CTNND2) is often overexpressed in prostatic adenocarcinomas but the mechanisms of its activation are unknown. To address this question, we studied the hypothesis that Hes1, human homolog of Drosophila Hairy and enhancer of split (Hes) 1, is a transcriptional repressor of δ-catenin expression and plays an important role in molecular carcinogenesis.Neuronal synaptic junction protein δ-catenin (δ-catenin promoter reporter assay, Hes1, but not its inactive mutant, significantly repressed the upregulation of δ-catenin-luciferase activities induced by E2F1. Hes1 binds directly to the E-boxes on δ-catenin promoter and can reduce the expression of δ-catenin in prostate cancer cells. In prostate cancer CWR22-Rv1 and PC3 cell lines, which showed distinct δ-catenin overexpression, E2F1 and Hes1 expression pattern was altered. The suppression of Hes1 expression, either by γ-secretase inhibitors or by siRNA against Hes1, increased δ-catenin expression. γ-Secretase inhibition delayed S/G2-phase transition during cell cycle progression and induced cell shape changes to extend cellular processes in prostate cancer cells. In neuroendocrine prostate cancer mouse model derived allograft NE-10 tumors, δ-catenin showed an increased expression while Hes1 expression was diminished. Furthermore, E2F1 transcription was very high in subgroup of NE-10 tumors in which Hes1 still displayed residual expression, while its expression was only moderately increased in NE-10 tumors where Hes1 expression was completely suppressed.We identified that, using a These studies support coordinated regulation of δ-catenin expression by both the activating transcription factor E2F1 and repressive transcription factor Hes1 in prostate cancer progression. Deregulation of gene expression is one of the most prominent features of cancer. Upregulated or downregulated genes interfere with signaling pathways leading to altered cell functions. Thus, the elucidation of different mechanisms responsible for changes in gene expression is essential for the understanding of tumorigenesis.CTNND2) or NPRAP  was first identified with its primary expression in neural and neuroendocrine tissues -S-phenylglycine t-butyl ester) , a peptidomimetic inhibitor of γ-secretase. Control plates were treated using the solvent DMSO (dimethylsulfoxide) (Sigma) with the same final concentration. For cell morphology and flow cytometry analyses, PC3 cells were incubated with 20 μM DAPT, 300 nM WPE-III-31C  ,35, or DE2F1, WT-Hes1 or DN-Hes1, luciferase gene, GAL) as indicated in the result section. Luciferase activity was measured 24 hours after transfection using the Luciferase Assay system (Promega) and normalized to β-galactosidase activity, which was used as control to determine transfection efficiency. All experiments were performed in quintuplicates. Whenever errors were displayed they represented standard deviation of mean (SDM) except in cell cycle measurements where SEM was employed.Cell cultures in 12-well plates were transiently transfected using Fugene 6 reagent with expression vectors for several genes (pcDNA-flag-WT-Hes1 using Fugene 6 reagent (Roche) 24 h before preparation according to instructions for NE-PER Nuclear and Cytoplasmic Extraction Reagents . Oligonucleotides containing the HLH motif derived in δ-catenin promoter (5'-GGGCGAAGGCCCAGAGGCCTTCCTTGGCACATGTTTTGGGTTTCGTTTTTCAACAAGACT-3') [the E-box/HLH motif is underlined]  [the E-box/HLH motif is underlined] and its reverse complement sequences were annealed and used as competitors. Mutant probe was produced when E-box sequence CACATG was mutated to GTCTCA. EMSA reactions were performed according to instructions of LightShift Chemiluminescent EMSA Kit . Nuclear protein extracts (2 μl) were incubated for 20 min at room temperature with 1 μl of non-specific competitor DNA Poly (dI·dC) and 20 fmol of biotin-labeled oligonucleotides. Competition assays were performed by mixing non-specific competitor DNA with 4 pmol unlabeled oligonucleotides (200-fold molar excess) and nuclear extracts before addition of probes. Hes-1 antibody  was used for disrupt-shift experiments. Normal goat IgG, Pax-6 antibody , and δ-catenin antibody  were used as a non-specific antibody. Antibodies were mixed with nuclear protein on ice for 20 min before the addition of probe DNA. Protein-DNA complexes were run on a 6% acrylamide gel, electrophoretically transferred to nylon membrane and visualized by Chemiluminescent Nucleic Acid Detection Module .Nuclear extracts were prepared from PC3 or CWR22-Rv1 cells transfected with δ-catenin promoter using the Abcam ChIP kit  according to the manufacturer's instruction. Cross-linking was performed by adding formaldehyde to a final concentration of 1% at room temperature for 10 minutes and reaction stopped by the addition of 125 mM glycine. Cells were washed with ice-cold phosphate buffered saline containing 0.1 mM PMSF. Cell pellets, collected by centrifugation at 2000 rpm at 4°C, were resuspended in 1 ml of ChIP sonication buffer. DNA was sheared by sonication and the cell debris was pelleted by centrifugation at 14,000 g for 15 minutes. The pre-cleared whole cell extract was incubated with or without the antibodies, as described in the legend, at 4°C overnight. Immunoprecipitated, immune complexes were collected using anti-Hes 1 conjugated-Protein A. Anti-Histone 3 (ab1791) antibody and IgG were used as positive control and negative control, respectively. The primer sets of real-time PCR for δ-catenin promoter were as follows: Forward: 5'-CCTTCCAGCTTTCGCCTA G-3', Reverse: 5'-TCCACTTCTCCTGGTTTTCG-3'; GAPDH was used as control, the primers were as follows: Forward: 5'-AGAAACAGGAGGTCCCTACTCCC-3', Reverse: 5'-AGAGCGCGAAAGGAAAGA AAG CGT -3'.PC3 cells were collected and ChIP assay was performed to test Hes1 protein regulation on The development of mouse neuroendocrine tumors (NE-10) was described before . Male CDFor NE-10 and CD-1 mice prostate tissue immunohistochemistry, 5 μm tissue sections of formalin-fixed, paraffin-embedded blocks were deparaffinized and rehydrated; endogenous peroxidase was blocked by incubation with hydrogen peroxide. The sections were immunostained using rabbit anti-δ-catenin R1152 (1:100)  followed by streptavidin-biotin peroxidase method for detection. The immunostaining was done in Dako Autostainer  according to the manufacture instruction.Total RNAs were extracted from cultured cells or mouse tissues using an RNeasy mini kit (Qiagen) with residual genomic DNA removed by RNase-free DNase (Qiagen) treatment. 1~2 μg of total RNA was reverse-transcribed using Retroscript reverse transcription kit (Applied Biosystems).δ-catenin, forward 5'-ATGTTTGCGAGGAAGCCGC-3' and reverse 5'-GTCTGGTTGCTATGGTAGCTGGC-3'; mouse δ-catenin, forward 5'-GAGCTATGCCTGTCCCAGAC-3' and reverse 5'-AGCTGAGAAGGGGCTGTGT-3'; human Hes1, forward 5'-CAGCGAGTGCATGAACGAGGTGA-3' and reverse 5'- AGGTGCCGCTGTTGCTGGTGTAGA-3'; mouse Hes1, forward 5'-AAGAGGCGAAGGGCAAGAATAAAT-3' and reverse 5'-CCGGGGATGGGCACAAG-3'; rat Hes1, 5'-ACAGCCTCTGAGCACAGAAAGTCA-3' and reverse 5'-TGAGGAAAGCAAATTGGCCGTCAG-3'; human Hey1, forward 5'- TCGAGTTCGACTGGTTTCGCATCT-3' and reverse 5'- AGGTCTATAGGGCTTGCCAAGGTT-3'; human E2F1, forward 5'-ACTCCTCGCAGATCGTCATCATCT-3' and reverse 5'-GGACGTTGGTGATGTCATAGATGCG-3'; mouse E2F1, forward 5'-GCATCCAGCTCATTGCCAAGAAGT-3' and reverse 5'-TGGTGACAGTTGGTCCTCTTCCAT-3'; human LEF-1, forward 5'-CCCGCTTCCGCCCGCTGTCC-3' and reverse 5'-CGGGGTGTTCTCTGGCCTTGTCGT-3'; human p53, forward 5'-AGACCGGCGCACAGAGGAAGAGAA-3' and reverse 5'-CCCCGGGACAAAGCAAATGGAAGT-3'; human CDK8, forward 5'-AGAAGCTGCTTACCATGGACCCAA-3' and reverse 5'- TGGTGGAACTTGGCTACTGGACAT-3'; human androgen receptor (AR), 5'-AGACGCTTCTACCAGCTCACCAA-3' and 5'- AGCTCTCTAAACTTCCCGTGGCAT-3'; human c-Myc, forward TCCACACATCAGCACAACTACGCA and reverse TCAGCCAAGGTTGTGAGGTTGCAT; human GAPDH (as control), forward 5'-GGGGAGCCAAAAGGGTCATCATCT-3' and reverse 5'- GACGCCTGCTTCACCACCTTCTTG-3'; mouse GAPDH (as control), forward 5'-AACTTTGGCATTGTGGAAGG-3' and reverse 5'- TGTGAGGGAGATGCTCAGTG-3'. PCR products were analyzed by electrophoresis in 1.0% agarose gel containing ethidium bromide and photographed under UV illumination.For RT-PCR, the reaction was initiated using Advantage 2 polymerase or Advantage-GC 2 polymerase (Clontech). The following forward and reverse primers were used to produce gene specific fragments: human GAPDH. The following primers for real time qPCR were used: mouse δ-catenin, forward 5'- ACCTCGGGAAATGATCAGCCTCAA-3' and reverse 5'- TAGTTCCGTGGTAAGTGGCGTTGT-3'; mouse Hes1, forward 5'- CAACACGACACCGGACAAACCAAA-3' and reverse 5'- TGGAATGCCGGGAGCTATCTTTCT-3'; mouse E2F1, forward 5'-TCATGCCAGGAGACATCC-3' and reverse 5'-CAATACTGCTTCTTGCTCCA-3'; mouse c-Myc, forward 5'- TGCTGCATGAGGAGACA -3' and reverse 5'-TCGGGATGGAGATGAGC-3'; mouse AR, forward 5'- CTGCCTGATCTGTGGAGA-3' and reverse 5'- CAATGGTACAATCGTTTCTGC-3'; mouse CDK8, forward 5'- ACATTCTGGTACCGAGCT-3' and reverse 5'- CACCCTATAGCCCAAATATCAA-3'; and mouse GAPDH (as control), forward 5'-TCAACAGCAACTCCCACTCTTCCA-3' and reverse 5'- ACCCTGTTGCTGTAGCCGTATTCA-3'. The real-time PCR primer set for human genes were: human δ-catenin, forward 5'-GCCTCAGTCAAAGAACAGGA-3'and reverse 5'-AGCTTGCATCGCTCCA-3'; human Hes1, forward 5'-CTGAAGAAAGATAGCTCGCG-3' and reverse 5'-ACTTCCCCAGCACACTT-3'; human E2F1, forward 5'- CTCCGAGGACACTGACA-3' and reverse 5'- CACCATAACCATCTGCTCTG-3'; human GAPDH, forward 5'-ACAGTCAGCCGCATCTT-3' and 5'-GCCCAATACGACCAAATCC-3'.For real time qPCR, reaction was initiated using IQ SYBR green supermix (Bio-Rad) in an iCycler iQ Multicolor Detection System (Bio-Rad). The relative mRNA levels of tested genes were calculated against control Cells on coverslips were fixed in 4% paraformaldehyde/phosphate buffered saline, permeabilized with 0.2% Triton X-100, blocked with 10% BSA in PBS, double immunostained with mouse anti-flag antibody M5 (1:600)/secondary antibody mouse Cy3 (1:400) and then rabbit polyclonal anti-δ-catenin antibody R1152 (1:50)/secondary antibody rabbit FITC (1:100). The nuclei were counter stained with Hoechst 33258 (2 μg/ml). The slides were mounted using Molecular Probe's Antifade medium and were analyzed under Zeiss Axiovert inverted fluorescent light microscope (Carl Zeiss) equipped with MetaMorph software (Molecular Devices).Cultured cells were lysed in 10 mM HEPES, pH 7.3, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.2% SDS with protease inhibitor cocktails. Equal amounts of protein samples were analyzed directly by SDS-PAGE. After proteins were transferred to nitrocellulose membranes (PGC Scientifics), Western blots were performed with appropriate antibodies and developed with ECL Western blotting detection reagents (Amersham).The authors declare that they have no competing interests.JL and JZ performed most of the experiments and contributed to the data analyses. KK provided δ-catenin promoter constructs. YHC provided real-time qRT-PCR analyses. JN performed immunohistochemistry experiments. TC and RM provided NE-10 samples and contributed to the design of the experiments. MW provided γ-secretase inhibitor and provided suggestions for the experiments. QL contributed to the design of the entire study and the editing of the manuscript. All authors read an approved the final draft.


