Cultural Differences in Perceptual Reorganization in US and Pirahã Adults

Visual illusions and other perceptual phenomena can be used as tools to uncover the otherwise hidden constructive processes that give rise to perception. Although many perceptual processes are assumed to be universal, variable susceptibility to certain illusions and perceptual effects across populations suggests a role for factors that vary culturally. One striking phenomenon is seen with two-tone images—photos reduced to two tones: black and white. Deficient recognition is observed in young children under conditions that trigger automatic recognition in adults. Here we show a similar lack of cue-triggered perceptual reorganization in the Pirahã, a hunter-gatherer tribe with limited exposure to modern visual media, suggesting such recognition is experience- and culture-specific

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Green tea catechins (GTC) have been shown to inhibit the activities of enzymes involved in folate uptake. Hence, regular green tea drinkers may be at risk of impaired folate status. The present experiments aimed at studying the impact of dietary GTC on folate concentrations and metabolism. In a human pilot study (parallel design) healthy men consumed for 3 weeks 6 capsules (~670 mg GTC) per day (2 capsules with each principal meal) containing aqueous extracts of the leaves of Camellia sinensis (n=17) or placebo (n=16). No differences in plasma folate concentrations were observed between treatments. We further fed groups of 10 male rats diets fortified with 0, 0.05, 0.5, 1, or 5 g GTC/kg for 6 weeks. Only at the highest intake, GTC significantly decreased serum 5-methyl-tetrahydrofolate concentrations in rats, while mRNA concentrations of reduced folate carrier, proton-coupled folate transporter/heme carrier protein 1, and dihydrofolate reductase (DHFR) remained unchanged in intestinal mucosa. Using an in vitro enzyme activity assay, we observed a time-and dose-dependent inhibition of DHFR activity by epigallocatechin gallate and a green tea extract. Our data suggest that regular green tea consumption is unlikely to impair folate status in healthy males, despite the DHFR inhibitory activity of GTC. K e y w o r d s : folates, catechins, bioavailability, human, rat MATERIAL AND METHODS Dihydrofolate reductase activity The inhibition of human dihydrofolate reductase (DHFR) activity by (-) epigallocatechin gallate (EGCG) and a standardized green tea extract (Polyphenon 60 (P60); Sigma Chemical Co., St Louis, MO, USA) was measured using a commercial dihydrofolate reductase assay kit (Sigma-Aldrich) according to the manufacturer's protocol. Methotrexate, a well-known competitive DHFR inhibitor was used as a positive control. EGCG and P60 were dissolved in ultra pure-water (containing 1% ascorbic acid (w/v) (Merck KGaA, Darmstadt, Germany) to stabilize the catechins) on the day of the experiments. DHFR was used at a final activity of 1.5 x 10 -3 units per reaction. Final concentrations of EGCG and methotrexate were 1000, 100 and 10 nmol/L per reaction. P60 was used at final concentrations of 1428 .57, 142.86, and 14.29 µg/L and, thus, contained 1060 , 106, and 10.6 nmol/L EGCG and 1427.3 nmol/L of the gallated catechins (EGCG, ECG and gallocatechin gallate), respectively. Rat study Fifty male Wistar rats (Harlan Winkelmann GmbH, Borchen, Germany) with an initial body weight of 99.8 ± 2.0 g (mean ± SEM) were randomized into 5 groups of 10 animals each and housed pair-wise with sawdust bedding under controlled environmental conditions (23 ± 2°C and 65 ± 5% relative humidity, 12 h dark-light cycle). The rats were kept for 5 days on a folate-adjusted rat diet for growing animals containing 2 mg of folic acid/kg (C1027; Altromin GmbH, Lage, Germany) and thereafter received their respective experimental diets consisting of the standard diet supplemented with 0, 0.05, 0.5, 1, or 5 g green tea catechins per kg diet using P60 as the source of catechins (see The animal experiment was conducted in accordance with the German Guidelines and Regulations on Animal Care (Deutsches Tierschutzgesetz, 2006) and was approved by the University of Kiel Ethics Committee on Animal Care. Human pilot study Healthy males were recruited by advertisement at the University and local community of Reading (United Kingdom) and amongst volunteers who previously participated in nutritional trials at the Hugh Sinclair Human Nutrition Unit. Inclusion criteria were: male gender, 18-55 y of age, and a BMI in the range of 22-32 kg/m 2 . Subjects were excluded from the trial if they were diagnosed with any illness or on long-term medication, used dietary supplements, participated in >5 h of aerobic exercise activity per week, or were involved in a clinical trial within 3 months prior to the study. The study protocol was approved by the University of Reading ethics committee and all subjects gave written informed consent before participation. A standardized aqueous green tea extract prepared from the leaves of Camellia sinensis L. (a kind gift of Cognis Deutschland GmbH & Co KG, Monheim am Rhein, Germany) was used to make the green tea extract (GTE) capsules. The composition of the GTE is given in The trial was designed as a double-blind placebo-controlled parallel study. Thirty-one volunteers were randomly assigned to one of two treatment groups (GTE, n=16 or placebo, n=15) with similar BMI and age (data not shown). Subjects took 6 capsules per day, two with each principal meal, for 3 weeks and were instructed