It is well-known that emotionally salient events are remembered more vividly than mundane ones. Our recent research has demonstrated that such memory vividness (Mviv) is due in part to the subjective experience of emotional events as more perceptually vivid, an effect we call emotionally enhanced vividness (EEV). The present study built on previously reported research in which fMRI data were collected while participants rated relative levels of visual noise overlaid on emotionally salient and neutral images. Ratings of greater EEV were associated with greater activation in the amygdala and visual cortex. In the present study, we measured BOLD activation that predicted recognition Mviv for these same images 1 week later. Results showed that, after controlling for differences between scenes in low-level objective features, hippocampus activation uniquely predicted subsequent Mviv. In contrast, amygdala and visual cortex regions that were sensitive to EEV were also modulated by subsequent ratings of Mviv. These findings suggest shared neural substrates for the influence of emotional salience on perceptual and mnemonic vividness, with amygdala and visual cortex activation at encoding contributing to the experience of both perception and subsequent memory. It is well-known that emotionally important events such as the birth of a child or September 11 hold a special place in memory  in part reflects a subjective richness of perceptual experience, or emotionally enhanced vividness (EEV)  with normal or corrected to normal vision and no history of psychiatric disorders participated in this study. Two participants were removed from subsequent fMRI analysis: one had excessive movement and one misunderstood the task, leaving 16 participants .In order to localize category-selective regions of visual cortex, we used a block design task that alternated blocks of line-drawings of objects and scrambled line-drawings to localize object-selective regions of the LOC. The task also included blocks of faces and houses to localize face and place selective regions of visual cortex respectively. Blocks alternated randomly to minimize category predictability and each image category was presented six times. Each 20 s block contained five images presented for 4000 ms each. In each block one of the images was repeated. To ensure that they were attending to the images presented onscreen, participants were asked to push a button with the index finger each time an image appeared if the image was not the same as the one immediately preceding it, and to press a second button with the middle finger if the image was identical to the one immediately preceding it. Because the behavioral task served merely as a vigilance task to maintain attention and maximize BOLD activation, behavioral results were not analyzed.Fs < 0.1. Positive, negative, and neutral images were selected to be equivalent on basic low-level image statistics, equated in log luminance, F < 1, and RMS contrast, F < 1. One of three levels of Gaussian all-color noise (15%) was superimposed over each image using adobe Photoshop 7.0.Twenty-five negative photos and 25 positive photos were taken from the International Affective Picture System (IAPS). Twenty-five neutral photos were retrieved from the internet as well as the IAPS. A separate set of participants rated all pictures to be equal in brightness, contrast, and visual complexity, To minimize variance associated with differences in luminance and contrast across images, a standard was created to match each corresponding comparison image Figure . StandarTrials were presented in five separate runs of 30 trials. In order to reduce time spent into the scanner in this slow event-related design, each image was presented twice at two out of three levels of standard noise for a total of 150 trials . Thus, each image was seen twice at 15% noise paired with a standard at two out of three possible noise levels . In each 12-s trial a standard was presented for 1500 ms, followed by a 500 ms ISI, followed by the picture which was presented for 1500 ms. Both standards and images subtended a visual angle of 13° × 9.5°. After a randomly jittered interval of 1500, 2000, or 2500 ms, a response meter appeared for 4000 ms, followed by a randomly jittered ITI of 2000, 2500, or 3000 ms. Participants clicked a button with the index finger to move the cursor to the left (less noisy than standard) and with the middle finger to move the cursor to the right (more noisy than standard), and a third button with the middle finger to indicate that the cursor was placed at the desired degree of noisiness. Fifty null trials (1/3), consisting of 12 s of fixation, were included at randomized intervals. After the scan participants were asked to log on to a website 1 week later.One week after performing the NE task, participants were instructed to log on to a website to complete a surprise memory task exactly 1 week after performing the NE task. An additional set of images, 12 positive, 12 neutral, and 12 negative, were matched with the Session 1 images for arousal, scene content, contrast, and luminance. Participants were shown the original 75 images and the 36 new images. After rating an image as new, familiar, or recollected, participants rated the vividness of the memory for recognized items on a scale of 1–7 (0 = new image), ranging from vague to detailed.Imaging data were collected with a 3T Siemens scanner using a 12-channel head coil. Both