The presence of non-simultaneous maskers can result in strong impairment in auditory intensity resolution relative to a condition without maskers, and causes a complex pattern of effects that is difficult to explain on the basis of peripheral processing. We suggest that the failure of selective attention to the target tones is a useful framework for understanding these effects. Two experiments tested the hypothesis that the sequential grouping of the targets and the maskers into separate auditory objects facilitates selective attention and therefore reduces the masker-induced impairment in intensity resolution. In Experiment 1, a condition favoring the processing of the maskers and the targets as two separate auditory objects due to grouping by temporal proximity was contrasted with the usual forward masking setting where the masker and the target presented within each observation interval of the two-interval task can be expected to be grouped together. As expected, the former condition resulted in a significantly smaller masker-induced elevation of the intensity difference limens (DLs). In Experiment 2, embedding the targets in an isochronous sequence of maskers led to a significantly smaller DL-elevation than control conditions not favoring the perception of the maskers as a separate auditory stream. The observed effects of grouping are compatible with the assumption that a precise representation of target intensity is available at the decision stage, but that this information is used only in a suboptimal fashion due to limitations of selective attention. The data can be explained within a framework of object-based attention. The results impose constraints on physiological models of intensity discrimination. We discuss candidate structures for physiological correlates of the psychophysical data. The intensity of an auditory stimulus is one of the most important basic attributes of auditory perception, besides pitch and spatial localization. We use information about auditory intensity in many different situations. If we want to cross a road, the acoustic intensity (change) provides information about the distance or the time-to-contact of an approaching car e.g., . We atteobject-based selective attention in an intensity discrimination task. Research on object-based attention in the visual domain  into separate objects  and targets was selected so that either (a) the masker(s) and the target presented in each interval could be expected to be grouped together on the basis of temporal proximity streaming condition where the two target tones were embedded in an isochronous sequence of eight masker tones. We expected the maskers to be perceived as one auditory stream, and the two target tones as separate auditory events. The remaining conditions were control conditions in which no regular masker rhythm was presented. Thus, we assumed the target and its adjacent masker(s) to be grouped together. Our hypothesis was that the masker-induced DL elevation would be smaller in the streaming condition than in the remaining conditions. The aim of the second experiment was simply to rule out the possibility that the effect of grouping the maskers and the targets as separate objects is idiosyncratic to the condition with three forward maskers studied in Experiment 1.In Experiment 2, we presented a Both experiments included the standard level as an additional variable to investigate the mid-level hump, i.e. the more pronounced effect of non-simultaneous masking for a mid-level standard compared to a low-level standard. We expected a stronger release from masking in conditions facilitating selective attention to the targets for midlevel standards compared to low-level standards.The experiments were approved by the ethics committee of the Department of Psychology, Universität Mainz. All subjects participated voluntarily after providing informed written consent. They received partial course credit or were paid €6 per hour. The experiments were conducted according to the principles expressed in the Declaration of Helsinki.Eight subjects  participated in Experiment 1. Seven subjects  participated in Experiment 2, one of them had already been tested in Experiment 1. All subjects were screened for normal hearing (within 10 dB) on the ear tested for frequencies between 250 and 4000 Hz. They were also screened for detection thresholds lower than 23 dB SPL in any masking condition, to ensure that the standard in the intensity discrimination task was always at least 7 dB SPL above threshold.2-ramps. In Experiment 2, the standard was a 1 kHz pure tone with a steady-state duration of 20 ms. The maskers were presented with a longer steady-state duration (50 ms) than the targets and at a different frequency  were marked by visual signals.In Experiment 1, the standard and the masker were 1 kHz pure tones with a steady-state duration of 20 ms, gated on and off with 5 ms cosfs = 44.1 kHz, 24-bit resolution), attenuated by a TDT PA5 attenuator, buffered by a TDT HB7 headphone amplifier, and presented to the right ear via Sennheiser HDA 200 circumaural headphones calibrated according to IEC 318 The stimuli were generated digitally, played back via one channel of an RME ADI/S D/A converter  were presented in quiet, and in three conditions presenting 90 dB SPL forward maskers. We included two different standard levels in order to investigate how perceptual grouping influences the midlevel-hump pattern, that is, a stronger masker-induced DL-elevation at the 60 dB SPL than at the 30 dB SPL standard level In the condition with one masker per observation interval 1 M, see Figure 1Areduction in masking in the intensity discrimination task in the 3 M condition cannot be attributed to a reduction in peripheral adaptation.Note that the frequency and the duration of the target and the maskers remained constant across conditions, as well as the temporal separation between the target and its adjacent masker. Therefore, according to current models for forward masking, in the auditory periphery at the most a weak summation of masking should occur in the 3 M condition streaming condition  were presented in quiet, and in four conditions including 85 dB SPL maskers. In the ondition Figure 2Aondition . We expeondition .forward masker  remained constant across all conditions. In both experiments, a within-subjects design was used. Each subject was tested in all experimental conditions.In the Intensity DLs were measured in a two-interval, two alternative forced-choice task using an adaptive procedure with a 3-down, 1-up rule 10(ΔI/I) = 8 dB. The step size was 5 dB until the fourth reversal, and 2 dB for the remaining reversals. A track ended when 12 reversals had been obtained or when 70 trials had been presented. It was discarded if the standard deviation of 10 log10(ΔI/I) at the counting reversals was greater than 6 dB.The initial level of the intensity increment was 10 log10(ΔI/I) at the fifth reversal up to the last even-numbered reversal was taken as the DL estimate. Per listener and condition, six blocks were obtained in separate sessions. The order of conditions was randomized. For a given listener and condition, DL estimates more than 1.5 times the interquartile range beyond the quartiles were classified as outliers In Experiment 1, only one adaptive track was presented per experimental block. The arithmetic mean of 10 log10(ΔI/I) at the fifth reversal up to the last even-numbered reversal was computed. The arithmetic mean of the two resulting values was taken as the DL estimate. A block was discarded if the standard deviation of 10 log10(ΔI/I) at the counting reversals was greater than 6 dB in either track. At least six blocks containing two tracks were obtained per listener and condition, in separate sessions.In Experiment 2, each block contained two adaptive tracks presenting the same condition, which were run in a randomly interleaved fashion in order to reduce the predictability of the sequence of target levels 2-ramps) were measured for the same experimental conditions as in the discrimination task. In one of the two observation intervals (selected randomly) the signal was presented. The other interval contained no signal. The observation intervals were marked by visual signals. The level of the signal was adjusted by a 3-down, 1-up adaptive rule. Listeners selected the interval containing the signal and were instructed to ignore the maskers. Visual trial-by-trial feedback was provided. The initial signal level was 30 dB SPL. The step size was 8 dB until the fourth reversal, and 2 dB for the remaining eight reversals. The arithmetic mean of the signal levels at the final eight reversals was taken as the detection threshold. A block was discarded if the standard deviation of the signal levels at the eight final reversals was greater than 6 dB. In Experiment 1, at least three blocks were presented per listener and condition. In Experiment 2, at least six blocks were obtained in separate sessions for each listener and each condition containing maskers, and at least four blocks in quiet.Detection thresholds for the standard presented in the discrimination task , under masking and the DL in quiet. As The data were analyzed in terms of the DL-elevation, which denotes the difference between the DL, measured in units of 10 log2 is reported as a measure of association strength. Post-hoc analyses of the differences between the masking conditions were computed by means of separate paired-samples t-tests  using a univariate approach with Huynh-Feldt correction for the degrees of freedom. Partial ηF = 5.11, p = .022, 2 = .422, confirming the descriptive differences between the three masking conditions. The DL-elevation was 3.15 dB  smaller in the 3 M condition than in the 1 M condition, 2.61 dB  smaller in the 3 M condition than in the 2 M condition, and 0.55 dB  smaller in the 2 M condition than in the 1 M condition. Post-hoc pairwise comparisons showed that the difference in the DL-elevation was significant between the 3 M and the 1 M condition, but not for the remaining pairs.The effect of masking condition was significant, F = 8.51, p = .022, η2 = .549, confirming the observed midlevel-hump pattern. The Masking Condition × Standard Level interaction was not significant, F = 1.97, p = 0.324. Thus, contrary to expectation, facilitating selective attention to the target via auditory grouping was not of greater help in the “midlevel” condition (standard level 60 dB SPL), although descriptively the data indicate such an effect.The effect of standard level was significant, F = .02, p = .983. This confirms our assumption that the 3 M condition does not produce stronger (or weaker) adaptation in the auditory periphery than the 1 M condition.The detection thresholds in quiet and in the three conditions presenting forward maskers are displayed in F = 8.91, p = .024, 2η = .60. The mean DL-elevation was minimal in the streaming condition at both standard levels, which is compatible with our hypothesis that the DL-elevation in the streaming condition is reduced because maskers and targets are perceived as two separated objects. This observation was confirmed by a significant effect of masking condition, F = 24.00, p<.001, 2η = .80. The Standard Level × Masking Condition interaction was also significant, F = 5.69, p = .007, 2η = .49, most likely owing to the unexpectedly high DL-elevation observed in the backward masking condition at the 30-dB SPL standard level. Note that several previous studies also reported extremely high DL elevations for some listeners