to limit their daily tea and coffee consumption to ≤ 3 cups, but to otherwise maintain their normal diet and exercise patterns. Compliance was determined by counting of the returned capsules at the end of the trial and was high (>98%). Blood samples (20 ml) were drawn into tubes containing 0.05 mL 15% K 3 EDTA (Vacutainer; Becton Dickinson UK Ltd., Oxford, UK) after an overnight fast on the first and last day of the intervention period. Plasma was immediately obtained by centrifugation (1,000 x g, 10 min) and 3 ml aliquots were stored at -80°C until analysis. Folate quantification by HPLC Procedures for extraction and purification of folates from human plasma and rat serum and liver samples by strong anion exchange solid-phase extraction were described previously by Witthoft et al. (18). Dialysed rat serum (500 µl/g) was used to ensure complete deconjugation of folate polyglutamates in liver samples; modified from Patring et al. (19). Analyses were performed using an HPLC system (Agilent 1100) consisting of a 104 10-formyltetrahydrofolate (10-HCO-H 4 folate), and 5,10-methenyltetrahydrofolate (5,10-CH + -H 4 folate) (a gift of Merck Eprova AG, Schaffhausen, Switzerland, except 10-HCO-H 4 folate, which was purchased from Schircks Laboratories, Jona, Switzerland). Quantification was based on a multilevel (n=7) external calibration curve with a linear range over 1.2-118.0 ng/mL for H 4 folate, 0.6-93.1 ng/mL for 5-CH 3 -H 4 folate, 0.9-184.1 ng/mL for 10-HCO-H 4 folate and 9.3-184.5 ng/mL for 5,10-CH + -H 4 folate. mRNA quantification RNA was isolated from rat duodenal mucosa using the RNeasy Lipid Tissue Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. DNA digestion was performed with RNase-Free DNase Set (Qiagen). RNA integrity was checked by electrophoresis on a denaturing agarose gel and ethidium bromide staining. The concentration and purity of isolated RNA was determined by measuring the absorbance (AB) at 260 and 280 nm in a spectrophotometer (DU800, Beckmann Instruments; Munich, Germany). A ratio of >1.8 between AB 260nm and AB 280nm was considered as acceptable. RNA aliquots were stored at -80°C until analysis. Primer pairs of β-actin, reduced folate carrier (RFC) and proton-coupled folate transporter/heme carrier protein-1 (PCFT/HCP1) were designed to the corresponding sequences of Rattus norvegicus mRNA with Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/ primer3_www.cgi; 03.05.2007) and purchased from MWGBiotech AG (Ebersberg, Germany). The sequences of primers used in this study were as follows: Sense primer for β-actin, 5´-GGGGTGTTGAAGGTCTCAAA-3´, antisense primer for β-actin, 5´-TGTCACCAACTGGGACGATA-3´; sense primer for RFC, 5´-GGCTCGTGTTCTACCTCTGC-3´, antisense primer for RFC, 5´-GGTAGTCGGTGAGCAGGAAG-3´; sense primer for PCFT/HCP1, 5´-TGAGCTAAGCACACCCCTCT-3´, antisense primer for PCFT/HCP1, 5´-TCCGTACCCTGTGAACATGA-3´. The product size was 90 base pair (bp) for β-actin; 183 bp for RFC and 217 bp for PCFT/HCP1. QuantiTect ® Primer Assay (Qiagen) was used for DHFR mRNA amplification, with a product size of 88 bp. For one-step quantitative reverse transcriptase polymerase chain reaction (one-step qRT-PCR) two aliquots of RNA were amplified. External relative standard curves of total RNA were determined with each run. Data was normalized by dividing the concentrations of RFC, PCFT/HCP1 or DHFR by the concentrations of β-actin mRNA. Each PCR reaction (final volume 20 µl) contained 0.5 µmol/L of each primer, 10 µl of 2x QuantiTect ® SYBR ® Green RT-PCR Master Mix (Qiagen), 0.2 µl QuantiTect RT-Mix (Qiagen), 8 µl of RNA dilution and 1.4 µl water. Real-time cycler conditions were set according to the manufacturers protocol to 40 cycles with annealing temperatures of 56°C for β-actin, 59°C for RFC, 56°C for PCFT/HCP1 and 55°C for DHFR, respectively. Quantification and melting curves of the amplified products were analysed using the RotorGene 6.0 software (Corbett Lifescience; Sydney, Australia). Melting curve analyses and agarose gel electrophoresis with ethidium bromide staining were performed to exclude non-specific products. Statistical analyses Statistical calculations were performed with GraphPad Prism 4 software (GraphPad Software Inc., San Diego, CA, USA). Analyses of the data from the rat study and the in vitro assay were performed by means of a one-way ANOVA followed by Dunnetts test for multiple comparisons of group means between animals receiving GTC or control diet. Analyses of the data from the human pilot study were performed by means of a paired Student's t-test for comparison of baseline vs. treatment and by means of an unpaired Student's t-test for comparisons between subjects receiving GTE or placebo. Reported values are means ± SEM and effects were considered significant at P<0.05. RESULTS Dihydrofolate reductase activity in vitro Both pure EGCG and P60, at concentrations of 1000 for EGCG and 1060 nmol/L for EGCG from P60, respectively, time-dependently inhibited DHFR activity Serum and liver folate concentrations in rats Feed consumption and final body mass (318.7 ± 4.8 g) of the Wistar rats were similar in all groups. Intake of diets containing 0.5% GTC over a period of 42 days significantly decreased the serum concentration of 5-CH 3 -H 4 folate compared to control rats, whereas the concentrations of H 4 folate remained unchanged ( Relative mRNA levels of reduced folate carrier and dihydrofolate reductase in rat duodenal mucosa The housekeeping gene β-actin was expressed at similar levels in all animals and no significant