the localizer and experimental tasks were programed in E-prime Version 1.2 . For each subject, a three-dimensional magnetization prepared rapid-acquisition gradient-echo pulse sequence (MPRAGE) was used to acquire a high-resolution T1-weighted structural volume: repetition time (TR) = 1760 ms; echo time (TE) = 2.2 ms; FOV = 256 × 256; slice thickness = 1mm; 176 slices; total acquisition time = 7:32 min.T2* weighted gradient-echo echo planar images were collected for two short field mapping series to correct for EPI distortion due to inhomogeneities in the magnetic field. Parameters for the field mapping series were: TR = 793 ms; TE1 = 5.19 ms, and TE2 = 7.65 ms; flip angle = 60; FOV = 211 mm. Thirty-five slices were acquired with a voxel size of 3.3 mm × 3.3 mm × 3.5 mm. EPI parameters for the two functional tasks were: TR = 2000 ms; TE = 25 ms; flip angle = 78°; FOV = 211mm. Thirty-five slices were acquired with a voxel size of 3.3 mm × 3.3 mm × 3.5 mm.http://www.fil.ion.ucl.ac.uk/spm/sofware/spm8). Slice timing correction of reconstructed images was performed after removing the first five time points from each functional run. Field maps were unwarped with the EPI time series and time series were realigned for motion and field distortion correction. T1-weighted structural images were co-registered to the EPI images, and then bias corrected and segmented using MNI template tissue probability maps. EPI images were then co-registered to the normalized segmented anatomical images. Finally, time series data were smoothed with a 6 mm full-width half maximum Gaussian kernel.Data were analyzed with Statistical Parametric Mapping software  . Our goal was to compare regions correlated trial by trial with Mviv ratings with previously patterns of parametric modulation by emotionally enhanced perceptual vividness. We thus examined parametric modulation of the BOLD response by both perceptual and mnemonic vividness ratings for each trial across all stimulus categories . For the experimental data, a delta function regressor was modeled for image onset and convolved with the canonical HRF for each trial in each analysis. For each subject, we created a first-level statistical parametric model (SPM). To capture variance due to low-level features of the images which could influence our results, we first entered four separate regressors modeling scene complexity, hue, contrast, and mean visual saliency   from each individual were entered into a one-way ANOVA, with condition as the single factor. Contrasts for (objects > scrambled objects) were used to specify category-selective activation in the LOC. An additional contrast was used to specify place-sensitive regions of parahippocampal cortex (houses > faces). Functionally defined masks were created using 10 mm spheres around maxima activations in the group maps  thresholded at p < 0.05 (FWE). Anatomical masks for right and left amygdala and right and left hippocampus were created from automated anatomical labeling (AAL)  at the group level. Initial SPMs were thresholded at a height threshold of p < 0.005, uncorrected, with a cluster extent threshold of 10 voxels  = 28.42, p < 0.001. A significant linear contrast, F = 32.01, p < 0.001, revealed noise ratings of the images to be relatively lower relative to the standard as standard noise levels got higher, again indicating that participants were accurate at gaging relative noise even when noise level was manipulated in the standard. There was also a main effect of emotion, F = 10.32, p < 0.001, with arousing images rated as less noisy – or more perceptually vivid – than neutral images, F = 12.50, p = 0.003. A modest standard noise × emotion interaction, F = 4.26, p < 0.05, revealed differences between positive and neutral images to be greater at the two lower levels of standard noise. Contrasts revealed that, as in previous studies both positive and negative images were rated as less noisy, or more vivid, than neutral images, ps < 0.05; unlike in previous studies, negative images were also rated as more vivid than positive images, p < 0.05.A two factor repeated measures ANOVA was performed on NE with standard noise (3) and emotion (3) as factors. Results showed a main effect of standard noise, F = 9.33, p = 0.001. Contrasts revealed that negative images were remembered more vividly than neutral or positive images, F = 20.82, p = 0.001. There was no effect of emotion category on memory accuracy.A one-way ANOVA was performed on Mviv scores with emotion as a repeated measure. There was a main effect of emotion category, −1 were associated with more activation in the left amygdala  and left LOC . Regions where greater activation was associated with higher levels of Mviv similarly included the left amygdala  and left LOC . To more closely examine the relation between regions modulated by memory and perceptual vividness, the contrast for Mviv was masked inclusively by activation for NE−1at a threshold of p = 0.005, uncorrected. All voxels activated by memory in the left amygdala and LOC fell within regions that were activated by perceptual vividness. As Figures −1 in the left amygdala and left LOC. These voxels were also activated significantly in both conditions when analyzed separately.As previously reported, higher levels of NE−1 revealed that a region of left hippocampus  and a place-sensitive region of left parahippocampal gyrus  were activated more for Mviv than NE−1  are more likely to be remembered than neutral