if a low-level standard was combined with an intense masker  = 21.50, p = .001, 2η = .78. Compared to the forward masking condition, the streaming condition resulted in a release from masking of 4.02 dB . The streaming condition caused an even stronger release from masking of 5.79 dB  if compared to the forward-backward masking condition. As expected, in the forward-backward condition the DL-elevation was 1.77 dB  higher than in the forward masking condition. Post-hoc pairwise comparisons indicated that the DL-elevation in the streaming condition differed significantly from the forward masking and the forward-backward condition, while the difference between the latter two conditions was not significant. The significant difference between the DL-elevation in the streaming and in the forward-backward masking condition shows that the smaller masking effect in the streaming condition cannot be attributed to the presence of two rather than one adjacent maskers. In the ANOVA with the backward masking condition excluded, the standard level × masking condition interaction was non-significant, F = 0.36, p = .703.The mean DL-elevations observed in Experiment 2 are displayed in F = 10.40, p = .001, 2 = .63. Post-hoc pairwise comparisons between all pairs of masking condition including the in quiet condition were conducted. For the total of 10 pairs tested, only four tests were significant . In all masking conditions expect backward masking, the thresholds were significantly higher than in quiet. This pattern is compatible with current models of forward masking . One listener reported to have perceived maskers and targets to be grouped together at the 30 dB SPL standard level, but as segregated at the 60 dB SPL standard level. One other listener made use of the opportunity to draw in the perceived organization of stimuli at both standard levels. Unfortunately, we were not able to classify the drawings as either indicating integration or segregation of maskers and targets. Taken together, the subjective ratings are compatible with our assumption that the streaming condition favored the processing of the maskers and the targets as two separate auditory objects, despite the fact that we used a rather short duration of the masker sequence e.g., . The resWe measured intensity resolution for pure tones in the presence of non-simultaneous maskers. By comparing conditions either favoring the perception of masker and target as one unitary object, or as two separate objects, we tested the hypothesis that limitations of selective attention play an important role for intensity discrimination performance. Compatible with our hypothesis, compared to the usual forward masking setting we found a significant release from masking of 3 to 4 dB if the maskers could be grouped together on the basis of temporal proximity (Experiment 1), or could be perceived as a separate auditory stream (Experiment 2). These results are compatible with the assumed role of object-based attention for intensity resolution under non-simultaneous masking.Two potential problems with this proposed interpretation of our results should be noted. First, although we presented conditions likely to favor the grouping of maskers and targets into separate objects, these manipulations may not have had the desired effect. Second, the question could be asked whether processes different from selective attention might account for the results, for example because the masking conditions may have differed in terms of peripheral adaptation.Concerning the question of whether our manipulations of the masker-target sequence indeed caused different perceptual organizations, the reports of the subjects in Experiment 2 indicated that the streaming condition favored the perceptual of maskers and targets into separate objects, compared to the remaining conditions. In Experiment 1, unfortunately, we had failed to collect subjective ratings of the perceived organization. Thus, while it appears likely that in the 3 M conditions the three maskers were perceived as one object due to their temporal proximity reduction in the masker-induced DL-elevation in the 3 M condition compared to the 1 M condition could be attributed to a reduction in adaptation at early stages. Additionally, the three maskers presented in the 3 M condition did not render the standard undetectable, which might have led to different decision strategies in the intensity discrimination task Could the observed release from masking in the 3 M condition (Experiment 1) and the streaming condition (Experiment 2) be attributed to other mechanisms than the facilitation of selective attention to the targets? Evidently, it is critical that experimental manipulations intended to either favor or discourage perceptual grouping of maskers and targets into separate objects should not cause effects in the auditory periphery that could confound the changes in intensity resolution expected due to object separation. For example, frequency differences are one of the strongest cues favoring auditory stream segregation In both experiments of the present study, the smallest masker-induced DL-elevation was observed in the condition presenting the highest numbers of maskers. Could the DL-elevation simply decrease with the number of maskers? The data from Experiment 2 argue against this explanation. The effects of masking first increased with number of maskers when going from the forward masking condition to the forward-backward condition, but then decreased again in the streaming condition presenting the highest number of maskers. Additionally, if the intensity resolution would increase with the number of tones per trial, then the worst performance should be observed in quiet, which is clearly not the case. From a physiological point of view, while neural response enhancement by previous tones has been reported e.g., , some strelease from masking.If one compares the temporal sequence of stimuli between the forward-backward masking condition and the streaming condition, it is difficult to imagine that in the forward-backward condition the missing “inducing” tones or the missing middle masker should have had an effect other than on the formation of a “masker stream”. These tones missing in the forward-backward condition were separated from the target by more than 350 ms in the streaming condition. For this temporal separation, the maskers’ effect on the neural representation of the target in the auditory nerve should be very weak perceptual similarity between the masker and the target is an important factor in predicting the masker-induced DL elevation  is directly compatible with reports that the on e.g., . For inste e.g., . Note thThe results from Experiment 1 and 2 are compatible with recent data from our lab where the lateralization a binaural forward masker was varied via the inter-aural time difference (ITD) so that it was lateralized either on the same side of the head as the binaural target, or on the opposite side It is interesting to note that in Experiment 2 we observed effects of grouping despite the fact that the stimuli may have favored stream segregation only to a rather limited extent. Sequential stream segregation is typically studied with pitch differences of several semitones e.g., . HoweverAs stated in the Attempts to identify neural correlates of auditory stream segregation have focused on the auditory cortex e.g., . HoweverMost previous studies on neural correlates of stream segregation looked for differential enhancements or reductions in the physiological responses to the two types of tones presented in the alternating type of sequences  frequently used for investigating auditory stream segregation Electrophysiological data would also provide an opportunity to gain a deeper insight into the mechanisms responsible for the observed effects. Specifically, the mismatch-negativity MMN; cf.  has been(…) similarity can cause the target and masker to be perceived as part of the same, larger perceptual object (…)”.Our results are in accord with evidence for effects of object-based attention on informational masking, which by definition also represents the effects of processes beyond the auditory periphery cf. . SequentA recent study by Dyson and Ishfaq


The Wnt/β-catenin signaling pathway is essential for controlling bone mass; however, little is known about the variable effects of the constitutive activation of β-catenin (CA-β-catenin) on bone growth and remodeling at different postnatal stages. The goal of the present study was to observe the effects of CA-β-catenin on vertebral bone growth and remodeling in mice at different postnatal stages. In particular, special attention was paid to whether CA-β-catenin has detrimental effects on these processes.Catnblox(ex 3) mice were crossed with mice expressing the TM-inducible Cre fusion protein, which could be activated at designated time points via injection of tamoxifen. β-catenin was stabilized by tamoxifen injection 3 days, and 2, 4, 5, and 7 months after birth, and the effects lasted for one month. Radiographic imaging, micro-computed tomography, immunohistochemistry, and safranin O and tartrate-resistant acid phosphatase staining were employed to observe the effects of CA-β-catenin on vertebral bone growth and remodeling.CA-β-catenin in both early (3 days after birth) and late stages  increased bone formation and decreased bone resorption, which together increased vertebral bone volume. However, when β-catenin was stabilized in the early stage, vertebral linear growth was retarded, and the mice demonstrated shorter statures. In addition, the newly formed bone was mainly immature and located close to the growth plate. In contrast, when β-catenin was stabilized in the late stage, vertebral linear growth was unaffected, and the newly formed bone was mainly mature and evenly distributed throughout the vertebral body.CA-β-catenin in both early and late stages of growth can increase vertebral bone volume, but β-catenin has differential effects on vertebral growth and remodeling when activated at different postnatal stages. Both canonical and non-canonical signaling pathways participate in many physiological and pathological processes [The evolutionarily conserved Wnt signaling pathway mainly includes the canonical Wnt/β-catenin and non-canonical pathways that can be further subdivided into planar cell polarity and Wnt/Carocesses -4. Severrocesses . Activatrocesses ,10. Studrocesses . Overexprocesses . In controcesses . These rWnt/β-catenin signaling involves multiple steps that may be considered for pharmacological intervention ,14. EnhaTM as the promoter, which could be activated at designated time-points via injection of tamoxifen (TM) [The large number of Wnt proteins, receptors, coreceptors, and soluble inhibitors of Wnt signaling makes it difficult to define the specific functions of Wnt /β-catenin signaling. Fortunately, a molecular node of canonical Wnt signaling involves β-catenin with only one encoding gene, which offers the possibility of genetic manipulation. Constitutive activation of β-catenin (CA-β-catenin) can thus be induced in mice expressing a β-catenin mutant allele in which all the serine and threonine residues of exon 3 are phosphorylated by GSK-3β . In the fen (TM) ,28.The experimental protocol was reviewed and approved by the Ethical Committee of the Daping Hospital, Third Military Medical University (China).TM, under the control of a 3.2 kb mouse pro-collagen 1 promoter (3.2 kb Col1-Cre ERTM) active in osteoblasts, odontoblasts and tendon