differences in the relative mRNA levels of RFC, PCFT/HCP1 or DHFR in the duodenal mucosa were observed Plasma folate concentrations in humans Consumption of 670 mg of GTC per day or placebo did not affect plasma folate concentrations in healthy male volunteers. No significant differences in plasma concentrations of 5-CH 3 -H 4 folate were observed between the treatment groups at baseline (placebo, 16.3 ± 2.6 nmol/L; GTE, 19.1 ± 2.4 nmol/L) or after intervention (placebo, 15.5 ± 2.1 nmol/L; GTE, 17.6 ± 2.4 nmol/L). DISCUSSION Green tea is a widely consumed beverage in many countries and contains appreciable amounts of polyphenols. Catechins (flavanols) are the major subclass of bioactive compounds within the polyphenol fraction of green tea. Epidemiological studies associated a high dietary intake of catechins with a reduced risk to suffer from a variety of diseases (reviewed in 20), including certain forms of cancer (21). The underlying molecular and cellular mechanisms by which green tea catechins may mediate anticarcinogenic acitivty seem to be diverse: Cell culture experiments as well as studies in rodents indicate that green tea catechin may inhibit angiogenesis via a down-regulation of vascular endothelial growth factor (reviewed in 22). Furthermore it has been suggested that the anticancer activity of green tea catechins against different kind of cancers may find an explanation in direct targeting of lipid rafts (23). Recent in vitro studies have shown that epigallocatechin gallate (EGCG), the predominant catechin in green tea, competitively inhibits the enzyme dihydrofolate reductase (DHFR) (9, 13). DHFR inhibition is the mechanism by which so-called antifolates, such as the cytostatic drug methotrexate, inhibit cell division and reduce tumor growth (15, 24). Co-administration of folic acid and the DHFR inhibitors methotrexate and pyrimethamine, respectively, reduced plasma folate concentrations in rats The commercial green tea extract Polyphenon 60 (P60) used in the rat study and its principle bioactive ingredient EGCG inhibited DHRF activity time-and concentration-dependently in vitro In order to study whether or not the effects observed in vitro bear a meaning for the more complex physiological processes in vivo, Wistar rats were fed for 42 days with diets fortified with increasing concentrations of green tea catechins (GTC) using a standardized green tea extract (P60). The diets contained 2 mg folic acid per kg, which is equivalent to twice the dietary recommendations for laboratory rats as given by the National Research Council (28). It is noteworthy that folates synthesized by the microflora of the large intestine are absorbed and may significantly contribute to blood folate concentrations (reviewed in 29). The diet used in this study was therefore formulated to provide a minimum of substrate to the intestinal microflora to limit bacterial folate synthesis. Only in those animals fed the highest concentrations of the green tea extract (0.5% GTC), did we observe a significant decrease in serum 5-CH 3 -H 4 folate concentrations as compared to the control group ( At a given substrate affinity and substrate concentration, the capacity of enzymatic turnover of folates as well as the amount of their carrier-mediated transport across cellular membranes is mainly affected by the amount of enzymes/carriers present at the tissue level. Because catechins are known to alter the gene expression for a variety of proteins (35), we quantified relative mRNA concentrations of the RFC, PCFT/HCP1, and DHFR in the duodenal mucosa of rats fed GTC. No significant differences in mRNA concentrations of RFC, PCFT/HCP1, and of DHFR were found between the experimental groups The current findings suggested that GTC might decrease serum folate concentrations only if supplied at supra-nutritional doses. A 70 kg human would have to drink almost 100 cups of green tea infusion per day to match the highest dose fed to rats in the present study. Because such a human study would be unfeasible as well as unrealistic, we designed a pilot study with a standardized green tea extract to assess whether or not regular consumption of high doses of GTC might affect plasma folate concentrations in humans. The intake of 670 mg of GTC per day, which corresponds to about 20 cups of green tea, caused no significant differences in plasma concentrations of 5-CH 3 -H 4 folate between the treatment and placebo groups, both of which consuming a normal diet containing on average ~328 ± 26 µg folate/d. Insufficient dietary intake of folates for as short as 2-3 weeks has been reported to result in reduced blood concentrations of the vitamin (30). Our findings therefore suggest that green tea drinking is unlikely to affect plasma folate concentrations in healthy, free-living subjects and that a longer treatment period and/or even higher doses of dietary GTC may be necessary to induce changes in folate concentrations, if possible at all. Further human studies with GTC and a standardized supply of folic acid (in the absence of naturally occurring reduced folates) are warranted to investigate the influence of GTC on DHFR activity in vivo. In addition, the measurement of (oxidized) serum folic acid should be considered because folic acid has been found in serum of subjects consuming folic acid-fortified foods for 5 d (11). Based on the experiments presented here, it appears unlikely that daily green tea consumption, even at high levels, may affect folate concentrations in healthy humans. Acknowledgement

