promotion of local community involvement in understanding ‘responsible pet ownership’ as well as increased education on how to prevent rabies. In Bohol (one of the Visayas islands, with a total population of 1.4 million), the Rabies Prevention and Eradication Program is already in progress. This

4-year project (2007–2010) is supported by the national government and the Bohol Provincial Government, all the Alliance for Rabies Control and a private Swiss foundation. Bohol was the first region in recent years to successfully utilize a “one health approach” to prevent and control rabies in the Philippines. A survey of progress to date indicates that specific education about how to prevent rabies has been successfully integrated in the Modulators elementary school curriculum; 71% of the dogs in the province have been vaccinated; and 85% of the households are aware of activities related to dog rabies control. As a result of the implementation of the program, no human rabies case was reported in Bohol in 2009, whereas approximately 10 human deaths were reported annually before the program was initiated.

All procedures relating to animal care and treatment conformed to institutional and NIH guidelines. Whole-mount tyrosine hydroxylase immunohistochemistry was performed on E16.5–E18.5 mouse embryos, as previously described (Kuruvilla et al., 2004). For NFAT immunostaining, sympathetic neurons were treated with 100 ng/ml NGF for 30 min, and neurons were fixed and immunostained using pan-NFAT antibody, β-III-tubulin, and DAPI (4′,6-diamidino-2-phenylindole). Images representing 1 μm optical slices were acquired using a Zeiss LSM 510 confocal scanning microscope equipped with diode (405 nm), Ar (458–488 nm),

and He/Ne (543–633) lasers. Sympathetic neurons were harvested from P0.5 Sprague-Dawley rats and were grown in mass cultures or compartmentalized cultures, as described previously (Kuruvilla et al., 2004). Dissociated DRG neurons were isolated from E15–16 rats and were grown in mass cultures or compartmentalized cultures, using CFTR activator culture conditions similar to that described for sympathetic neurons. Plasmids, adenoviral vectors, pharmacological reagents, and see more antibodies used in this study

are described in detail in Supplemental Experimental Procedures. Axon growth in compartmentalized cultures was quantified by capturing phase contrast images of the distal axon compartments over 8 hr or consecutive 24 hr intervals using a Zeiss Axiovert 200 microscope with a Retiga EXi camera. Rate of axonal growth (μm/day) was measured using Openlab 4.04. For all neurite growth assays in mass cultures, images were taken using an Axio Imager M1 (Zeiss) microscope, and length of the longest neurite was measured using Axiovision software (Zeiss). Measurements from 30 to 50 neurons were averaged for each condition for a single experiment. Details of analyses of neurotrophin-dependent neurite growth with dynamin1 phosphopeptides and short-term changes in growth cone morphologies are described in Supplemental Experimental

Procedures. Sympathetic neurons were Phosphatidylinositol diacylglycerol-lyase infected with NFAT-luciferase reporter adenovirus for 24 hr, and then neurons were stimulated with control media, NGF, or NT-3 (100 ng/ml) for 2, 8, and 24 hr; reporter gene activity was assessed with Luciferase Reporter Assay System (Promega, E1910). Similar analyses were used to report NFAT transcriptional activity in DRG neurons. Cell-surface biotinylation assays were performed in cultured sympathetic neurons as previously described (Kuruvilla et al., 2004). Live cell antibody feeding assays were performed as previously described (Ascano et al., 2009). For analysis of tyrosine phosphorylation of PLC-γ, sympathetic neurons were treated with NGF or NT-3 (100 ng/ml) for 30 min at 37°C. Cells were lysed with RIPA solution, and lysates were subjected to immunoprecipitation with anti-phosphotyrosine (PY-20; Sigma) and were incubated with Protein-A agarose beads (Santa Cruz Biotechnology). Immunoprecipitates were then immunoblotted for PLC-γ.