fibroblasts [5'-CCCGCAGAACCTGAAGATG-3' (sense) and 5'-GACCCGGCAAAACAGGTAG-3' (anti-sense), and the PCR primers for Catnb+/lox(exon 3) mice were 5'-AGGGTACCTGAAGCTCAGCG-3' (sense) and 5'-CAGTGGCTGACAGCAGCTTT-3' (anti-sense).Mice expressing the TM-inducible Cre fusion protein, Cre-ERroblasts ,28 were roblasts . GenotypTM  was dissolved in a small volume of ethanol and diluted with corn oil at a concentration of 10 mg/ml ,28. CA-βTM was examined by crossing Col1-Cre ERTM mice with ROSA26 reporter mice. Wild-type mice were also crossed with ROSA26 mice to serve as controls. TM was injected as aforementioned. The specificity of Col1-CreERTM expression was confirmed by β-gal staining.Site-specificity of Col1-Cre ERAt the designated time-points , mice were deeply anesthetized and radiographic images of entire skeletons obtained using a small animal X-ray machine  . After wAfter whole-body x-ray was performed and whole blood was taken, the mice were sacrificed by overdose of anesthesia and the spines were removed and fixed in 4% paraformaldehyde at 4°C overnight. The fifth lumbar vertebra was cut and saved for micro-computed tomography (microCT) examination and real-time PCR. The samples that were left were decalcified in 10% ethylenediaminetetraacetic acid at 4°C for 7 days. X-ray imaging was used to determine if the samples were adequately decalcified. Then, the third and fourth lumbar vertebrae were tissue-processed, embedded in paraffin, and sectioned at a thickness of 5 µm horizontally or coronally. The sections were de-paraffined and rehydrated, and were used for hematoxylin and eosin (H&E) staining, immunohistochemistry, TRAP staining, and safranin O staining.TM  according to the manufacturer’s instructions. The amount of RNA was quantified by spectrophotometry, after which cDNA was prepared from 1.0 µg RNA. The RNA was digested with DNase to eliminate any contaminating genomic DNA before quantitative real-time RT-PCR was performed. Then SYBR green detection method was used to examine the expression of receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), TRCAP, tissue non-specific alkaline phosphatase (TNAP), RunX2, and bone sialoprotein (BSP). Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) served as a control and the expression of a given gene was expressed as proportion relative to the average value of GAPDH. The relative ratio of RANKL:OPG expression was calculated and used for statistics. The primers used were sythesized by Sangon Biotech  and are shown in Total RNA was extracted from 100 mg of the vertebral trabecular bone using Tripure3. Images were reconstructed and analyzed with EVS Beam software using a global threshold of 1400 Hounsfield units. Total vertebral bone volume fraction was calculated as the percentage of bone volume to total volume (BV/TV) of fifth lumbar vertebra. The mean trabecular thickness (Tb.Th), trabecular separation (Tb. Sp), and trabecular number (Tb.N) were also calculated [The fifth lumbar vertebrae and tibia of mice from each group were dissected and scanned in a micro-CT imaging system , as described previously . The scalculated . As for 2O2 at room temperature for 10 min, followed by treatment with 3% bovine serum albumin (BSA) at room temperature for 1 h. Sections were incubated with primary antibodies diluted at the appropriate concentrations in 2% BSA/0.1 M phosphate buffered saline (PBS) overnight at 4°C, washed in PBS, and further incubated with a biotinylated IgG antibody at room temperature for 1 h. Next, slides were treated with ABC reagents , and signals for antibody binding visualized with diaminobenzidine (Boster) substrate. Counterstaining was performed with Methyl Green. Slides were fully rinsed with 0.1 M PBS or distilled water between individual steps.Immunohistochemistry was performed according to a previous report . BrieflyThe primary antibodies used included goat TNAP (1:200), goat polyclonal Runx2 (1:300) and rabbit anti- BSP (1:300). All primary antibodies were purchased from Santa Cruz Biotechnology, Inc , and biotinylated goat anti-mouse, rabbit anti-goat, and goat anti-rabbit IgG acquired from Boster. To obtain the percentage of cells expressing a given marker protein, pictures of each section were taken by an Olympus microscope and digital camera under a 200x magnification. The number of specific antigen-positive cells was counted in five random fields. The average percentage of positive cells and standard deviation were calculated in each group and used for statistics.Rehydrated sections were stained with Weigert’s iron hematoxylin for 1 min and rinsed in distilled water until clear. Sections were stained sequentially with 0.02% Fast Green for 5 min, 1% acetic acid for 30 s, and 0.1% Safranin O for 20 min. Slides were not rinsed between steps. Subsequently, slides were rinsed in 95% alcohol for 2 min, 100% alcohol for 3 min (twice), xylene for 2 min (twice), and coverslipped with Permount. All chemicals were purchased from Chuandong Corporation .Two coplin jars (A and B) were pre-heated to 37°C with 50 ml stock basic incubation medium . Then, 50 µl of 2% naphthol AS-BI phosphate substrate in ethylene glycol monoethyl ether was added to jar A, into which the slides were added, and incubated at 37°C for 45 min. A few minutes before the 45 min elapsed, 1 ml of 5% pararosaniline chloride and 1 ml of 4% sodium nitrite was mixed for 30 sec, incubated at room temperature for 2 min without mixing, and then transferred into jar B and mixed well. The slides from jar A were then moved into jar B without rinsing. Incubation at room temperature was done for 1-3 min until the color developed, followed by rinsing and counterstaining with methyl green for 5 min, dehydration, and covering with Permount. All chemicals were purchased from the Chuandong Corporation .Pictures of each section were taken under a magnification of 200×, and the number of TRAP-positive cells was counted in five random fields. The average number and standard deviation of TRAP-positive cells were calculated and used for statistical analysesP<0.05.All data were expressed as means ± standard error. Statistical significance was evaluated using ANOVA with SPSS11.0 software. Data were considered significant at TM expression was confirmed by β-gal staining using ROSA26 mice. Positive staining was mainly found in osteoblasts, but not in the chondrocytes of the growth plate  were injected with tamoxifen. The phenotypes of Col-Cre and β-catenin exon 3 fx +/+ mice (without Col I Cre) were the same as wild-type mice (data not shown). We feared that this data would make the manuscript too complicated, so only data from wild-type mice were presented as control in the current study. When β-catenin was stabilized with Col1-CreERype mice .Growth plate is the structure responsible for linear growth in vertebrates. H&E and Safranin O staining showed that growth plates in mice with β-catenin stabilization from day 3 were longer than that in wild-type mice  and 3. TSimilar findings were observed in the long bone. When β-catenin was stabilized from day 3 of life, the lengths of the tibia and femur were shorter than those in wild-type mice, and the hypertrophic layer of the growth plate was enlarged . In contP <0.05, p<0.05, The Wnt/β-catenin signaling pathway plays an important role in controlling bone volume. Our experiments disclosed significantly increased trabecular bone volumes in mice with constitutive activation of β-catenin at both early (day 3) and late stages , compared to wild-type mice P <0.05, . In addiHowever, the location and extent of maturation of excessive formed bone varied with different stages of β-catenin stabilization. Upon induction of β-catenin stabilization from day 3 to month 1 of life, excessive formed bone was located in close proximity to the growth plate, while mice with β-catenin stabilization at the late stages  displayed even distribution of excessive bone in the vertebral body .Safranin O is commonly employed for proteoglycan staining. During rapid bone formation, immature bone stains positive for Safranin O ,33. Our Similar findings were observed in the long bone . Specifip<0.05, p<0.05, p<0.05, The balance of bone formation and bone resorption controls bone volume. TRAP staining showed that the number of osteoclasts in CA-β-catenin mice was lower than that in wild-type mice in both early and late postnatal stages p<0.05, . RANKL wp<0.05, The results of real-time PCR and ELISA further supported the histology findings. As shown in Taken together, these results indicate that stabilization of β-catenin in both early and late postnatal stages increases vertebral bone formation and decreases bone resorption.Accumulating evidence supports the significance of Wnt/β-catenin signaling in bone formation and bone resorption. However, no data are available about the effect of the Wnt/β-catenin signaling pathway on bone growth and remodeling at different stages after birth. In the current study, we showed that constitutive β-catenin activation at both the early and late stages of growth results in increased bone formation and decreased bone resorption, which concertedly increase vertebral bone volume. However, the stabilization of β-catenin at early and late stages had differential effects on vertebral growth.The current study found that CA-β-catenin in both the early and late stages of growth increases bone volume by increasing bone formation and decreasing bone resorption. However, controversies exist regarding the role of the Wnt/β-catenin signaling pathway in osteoblastogenesis and osteoclastogenesis -26. UsinAlthough controversy exists, the effects of Wnt/β-catenin on bone formation are generally considered positive ,25,34,35TM, is mainly expressed in osteoblasts, but not in chondrocytes, the effect of CA-β-catenin on the growth plate must be indirect. Our preliminary study showed that CA-β-catenin mice demonstrated hypophosphatemia, and a high phosphate diet could partially rescue the phenotype of enlarged growth plate in mice when β-catenin was stabilized at early stage (data not shown). These data indicated that hypophosphatemia might be the reason for retarded linear growth in CA-β-catenin mice. Future studies are needed to investigate the reason for these differential effects. However, regardless of the underlying mechanisms, these discrepancies should be addressed before therapeutics are used that target the Wnt/β-catenin signaling pathway.Another finding of the present study is that stabilization of β-catenin at early and late stages has differential effects on vertebral growth. When β-catenin was activated at the early stages (from day 3 after birth up to 1 month), maturation of vertebral growth plate was retarded in addition to increased bone formation, and mice displayed shortened statures. In contrast, when β-catenin was activated at the late stages , growth plates were not significantly affected. Our results indicate that constitutive activation of β-catenin at the early stages has a detrimental effect on vertebral growth. Furthermore, excessive formation of bone in early activation mainly occurs close to the growth plate and involves immature bone (staining positive for Safranin O), while excessive bone formed in the late stages is evenly distributed in the vertebral body and mature (staining negative for Safranin O). Until now, the precise mechanisms underlying the differential effects on vertebral growth by CA-β-catenin at different postnatal stages have remained unknown. Since the promoter used in the current study, pro-collagen I Cre-ERIn conclusion, the present study shows that CA-β-catenin at different stages after birth can increase vertebral bone volume. This increased bone volume was the result of increased bone formation and decreased bone resorption caused by CA-β-catenin. However, CA-β-catenin induced at different stages has differential effects on vertebral linear growth, as well as distribution and maturation of the newly formed bone.Figure S1TM.Site-specificity of Col1-CreERa Col 1 Cre expression was not found in growth plate in wild-type mice crossed with ROSA26 mice. b Col 1 Cre expression was not found in growth plate in Col1-CreERTM mice crossed with ROSA26 mice. c Col 1 Cre expression was not found in osteoblasts in wild-type mice crossed with ROSA26 mice. d Col 1 Cre expression was found in osteoblasts in Col1-CreERTM mice crossed with ROSA26 mice. Bars: 200 µm.