Pseudo-Synesthesia through Reading Books with Colored Letters

Background Synesthesia is a phenomenon where a stimulus produces consistent extraordinary subjective experiences. A relatively common type of synesthesia involves perception of color when viewing letters (e.g. the letter ‘a’ always appears as light blue). In this study, we examine whether traits typically regarded as markers of synesthesia can be acquired by simply reading in color. Methodology/Principal Findings Non-synesthetes were given specially prepared colored books to read. A modified Stroop task was administered before and after reading. A perceptual crowding task was administered after reading. Reading one book (>49,000 words) was sufficient to induce effects regarded as behavioral markers for synesthesia. The results of the Stroop tasks indicate that it is possible to learn letter-color associations through reading in color (F(1, 14) = 5.85, p = .030). Furthermore, Stroop effects correlated with subjective reports about experiencing letters in color (r(13) = 0.51, p = .05). The frequency of viewing letters is related to the level of association as seen by the difference in the Stroop effect size between upper- and lower-case letters (t(14) = 2.79, p = .014) and in a subgroup of participants whose Stroop effects increased as they continued to read in color. Readers did not show significant performance advantages on the crowding task compared to controls. Acknowledging the many differences between trainees and synesthetes, results suggest that it may be possible to acquire a subset of synesthetic behavioral traits in adulthood through training. Conclusion/Significance To our knowledge, this is the first evidence of acquiring letter-color associations through reading in color. Reading in color appears to be a promising avenue in which we may explore the differences and similarities between synesthetes and non-synesthetes. Additionally, reading in color is a plausible method for a long-term ‘synesthetic’ training program

Illusions of Visual Motion Elicited by Electrical Stimulation of Human MT Complex