Moreover,

fluctuations in the effects of feature-based but not spatial attention were coordinated across hemispheres. This suggests that spatial attention acts locally within a hemisphere, while feature-based attention operates globally across hemispheres (Figure 1B, bottom panel). It is unknown how this global feature-based modulation is implemented, but it likely involves a common input into areas V4 of both hemispheres from neurons that are feature selective. Zhou and Desimone’s study, previously discussed in this article, may provide an answer to this question. Projections from feature selective neurons in FEF may target sensory neurons in visual cortex with similar preferences and produce the observed FSG effects. This would AZD2014 imply a role of the FEF in the origins of both FM and FSG effects. Another possibility is that other areas containing selectivity for stimulus features such as the neighboring dorsolateral prefrontal cortex (Zaksas and Pasternak, 2006) may DNA Damage inhibitor provide top-down signals for the FSG modulation, since this type of attentional modulation does not seem to require the finer spatial resolution of the FEF map. These are important issues that need to be further investigated in future studies. In summary, from these two studies

we have learned that the mechanisms of feature-based attention are diverse and include different subtypes likely triggered by different task demands (e.g., FM during visual search, and FSG during detection/discrimination involving sustained covert attention). Moreover, the FEF, a structure involved in spatial attention, seems to play a role in FM during visual search. Histamine H2 receptor The mechanisms producing the global effects of FSG remain, so far, unknown. “
“The year 2011 marks 100 years since Marie Curie, one of the most notable scientists of the 20th century, was awarded her second Nobel Prize. In an era when it was still unthinkable for a woman to have a career, let alone one as a scientist, Dr. Curie faced—and overcame—insurmountable odds in her quest for knowledge. Dr. Curie was one of many pioneers whose courage, tenacity, and groundbreaking achievements spurred generations

of young women to follow in her inspiring footsteps, and today, the notion that women do not belong in the sciences is as antiquated as corsets and foot binding. Women across the globe have come a long way since the days of Dr. Curie, and more are opting to pursue careers in the life sciences. In Asia, changing mindsets and the recent growth in the bioscience sector has also enhanced career prospects in recent years. For instance, a decade or more ago, educational and career opportunities in the biosciences in this region were few and far between. Hence, it was not unusual for a young woman with a keen interest in the life sciences to head west to the US or Europe for education and training, as that was where pioneering research and exciting new advancements and breakthroughs were occurring.

During the “early phase” of the response (40–140 ms after stimulus onset), the population-response (Figures 2A and 2B) and activation maps (Figure 2C) were similar among the contour and noncontour trials. Maps measured from both conditions showed clear activation patches

corresponding to the individual Gabor elements comprising the stimuli. That is, the population response in the early phase appeared to encode mainly the representation of individual Gabor elements without any obvious circle/background segregation (see also Figures S1A–S1D available online). To further analyze this, we made a scatterplot of the population response in individual V1 pixels for the two conditions (Figure 2D). The red lines depict the activity differences Palbociclib between contour and noncontour trials before stimulus onset, i.e., the 1% and 99% percentile of the differences histogram (these values were then extrapolated to later times of stimulus presentation). Most pixels in the circle and background areas showed similar response amplitude and therefore lie within the red boundaries (Figure 2D). The pixel differences DNA Damage inhibitor histograms (contour-noncontour; Figure 2E) are centered on zero (d′ = 0.04 between circle and background histograms. This is not significantly different from d′ computed for trials with shuffled labels,

mean d′ = 0.04, p = 0.53, 100 iterations). This means that from 60 to 80 ms the population response in V1 pixels did not differ between the contour and noncontour conditions. This situation changed completely in the “late phase” of the response (150–250 ms after stimulus onset). Whereas the population response in the circle area was only slightly higher for the contour condition (Figure 2A, late phase), the time course of the population response in the background area showed suppression (Figure 2B). This suppression was prominent in the contour condition, starting∼140 ms after stimulus onset and reaching minimal amplitude at

∼250 ms after stimulus onset. Remarkably, the neural activation isothipendyl map of the late phase in the contour condition showed a clear amplitude segregation of the circle contour from the background (Figure 2F), with the high activation in the circle area simply “popping out” from the suppressed activation in the background area (see also Figure S1E, available online, for similar results in monkey S). To further analyze this, we made a scatterplot of the population response of individual V1 pixels for the two conditions (Figure 2G; red lines as in 2D). Fifty percent of V1 pixels lie above the upper boundary in the circle area (Figure 2G, left; cf. early phase Figure 2D, left). In the background area, 66% of the pixels lie below the lower boundary (Figure 2G, right cf. early phase Figure 2D, right). The pixel differences histograms (contour-noncontour; Figure 2H) are shifted from zero (d′ = 2.02 between circle and background histograms.