(TIF)Click here for additional data file.Figure S2H&E staining of coronal sections of the tibia in wild-type and CA-β-catenin mice.Boxes in a, c, e, g, I, k, m, o, q and s are magnified in b, d, f, h, j, l, n, p, r and t, respectively. Bars: 200 µm  and 100 µm .(TIF)Click here for additional data file.Figure S3MicroCT examination of trabecular bone of proximal tibia in each group.a-j representative, transverse MicroCT images of the trabecular bone in CA-β-catenin and wild-type mice at 1 month (a and b), 3 months (c and d), 5 months (e and f), 6 months (g and h) and 8 months (i and j) of age. k MicroCT analysis of BVF  of the proximal tibia in CA-β-catenin mice and wild-type mice. BV trabecular bone volume (mm3); TV total volume selected for analysis (mm3). l Trabecular number (Tb.N) of the proximal tibia in CA-β-catenin and wild-type mice. m Mean trabecular thickness (Tb.Th) of the proximal tibia in CA-β-catenin and wild-type mice. n Trabecular separation (Tb. Sp) of the proximal tibia in CA-β-catenin and wild-type mice. Bars represent the mean ± SEM (n=6 for each group). * p<0.05.(TIF)Click here for additional data file.Figure S4Safranin O staining of tibia in each group. a-h Safranin O staining of coronal sections of tibia in wild-type and CA-β-catenin mice at 1 month (a and b) 3 months  5 months (e and f), 6 months (i and j) and 8 months (k and l) of age. Boxes in c and d are magnified in g and h, respectively. Bars: 200 µm  and 100 µm (g and h).(TIF)Click here for additional data file.Figure S5MicroCT examination of cortical bone of tibia in each group. a-j representative, transverse MicroCT images of the cortical bone in CA-β-catenin and wild-type mice at 1 month (a and b), 3 months (c and d), 5 months (e and f), 6 months (g and h) and 8 months (i and j) of age. k Total cross-sectional area (Tt.Ar) in CA-β-catenin and wild-type mice. l Cortical bone area (Ct.Ar) in CA-β-catenin and wild-type mice. m Cortical area fraction (Ct.Ar/Tt.Ar) in CA-β-catenin and wild-type mice. n Average cortical thickness (Ct.Th) in CA-β-catenin and wild-type mice. Bars represent the mean ± SEM (n=6 for each group). *p<0.05.(TIF)Click here for additional data file.Figure S6TRAP staining of tibia in each group. a-h TRAP staining of coronal sections of tibia in wild-type and CA-β-catenin mice at 1 month (a and b), 3 months (c and d), 6 months (e and f), and 8 months (g and h) of age.(TIF)Click here for additional data file.


To protect against aneuploidy, chromosomes must attach to microtubules from opposite poles (‘biorientation’) prior to their segregation during mitosis. Biorientation relies on the correction of erroneous attachments by the aurora B kinase, which destabilizes kinetochore-microtubule attachments that lack tension. Incorrect attachments are also avoided because sister kinetochores are intrinsically biased towards capture by microtubules from opposite poles. Here, we show that shugoshin acts as a pericentromeric adaptor that plays dual roles in biorientation in budding yeast. Shugoshin maintains the aurora B kinase at kinetochores that lack tension, thereby engaging the error correction machinery. Shugoshin also recruits the chromosome-organizing complex, condensin, to the pericentromere. Pericentromeric condensin biases sister kinetochores towards capture by microtubules from opposite poles. Our findings uncover the molecular basis of the bias to sister kinetochore capture and expose shugoshin as a pericentromeric hub controlling chromosome biorientation.DOI:http://dx.doi.org/10.7554/eLife.01374.001 When a cell divides to create two new daughter cells, it must produce a copy of each of its chromosomes. It is important that each daughter cell gets just one copy of each chromosome. If an error occurs and one cell gets two copies of a single chromosome, it can lead to cancer or birth defects. Fortunately, there are multiple checks to ensure that this does not happen.During cell division the chromosomes line up in a way that increases the likelihood that each daughter cell will have one copy of each chromosome. After this process—which is called biorientation—is completed, microtubules pull the chromosomes to opposite ends of the cell, which then divides.Proteins called shugoshin proteins are known to be involved in biorientation in many organisms. These proteins are found in a region called the pericentromere, which surrounds the area on the chromosomes that the microtubules attach to, but the details of their involvement in biorientation are not fully understood. Now Verzijlbergen et al. have exploited sophisticated genetic techniques in yeast to explore how shugoshin proteins work.These experiments showed that the shugoshin protein helps to recruit condensin—a protein that keeps the DNA organized within the chromosome—to the pericentromere to assist with biorientation. It also keeps aurora B kinase—one of the enzymes that helps to correct errors during cell division—in the pericentromere when a microtubule attaches to the wrong chromosome. These results help us understand how a ‘hub’ in the pericentromere ensures biorientation. The next challenge will be to understand how this hub is disassembled after biorientation to allow error-free cell division to proceed. As shugoshins have been found to be damaged in some cancers, understanding the workings of this hub could also shed new light on how they contribute to disease.DOI:http://dx.doi.org/10.7554/eLife.01374.002 The accurate segregation of chromosomes during mitosis relies on the capture of newly duplicated sister chromatids by microtubules emanating from opposite poles. This is possible because on each chromosome a multi-subunit kinetochore assembles on a region of DNA, known as a centromere, to mediate attachment to microtubules. The proper attachment of sister kinetochores to opposite poles, called biorientation, generates tension owing to sister chromatid cohesion . Sister Though fundamental for accurate chromosome segregation, the role of shugoshin in biorientation has remained unclear. Budding yeast has a single shugoshin, Sgo1, which protects pericentromeric cohesin during meiosis but does not regulate cohesion during mitosis . We havesgo1 null cells (sgo1Δ) together with three missense mutants: sgo1-100, sgo1-700 and sgo1-3A. The sgo1-3A mutant was engineered to disrupt the binding site for PP2A-Rts1 (sgo1-100 and sgo1-700 were isolated in a screen due to their inability to sense a lack of tension (sgo1Δ cells have previously been reported to affect biorientation after microtubule perturbation (sgo1-100 and sgo1-700 mutants is similar to that of wild-type cells (SPC42-tdTomato), the centromere of chromosome IV labeled with GFP (CEN4-GFP) and with CDC20 under control of the methionione-repressible promoter (pMET-CDC20), to enable metaphase arrest by addition of methionine. All strains were released from a G1 arrest into nocodazole- and methionine-containing medium to depolymerize microtubules and induce a metaphase arrest before nocodazole was washed out to allow metaphase spindle formation  containing aurora B kinase with centromeres . BuddingCEN4 . Early ICEN4 , the homCEN4 . The recCEN4 . Sgo1-inCEN4  can explrization . The Iplrization  and centrization  were alstub2-401 ) where E represents the specific primer efficiency value. Enrichment/ input value was obtained from the following formula: E^−ΔCT. qPCR was performed in triplicate, typically for each of three or more independent experimental repeats. Error bars represent standard error. For ChIP-Seq, purified chromatin was recovered using a PCR purification kit  followed by drying in a speedvac. Samples were sequenced on a HiSeq2000 instrument  by the EMBL Core Genomics Facility . The summary of data obtained is given in ChIP was performed as described using anti-HA 12CA5, anti-Myc 9E10 or anti-Pk(V5) antibody . Primershttps://github.com/AlastairKerr/Marston2013. Single reads were mapped using BWA (Version: 0.6.1-r104)   to the s.1-r104)  to remov.1-r104) . As thissgo1Δ cells, we were concerned that the small number of precipitating sequences would be biased for amplification. To avoid this problem, we removed PCR replicates from our analysis. However, since this would also remove bona fide identical sequences from our dataset, we simultaneously analyzed the data with these reads present and compared the two data sets where all 16 centromeres were considered together. To do this, 100 bp windows were examined that extended 25 kb in each direction of every centromere, and a local maximum of the number of mapped reads was taken. The comparison of this data with and without PCR replicates included can be seen in During the amplification step prior to sequencing, multiple identical reads will be generated. Due to the low yield of total DNA precipitating with Brn1-6HA in 600 = 0.2 and grown at room temperature for 6 hr. Cells were harvested by centrifugation and the cell pellet was resuspended in 2 μM of dithiobis(succunimidylpropionate)  crosslinker for 30 min at room temperature before cell pellets were drop-frozen in liquid nitrogen as described by tub2-401 allele, 4 L of media was inoculated to OD600 = 0.02, grown at 30°C for 8 hr with shaking and then the temperature was reduced to 18°C for 7 hr to induce the arrest before holding the cells at 4°C for up to 5 hr before harvesting, crosslinking, and freezing as described above. Pulldowns and mass spectrometry were performed as described in To grow cells for purification of Sgo1 from cycling cells, 4 L of YPDA culture were inoculated to ODFor co-immunoprecipitation of TAP-tagged Sgo1, 200 ml of nocodazole-treated culture was harvested and either firstly cross-linked using DSP as described in Western immunoblot was performed as described in Indirect immunofluorescence and FACS were performed as described in pMET-CDC20, CEN4-GFP and SPC42-tdTomato were arrested in G1 using alpha factor (4 μg/ml) in minimal medium lacking methionine (SD/-met) at room temperature for 3 hr. Alpha factor was washed out and cells were released from the G1 arrest into rich medium containing methionine and nocodazole (YPDA + Met + NOC) plus the appropriate drugs . Methionine represses pMET-CDC20 expression, resulting in a metaphase arrest and nocodazole depolymerizes microtubules. After 3 hr, a sample was extracted (t = 0) and nocodazole was washed out by filtering in the presence of methionine and cultures were released into YPDA + Met to allow spindles to reform while maintaining the metaphase arrest. Samples were taken at 20 min intervals. Samples were fixed in 3.7% formaldehyde for 10 min, before washing in PBS and resuspending in DAPI for microscopy. Samples with two separated SPC42-tdTomato foci were scored as