Human cortical area MT+ (hMT+) is known to respond to visual motion stimuli, but its causal role in the conscious experience of motion remains largely unexplored. Studies in non-human primates demonstrate that altering activity in area MT can influence motion perception judgments, but animal studies are inherently limited in assessing subjective conscious experience. In the current study, we use functional magnetic resonance imaging (fMRI), intracranial electrocorticography (ECoG), and electrical brain stimulation (EBS) in three patients implanted with intracranial electrodes to address the role of area hMT+ in conscious visual motion perception. We show that in conscious human subjects, reproducible illusory motion can be elicited by electrical stimulation of hMT+. These visual motion percepts only occurred when the site of stimulation overlapped directly with the region of the brain that had increased fMRI and electrophysiological activity during moving compared to static visual stimuli in the same individual subjects. Electrical stimulation in neighboring regions failed to produce illusory motion. Our study provides evidence for the sufficient causal link between the hMT+ network and the human conscious experience of visual motion. It also suggests a clear spatial relationship between fMRI signal and ECoG activity in the human brain

Folate content in strawberries (Fragina x ananassa): Effects of cultivar, ripeness, year of harvest, storage, and commercial processing.

Folate concentrations in strawberries and folate retention during storage and commercial processing of strawberries were investigated. No previous study has focused on the effects of cultivar, ripeness, and year of harvest of strawberries with respect to the folate content. This study showed the folate concentration in strawberries to significantly depend on all of these different factors. Total folate was quantified using a modified and validated radioprotein-binding assay with external calibration (5-CH3-H4folate). Folate content in 13 different strawberry cultivars varied from 335 g/100 g of dry matter (DM) for cv. Senga Sengana to 644 g/100 g of DM for cv. Elsanta. Swedish harvests from 1999 and 2001 yielded higher folate concentrations than did the harvest from 2000, and the grade of ripeness affected the folate content in strawberries. This study indicated high folate retention in intact berries during storage until 3 or 9 days at 4 C (71-99%) and also in most tested commercial products (79-103%). On the basis of these data fresh strawberries as well as processed strawberry products are recommended to be good folate sources. For instance, 250 g (fresh weight) of strawberries (~125 g of folate) supplies ~50% of the recommended daily folate intake in various European countries (200-300 g/day) or 30% of the U.S. recommendation (400 g/day)

Thinking for Seeing: Enculturation of Visual-Referential Expertise as Demonstrated by Photo-Triggered Perceptual Reorganization of Two-Tone “Mooney” Images

Two-tones (”Mooney”-esque transformations of grayscale photographs) can be difficult to recognize. However, after viewing the photographs from which two-tones were created, adults experience rapid “perceptual reorganization, ” and the two-tones become immediately recognizable. In contrast to the effortless post-cue recognition seen in adults, preschoolaged children are generally unable to recognize two-tone images even when the photograph is simultaneously available. While simple instructional and perceptual interventions were ineffective, a cognitive intervention in which children were convinced that the photo and two-tone images were transformations of the same physical object improved children’s recognition. We found a similar deficit in recognition in adults from a hunter-gatherer tribe (Pirahã) with a sparse visual symbolic culture and limited exposure to modern visual media. Photo-triggered perceptual reorganization of two-tone images may therefore be a product of prolonged enculturation, reflecting visual-referential expertise. As we gain skill in representing visual correspondences, one of the surprising consequences may be the ability to literally see things we couldn’t see before

Example of misaligned photo and two-tone image pair.

<p>This image shows the actual degree of misalignment, but participants were never informed about the degree or direction of misalignment or even if any misalignment or distortion occurred between the images. The intersection of the horizontal and vertical red lines shows the same geometric point relative to the frame of the images. Participants could identify these matching points even without reference to the underlying images (for example, if the cards were blank). Actual corresponding features are shown with the red dot, and were displaced 1.8 cm in a direction that varied from image pair to image pair.</p

Upper left: an example two-tone stimulus from the Pirahã study.

<p>Subjects were first presented the two-tone alone and asked to point to the location of an eye or person in the image. Red circles mark where Pirahã participants indicated an eye, and numbers indicate individual participants. Circles outside the image show responses of the form “there are no eyes here.” Only two participants (2,8) correctly pointed to an eye in this image during Stage 1. Upper right: Performance of Pirahã participants on the original photo, which was presented alone after the two-tone image was removed from view. All participants correctly pointed to one of the two eyes. Bottom row: performance of Pirahã participants on the two-tone image during Stage 3, when it was shown side-by-side with the photo. Two Pirahã participants succeeded uncued (2 & 8), two more succeeded with the photo present, indicating reorganization (3 & 4), and five did not show evidence of photo-triggered perceptual reorganization (1, 5, 6, 7, 9).</p