containing one or two CEN4-GFP foci. Typically 200, and at least 100, cells were scored for each timepoint.Cells carrying For live cell imaging, cells were loaded onto the Onix Microfluidic Perfusion system  and visualized using a Deltavision Elite  coupled to a Cascade 2 EMCCD camera with temperature control to 30°C. Frames were grabbed at the indicated intervals and images were processed in ImagePro software .CEN4-GFP dot to nearest Spc42-tdTomato were obtained using this automated system.For image analysis, a custom-written macro was developed in ImagePro software (Media Cybernetics), details of which are available upon request. In brief, yeast are selected from a reference DIC image taken at each time point. The yeast are automatically thresholded and the centre pixel from each red and green spot is then calculated. The distances between the spots can then be calculated and the measurements output to a text file for analysis using microsoft excel. Measurements of inter-centromere distance and distance from CEN4 (CEN4-GFP) and SPB (SPC42-tdTomato) markers together with CDC20 under control of a methionine-repressible promoter (pMET-CDC20) were arrested in G1 using alpha factor and then loaded onto the microfluidic chamber. Cells were released in the chamber into medium containing nocodazole (to depolymerize microtubules) and methionine (to deplete CDC20 and induce a metaphase arrest) for 30 min. The addition of nocodazole to G1 cells prevents SPB separation because this requires microtubules. Therefore, under these conditions, microtubules do not separate which leads to a high rate of monoorientation once microtubules are allowed to reform. This leads to a strong reliance on the error correction machinery to establish biorientation , we began imaging at ∼74s intervals for a total of 21 frames.To test the efficiency of error correction, cells carrying entation . After 3CEN4-GFP foci. The total percentage of cells with two SPB foci that contained two GFP foci was calculated for all frames for each strain. The number of cell images analyzed was 1116 (wild type), 697 (sgo1Δ), 1251 (YCS5-aid) and 968 (ipl1-as). To mFor determination of the overall ability of strains to biorient in CEN4-GFP foci. Cells in which two SPB foci were detected for at least four consecutive frames were scored for the number of times that the number of CEN4-GFP foci changed from one to two or vice-versa for the frames in which two SPBs were consecutively visible. The ‘switching rate’ was calculated by dividing the number of times a cell alternated between one and two CEN4-GFP foci by the total time in which two SPB foci were consecutively visible. The average switching rate was determined for all cells in which two SPB foci were detected for at least four consecutive frames. In sgo1Δ cells (551 frames), 102 YCS5-aid cells (1046 frames) and 86 ipl1-as cells (748 frames).As a measure of the efficacy of the error correction machinery, we determined the ability of cells to switch between one and two visible CEN4 (CEN4-GFP) and SPB (SPC42-tdTomato) markers together with CDC20 under control of a methionine-repressible promoter (pMET-CDC20) were arrested in G1 using alpha factor and then released into medium containing methionine to deplete CDC20 and induce a metaphase arrest. Cells were loaded onto the microfluidics chamber and after approximately 1.5 hr, when around 90% of cells had 2 SPBs, cells were treated with YPDA containing nocodazole for 30 min. After 30 min, nocodazole was washed out after which cells were immediately imaged every ∼94 s for a total of 21 frames. We scored the percentage of cells in which separated GFP dots were observed at least once during the observation period.To test the bias on sister kinetochores to biorient, cells carrying  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  is important to promote biorientation through two mechanisms: through binding to the chromosome passenger complex, and through a newly identified association with the condensin complex. The authors show that Sgo1 is able to recruit condensin to centromeres or to ectopic sites. The authors identify a mutation in Sgo1 that perturbs binding to condensin and that this association is important for subsequent biorientation. The authors conclude that condensin is required to form a rigid back-to-back structure such that sister kinetochores are biased towards microtubule capture from opposite spindle poles.Overall this is an interesting study that reveals new insights into how budding yeast chromosomes biorent, and uncover a new role for condensin in centromere and kinetochore behaviour.There are, however, a number of substantive concerns that preclude publication in its present form. In particular, the conclusion that condensin recruitment alters kinetochore geometry to promote orientation towards opposite poles requires further supporting evidence. Moreover, the authors themselves do not appear to be certain whether there is a direct link between Sgo1 and condensin recruitment. They argue that Sgo1 is sufficient for condensin targeting to the pericentromere by over-expressing Sgo1 and measuring Brn1 accumulation. However, they previously state that Sgo1's effect on condensin is indirect, mediated through Scc2/4 (cohesin). There are also concerns regarding the validity of the approach the authors use to measure bi-orientation and the presentation of the ChIP analyses. The latter prevents a critical evaluation of the data quality and missing information will need to be added.Concerns to be addressed:1) The evidence for a direct interaction between Sgo1 and condensin is limited. Only a small number  of condensin peptides can be identified in the mass-spec analysis of Sgo1 purifications. In fact, the highest scoring condensin subunit  ranks number 137 in the list of proteins identified . Moreover, only a minute fraction of condensin . The authors should at least test whether ectopic recruitment can be measured at both sides of the TetO repeats. Yet a much better test whether condensin recruitment by Sgo1 is significant would be to employ the TetO-TetR approach to tether to centromeres an Sgo1 mutant that can otherwise not bind  and assay whether this restores condensin levels and chromosome bi-orientation.An explanation for the altered binding profiles of condensin measured by ChIP-seq might be changes in cohesin accumulation at centromeres in the Sgo1 mutants. The authors need to check by ChIP-qPCR  whether cohesin levels at centromeres are affected in the Sgo1 mutant.2) The authors monitor the splitting of a sister centromere pair as a measure for sister chromatid bi-orientation . The redOne possibility to distinguish whether chromatid pairs have attached to spindle microtubules from only one spindle pole or whether chromatids have bi-oriented but their centromeres cannot separate would be to score centromere-spindle pole body (SPB) distances in the live cell microscopy assay . If centThe data in 3) Most of the graphs that represent the results from ChIP-qPCR experiments  lack infTaking into account the possible variability between individual ChIP-qPCR experiments, careful statistical analysis will be required to evaluate the significance of the observed differences. For example, the values measured at CEN4 for the same wild-type strain (AM3513) in two independent Ipl1 HA-ChIP experiments vary 4-fold compare , calling 1) The evidence for a direct interaction between Sgo1 and condensin is limited. Only a small number  of condensin peptides can be identified in the mass-spec analysis of Sgo1 purifications. In fact, the highest scoring condensin subunit  ranks number 137 in the list of proteins identified .The Excel file submitted with the original manuscript was unsorted and did not take the contaminant proteins that also co-purify with the “no tag” control into account. When contaminants are taken into account, the condensin subunits rank number 8 and 26 in the list of proteins identified, in cycling and arrested cells, respectively. We have revised the file with the list of proteins sorted to exclude contaminant proteins.Moreover, only a minute fraction of condensin (less than 0.01%? See) can be detected by Western blotting in Sgo1 immunoprecipitations. Since both Sgo1 and condensin are enriched at centromeres, it is very likely that a small amount of condensin co-precipitates with Sgo1 because both proteins are bound to the same stretch of chromatin. In addition, the authors add amine-reactive crosslinkers to the cell extracts in both experiments (a fact they should mention at least in the figure legends and not only in the methods section), which will result in the formation of protein-chromatin crosslinks. The authors will need to repeat the pull-down experiments after DNase digestion and in the absence of crosslinker in order to rule out these concerns.We thank the reviewers for raising this important point. We performed the experiment suggested by the reviewers and found that the amount of the Ycs4 condensin subunit co-purifying with Sgo1 was independent of treatment with cross-linker or DNase. This shows that the interaction between Sgo1 and condensin is not dependent on DNA. This data is now presented in A second argument for a role of Sgo1 in condensin loading is that centromeric condensin levels increase upon Sgo1 overexpression. The increase in condensin binding could, however, result from the delay in anaphase onset caused by Sgo1 overexpression. Would condensin levels remain unchanged if anaphase onset were delayed by other means?SGO1-overexpressing cells was a consequence of the metaphase arrest caused by high levels of Sgo1, we overexpressed SGO1 in cells arrested in mitosis by treatment with nocodazole. Measurement of Brn1 association with two centromeric sites and a pericentromeric site by ChIP-qPCR showed that SGO1 overexpression also increased Brn1 levels in cells arrested in mitosis, arguing against the idea that the centromeric enrichment of Brn1 is solely due to the cell cycle block caused by SGO1 overexpression. This data is presented as To exclude the possibility that Brn1’s chromosomal enrichment in Ectopic recruitment of condensin by Sgo1 fused to TetR seems rather inefficient, with a two-fold enrichment (and considerable variation?). The authors should at least test whether ectopic recruitment can be measured at both sides of the TetO repeats. Yet a much better test whether condensin recruitment by Sgo1 is significant would be to employ the TetO-TetR approach to tether to centromeres an Sgo1 mutant that can otherwise not bind  and assay whether this restores condensin levels and chromosome bi-orientation.tetO repeats. Following sonication during the ChIP protocol, the majority of fragments are much shorter than this (in the range of ∼200 bp), so a primer set at this distance is expected to only inefficiently detect proteins recruited to the tetO repeats themselves. As suggested by the reviewers, we have now analyzed a site on the other side of the tetO repeats which is much closer (∼50bp) to the tethering site. As can be seen in In the original manuscript, we used a primer set ∼800 bp to the left of the SGO1. As we showed in sgo1-700 mutant loses the bias to sister kinetochore biorientation , it was necessary to perform this experiment in diploid cells and therefore we cannot presently test the ability of these tethered constructs to retain sister kinetochore bias (we cannot easily arrest and release the cells from G1 as they are not sensitive to mating pheromone). Moreover, other unpublished findings in the lab have suggested that the dynamicity of Sgo1 on centromeres is key to its regulation and important for accurate segregation so it is likely that Sgo1 tethering affects its function. Indeed, even tethered wild type Sgo1-tetR-GFP is not fully able to rescue the mis-segregation of chromosome IV in our experiment  whether cohesin levels at centromeres are affected in the Sgo1 mutant.sgo1? cells.We performed both these experiments and the data is shown in Overall, we feel our study now argues strongly for a direct role of Sgo1 in condensin recruitment to the pericentromere.2) The authors monitor the splitting of a sister centromere pair as a measure for sister chromatid bi-orientation . The reduction of sister centromere splitting observed in the Sgo1 and condensin mutants could, however, equally well result from an increase in centromeric sister chromatid cohesion. An increase in centromeric cohesion would prevent splitting of bi-oriented sister chromatids.One possibility to distinguish whether chromatid pairs have attached to spindle microtubules from only one spindle pole or whether chromatids have bi-oriented but their centromeres cannot separate would be to score centromere-spindle pole body (SPB) distances in the live cell microscopy assay . If centromeres were bi-oriented but merely failed to split, they should be positioned at equal distances to both SPBs. If sister centromeres failed to bi-orient, they should frequently be found close to one SPB. Alternatively, the authors could release cells from the Cdc20 arrest and allow them to enter anaphase, then score whether sister centromeres segregate to one pole or to opposite poles .sgo1? and YCS5-aid cells is not significantly different to that of wild type cells . The situation is further complicated by the fact that due to the partial functionality of the error correction machinery in condensin mutants  Most of the graphs that represent the results from ChIP-qPCR experiments  lack information that is essential to evaluate the data. What do the column plots represent? Do they show mean values, and if yes, from how many PCR runs and how many independent immunoprecipitation experiments? What do the error bars represent? The legend foris missing completely.We apologize for this oversight. We have now included a more detailed description of the procedures used in the Materials and methods and detailed figure legends throughout, explaining how each experiment was carried out, the procedures used to process the data and the meaning of the figures presented. As detailed in the figure legends, the vast majority of ChIP-qPCR experiments show mean values of three independent repeats. qPCR was always performed in triplicate . The number of independent immunoprecipitation experiments is given in the figure legends . Error bars represent standard error. The exception to this are time course experiments where the day-to-day variability of cell cycle progression means data from different days cannot be combined usefully. Here, experiments are repeated, typically 3 times in total, and a representative experiment is shown. Overall, the pattern does not vary, though the absolute values at each timepoint can.Taking into account the possible variability between individual ChIP-qPCR experiments, careful statistical analysis will be required to evaluate the significance of the observed differences. For example, the values measured at CEN4 for the same wild-type strain (AM3513) in two independent Ipl1 HA-ChIP experiments vary 4-fold (compare), calling into question whether the 2-fold reduction in ChIP signals in the Sgo1 mutants  is significant. Similarly, the ChIP signal at CEN4 measured for the Sgo1-100 mutant in G1 phase is more than 2-fold higher than the signal for wildtype Sgo1 . Is the 4-fold increase in Sgo1 ChIP signals during mitosis  then still significant?Sgo1-100 mutant proteins is likely due to slight changes in the timing of alpha factor release in the handling of these cultures (see above about the interpretation of time course experiments). The main point of these experiments was to show that residual association to centromeres occurs even though it is not maintained in nocodazole-arrested cells. We do not think it is appropriate to take any further interpretation based on the absolute values from these experiments as due to the inherent variability of time course experiments; to do so would require exquisitely controlled conditions and much more closely spaced sampling.Again, we apologize: these discrepancies are due to our failure to provide adequate figure legends describing the procedures used to obtain the data and can all be accounted for by different growth conditions. In particular, the ChIP-qPCR signals obtained in experiments where nocodazole was directly added to cycling cells are typically much higher than experiments where cells were first arrested in G1 before being released into nocodazole. The fraction of cells that were approaching, or in, mitosis at the time of nocodazole will spend a much greater time in mitosis, where our proteins of interest are presumably continually recruited, thereby accounting for these differences. Of course, repeats are carried out using exactly the same conditions, with all strains within one repeat grown and processed on the same day, allowing direct comparison for those conditions. Specifically, for the experiment shown in


Attentional blink (AB) is a phenomenon that describes the difficulty individuals have in reporting the second of two masked targets if the second target (T2) arrives 200–500 ms after the first target (T1). Recent studies explain the AB from cognitive resources limitation to distractors interference. For example, the temporary loss of control (TLC) hypothesis suggests that the AB is conduced by distractors disrupting the input filter for target processing. The inhibition models suggest that the T1+1 distractor triggers a suppression mechanism which could be beneficial for T1 processing but would suppress T2 at short T1–T2 lags. These models consider that the AB is caused by the appearance of distractors. However, in the present study, two methods were taken to help individuals to detect the distractors more effectively. An attenuated AB deficit was found when the distractors could be excluded or suppressed in time. We consider that under an appropriate condition the distractors detection and suppression have a beneficial effect on attentional blink.Two methods were employed to help individuals to detect the distractors more effectively: enlarging the low-level-physical characteristic difference between targets and distractors  and restThe present study found when the distractors are detected or identified quickly, they could be effectively suppressed, in order to reduce the interference from the targets and result in a smaller AB deficit. We suggest that the suppression mechanism for distractors have a beneficial effect on AB. Human attention is limited with respect to time, as demonstrated by tasks with the rapid serial visual presentation (RSVP) of stimuli. In these tasks, a rapid stream of visual stimuli is presented in the center of the screen, typically at a rate of 6–20 items per second. Participants have to monitor two targets in the stream (T1 and T2). The first target (T1) is often identified correctly, but the ability to identify the second target (T2) is impaired when it follows T1 within an interval of 200–500 ms During the past two decades, many theories have been introduced to explain AB. Most To overcome this shortcoming, several recent studies have shifted their attention from T1 consumption to distractors interference in the AB The results of Di Lollo et al. In the first experiment, we add the extra source of categorical information . We expect that the extra dimension potentially increases distance between letter and digit categories, and then letter targets can be distinguished from colored digit distractors at an early processing stage, possibly on the basis of perceptual features. Under this condition, fewer cognitive resources may be needed for suppressing the distractors, and the interference with the targets processing is reduced and more cognitive resources could be used for processing T2. Although similarity models In the second experiment, we attempt to reduce the candidate digit distractors so that participants may suppress the digit distractors effectively. Landauer and Freedman The inhibition models, such as the gating theory suggested by Raymond et al., In Fifteen university students  participated in the experiment. All reported normal or corrected-to-normal vision.The generation of stimuli and response recording were done with E-Prime 1.1 (SP3). The basic alphanumeric AB paradigm was used. Black letter and digit stimuli were set in 30 point Courier New font and presented on a white background. The stimuli were located in the center of a 17 inch monitor, and viewed at a distance of 60 cm. Each trial started with a fixation presented for 1,000 ms in the center of the display followed by a rapid serial presentation of 11 to 21 digits. Each digit was presented for 50 ms, followed by a blank screen for 30 ms. In each trial, two of the digits were replaced with letters. The second target (T2) was presented with four to six temporal positions from the end of the stream. T2 was the first, second, third, fourth, fifth, seventh or ninth item following T1, corresponding to lags of 80, 160, 240, 320, 400, 560 and 720 ms . At the end of each trial, participants could report the two targets in any order by pressing the corresponding keys on the keyboard. After 1,000 ms, a new trial started. In both the control condition and the experimental condition, T1 and T2 were randomly chosen from 21 letters without replacement . In the control condition see , the disTo assess performance on the RSVP task, average T1 and T2 identification accuracy data were submitted to an ANOVA with condition and lag as within-subject factors. Trials on which T1 and T2 were accurately identified but in the wrong order were treated as correct.F = 25.15, MSE = 0.06, p<0.001. The effect of the condition was significant, F = 7.71, MSE = 0.07, p<0.001. The T1 average correct percentage in the control condition is significantly lower than in the colored digits experimental condition (p = 0.013) and in the black digits in colored circles experimental condition (p<0.001). The performance between the two experimental conditions was not significantly different, p = 0.242. In addition, there was no Condition x Lag interaction (F <1).In F = 4.87, MSE = 0.08, p<0.001. The effect of condition was also significant, F = 21.69, MSE = 0.11, p<0.001. Furthermore, the Lag x Condition interaction was significant, F = 13.83, MSE = 0.09, p<0.001. The drop in accuracy after Lag 1 was shallower in the colored digits experimental condition than in the black digits in colored circles experimental condition and in the control condition. Separate pairwise comparisons revealed that performance in the colored digits experimental condition was better than in the control condition, p<0.001. The performance in the black digits in colored circles experimental condition was better than in the control condition too, p<0.001. The performance between the two experimental conditions was not significantly different, p = 0.118. However, the performance at Lag 2 was better in the colored digits experimental condition than in the black digits in colored circles experimental condition. The performances of two experimental conditions at other Lags did not differ significantly (Fs <1). And the performances in the control condition at all Lags were significantly worse than in the two experimental conditions.In The AB effect was significantly reduced when the distractors were changed from the black digits into colored digits or black digits in colored circles. Based on these findings, it seemed that the color information from the distractors in the two experimental conditions could be a useful clue to help participants to effectively detect and exclude the distractors in comparison to the black digits in the control condition. One reason may be that the distinguishing process occurs at an early processing stage and the distractors have not been thoroughly processed. Under this circumstance, less cognitive resources are needed to suppress the interference caused by the error processing of distractors.Since Landauer and Freedman Fifteen university students  participated in the experiment. All reported normal or corrected-to-normal vision.The generation of stimuli and response recording were the same as used in p<0.05 and analyzed performance on T1 and T2 separately. The average T1 and T2 identification accuracy data were submitted to an ANOVA with condition and lag as within-subject factors. Trials in which T1 and T2 were accurately identified but in the wrong order were treated as correct.We adopted a significance level of F = 28.40, MSE = 0.06, p<0.001. The effect of the condition was significant, F = 7.12, MSE = 0.05, p<0.001. The T1 average correct percentage in the control condition is significantly lower than in the three candidate digits experimental condition (p<0.001) and in the five candidate digits experimental condition (p = 0.035). Although the performance of the three candidate digits experimental condition looks like better than the five candidate digits experimental condition, the performance between the two experimental conditions was not significantly different, p = 0.085. In addition, there was no Condition x Lag interaction (F <1).The results for T1 performance  = 47.36, MSE = 0.11, p<0.001, which demonstrated a classic ‘U’ curve. The effect of condition was also significant, F = 59.71, MSE = 0.10, p<0.001. The Lag x Condition interaction was significant, F = 4.68, MSE = 0.10, p<0.001. Separate pairwise comparisons revealed that performance in the three candidate digits experimental condition was better than in the five candidate digits experimental condition, p<0.001. And performance in the five candidate digits experimental condition was better than in the control condition, p<0.001. However, the T2 performances at Lag 1 and Lag 2 of the two experimental condition were not significantly different, correspondingly p = 0.541 and p = 0.111. Performance was firstly promoted heavily at Lag 3 in the three candidate digits experimental condition, which was significantly higher than in five candidate digits experimental condition (p = 0.042) and in control condition (p<0.001).The results for T2 performance when T1 was identified correctly (see In this experiment, only the candidate number of the digit distractors was changed in the three conditions. According to the view of TLC hypothesis, the AB deficit should be the same in three conditions as digits in three conditions had the same chance or ability to disrupt the input filter. However, the result of our experiment showed that the AB deficit gradually minimized when the number of digit distractors reduced from eight to five and three. It seemed that the participants in the restricted set, in comparison to the unrestricted set, could identify the digit distractors more easily and quickly. However, the T2 performance at Lag 2 in two experimental conditions was not significantly different. Comparing with the five candidate digits experimental condition, performance in the three candidate digits experimental condition was promoted heavily at Lag 3. One reason why the there was no a significant difference at Lag 2 may be that distractors in the two experimental conditions of Throughout the present work, we found that enlarging the low-level-physical characteristic difference between targets and distractors as adding extra source of categorical information - the presence of a color in the digit distractors or using restricted distractor set, the AB deficit was significantly reduced. One common feature between the two experiments was that the two methods were used for reducing the distractors interference with targets processing. However, the specific process of how to reduce the interference was different between the two experiments. In Our experimental results suggest that distractors detection and suppression have a beneficial effect on AB. However, the TLC hypothesis As been discussed above, we consider that the cognitive resources can be separated into two parts. One part is used to monitor and process the targets, while the other part is used to detect and suppress the distractors. If the targets and the distractors are too similar, it is harder for participants to distinguish the distractors from the targets and the more possibility distractors will get further processing. If so, more interference will be induced and more cognitive resources will be distributed to suppress the interference. Then, fewer resources are left to process the targets. However, if the targets and the distractors are very different from each other e.g.,  or the dOur hypothesis can also explain the Lag 1 sparing effect. As the T1 is followed by T2 directly and there is no distractor between them, in a short time, no cognitive resource needs to be left to suppress the distractor and this can then be used to process T2. However, if there is a distractor between T1 and T2, a part of the resources are busy processing the T1 and other resources are busy suppressing the distractor. Therefore, no more resources are left to process the T2, resulting in an AB deficit. When the T1 processing is over, the resources used for processing targets can be utilized again in processing T2. The percentage of the T2 correct response gradually increases along with the lags, which means that the processing for T1 is gradually over and T2 can obtain more resources.In conclusion, we suggest that the distractors can be suppressed actively. The AB occurs because the distractors are not well suppressed and interfere with target processing. Although our hypothesis emphasizes the distractor detection and suppression in AB, we also acknowledge the limitation of the cognitive resources. The cognitive resources limitation cannot be surmounted, but can be legitimately distributed to obtain a better cognitive performance.


Here, we discovered a novel regulatory network between AFB1, miR-33a and β-catenin in human carcinoma cells. The level of miR-33a was up-regulated in hepatocellular carcinoma (HCC) cells treated with AFB1, while in the same cells causing the decrease in β-catenin expression when treated at their IC50 values. miR-33a, specifically miR-33a-5p, was demonstrated to down-regulate the expression of β-catenin, affect the β-catenin pathway, and inhibit cell growth. Also, by employing a luciferase assay, we found that miR-33a down-regulated β-catenin by directly binding to the 3’-UTR of β-catenin. These results suggested that AFB1 might down-regulate β-catenin by up-regulating miR-33a. This understanding opens new lines of thought in the potential role of miR-33a in the clinical therapy of cancer.MicroRNAs are known to play an important role in modulating gene expression in various diseases including cancers and cardiovascular disorders, but only a few of them are associated with the pathology of aflatoxin B Aspergillus parasiticus  and Aspergillus flavus (aflatoxins B1 and B2) with aflatoxin B1 (AFB1) being the most prevalent toxin. These aflatoxins producing by members of Aspergillus commonly contaminate food, especially peanuts and corn. In humans, evidence has shown that acute aflatoxicosis could cause vomiting, disease of the liver and heart, pulmonary edema, coma and even death  curves, and then used to obtain IC50 values.5 mg of AFB1 exposure or miRNA overexpression for 48 h, cells were lysed and separated in 7.5% SDS-PAGE, and then transferred to PVDF membranes  by semi-dry electrotransfer. The PVDF membranes were blocked by 5% fat-free milk powder dissolved in TBS buffer containing 0.1% Tween-20. Antibody against β-catenin  and BeyoECL Plus  were used for detection. Band intensity of western blots was analyzed with Scion Image software  and normalized to β-actin or GADPH .After AFB1 exposure for 48 h. cDNA synthesis was carried by a First Strand cDNA Synthesis Kit . Primers for the real time PCR were listed in ®Premix Ex TaqTM  according to the manufacturer’s instructions in Mastercycler® ep realplex real-time PCR system .The total RNA of cells was extracted with TRIpure  as suggested by the manufacturer after miRNA mimics transfection or AFBCell viability was determined by the MTT Cell Proliferation and Cytotoxicity Detection Kit  following the manufacturer’s instructions. In brief, cells were transfected in a 96-well plate for 4 d, and the cell viability was determined at 550 nm each day. For colony formation assay, cells were digested by trypsin  after transfection for 48 h, and 500 cells per well were replated on a fresh 6-well plate. Cells were maintained in RPMI medium 1640 supplemented with 10% FBS, penicillin and streptomycin for 2 weeks. The cells were then fixed with methanol and stained with 0.1% crystal violet and the colony numbers per well were counted.Luciferase assay was performed by RiboBio Co., Ltd . Details were presented below. miR-33a mimics (50 nM) or negative control were cotransfected with pMIR-luciferase reporter vectors  and pRL-TK vectors  into 293T cells in 96-well plates by using Lipofectamine 2000. Dual-Luciferase® Reporter Assay System  was employed to measure the luciferase activities at 24 h after transfection. All measurements were performed in triplicates and repeated in independent experiments at least thrice.Data were presented as mean ± SD. The statistical significance of differences was assessed using one way ANOVA in Microsoft Excel. P < 0.05 meant that the differences were statistically significant. Table S1The table contains 5 miRNA candidates predicted by three onlines computational algorithms. They are ranked based on the number of binding sites found in the 3’-UTR of human β-catenin and the frequencies of prediction by three computational algorithms.(DOC)Click here for additional data file.Table S2http://www.mirbase.org/). Two miRNA mimics and their miRNA mimics negative control are purchased from RiboBio Co. Ltd., Guangzhou, China.miR-33a-5p and miR-33a-3p are synthezied according to miRBase ((DOC)Click here for additional data file.Table S3Primers are designed by Primer Premier 5.0 software and synthezied by Biotechnology Co. Ltd., Shanghai, China.(DOC)Click here for additional data file.Table S4Primers of the wild-type 3’-UTR of β-catenin are designed by Primer Premier 5.0 software and used to amplified the wild-type 3’-UTR of β-catenin. The primers and three mutated 3’-UTR of β-catenin sequences are synthezied by Biotechnology Co. Ltd., Shanghai, China.(DOC)Click here for additional data file.Table S5Primers of U6 and miR-33a-5p are designed by Primer Premier 5.0 software and other primers indicated with * are gotten from RTPrimerDB. All primers are synthezied by Biotechnology Co. Ltd., Shanghai, China. (DOC)Click here for additional data file.