images Ochsner, . In partYet this finding is in contrast to a levels of processing framework (Craik and Lockhart, In previous studies we found no differences between noise estimation ratings for positive and negative images (Todd et al., As previously reported, amygdala activation accounted statistically for the relation between LOC activation and EEV at the time of encoding (Todd et al., after encoding influences memory consolidation to alter memory retention, which by definition cannot be explained by vivid perceptual encoding. Phasic arousal related to perceptual vividness may interact with more tonic arousal extending beyond initial encoding to alter memory consolidation (Cahill and Alkire, It is important to highlight that our previously reported behavioral findings suggested that enhanced perceptual vividness contributes to, but does not entirely account for, the heightened salience of emotional memories (Todd et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Post-mortem studies have suggested a link between the thalamus, psychiatric disorders, and suicide. We evaluated the thalamus and anterior thalamic radiations (ATR) in a group of Veterans with and without a history of suicidal behavior (SB) to determine if thalamic abnormalities were associated with an increased risk of SB. Forty Veterans with mild traumatic brain injury (TBI) and no SB (TBI-SB), 19 Veterans with mild TBI and a history of SB (TB + SB), and 15 healthy controls (HC) underwent magnetic resonance imaging scanning including a structural and diffusion tensor imaging scan. SBs were evaluated utilizing the Columbia Suicide Rating Scale and impulsivity was measured using the Barratt Impulsiveness Scale (BIS). Differences in thalamic volumes and ATR fractional anisotropy (FA) were examined between (1) TBI + SB versus HC and (2) TBI + SB versus combined HC and TBI-SB and (3) between TBI + SB and TBI-SB. Left and right thalamic volumes were significantly increased in those with TBI + SB compared to the HC, TBI-SB, and the combined group. Veterans with TBI + SB had increased FA bilaterally compared to the HC, HC and TBI-SB group, and the TBI-SB only group. Significant positive associations were found for bilateral ATR and BIS in the TBI + SB group. Our findings of thalamic enlargement and increased FA in individuals with TBI + SB suggest that this region may be a biomarker for suicide risk. Our findings are consistent with previous evidence indicating that suicide may be associated with behavioral disinhibition and frontal-thalamic-limbic dysfunction and suggest a neurobiologic mechanism that may increase vulnerability to suicide. Suicide represents a major global health problem as well as a significant personal tragedy. The return of Veterans from Operation Enduring Freedom/Operation Iraqi Freedom (OEF/OIF) has emphasized the crucial importance of understanding the social, environmental, and neurobiological factors associated with suicidal behavior (SB) in both a Veteran and civilian population. However, the identification of relevant factors has often been complicated by the presence of both state and trait factors, many of which may be related to psychiatric disorders. Over the past decade, several authors have suggested that risk for suicide may be independent of a specific psychiatric disorder and may have distinct trait neurobiological expressions –5. For iNeuroimaging studies have also found regions within the fronto-striatal-limbic circuit that may be associated with SBs, irrespective of diagnosis. For example, Monkul and colleagues reported reduced orbitofrontal cortex (OFC) and increased amygdala gray matter volumes in individuals with depression and SB compared to individuals with depression alone . In a reEarly work from Ahearn et al.  applied Currently there exists a paucity of conclusive findings regarding the relationship between suicide risk and traumatic brain injury (TBI). Of particular concern is the influx of TBI among Veterans returning from war zones in which they are frequently exposed to blast forces frequently resulting in disability. To date, over 320,000 soldiers have reported a possible TBI from OIF/OEF, and it has been estimated that as many as 60% of returning war Veterans have sustained a TBI , 20. In A post-mortem study of a mixed patient population, including MDD, BD, and schizophrenia, in which half died by suicide found enlarged thalamic volumes in those individuals who had an MDD diagnosis, died by suicide, or had the short allele (ss) for a genetic variant for the SERT gene . IndividThe association of impulsivity, aggression, and suicide risk has been reported in the literature , 28, 29.The overall aim of the proposed study was to examine the neurobiological correlates of trait related risk factors for suicide in a cohort of Veterans with TBI with and without SB, matched for depression, posttraumatic stress disorder (PTSD), and TBI severity indices. Given the recent post-mortem study of enlarged thalamic volumes in individuals who committed suicide and the paucity of data examining the morphological changes of the thalamus in individuals with SB, we performed a volumetric analysis of thalamic volumes in Veterans with TBI with and without SB. We hypothesized that individuals with SB would have enlarged thalamic volumes compared to a HC population and to individuals with TBI without SB. In addition, we examined the FA of the ATR to determine if FA would have a corresponding increase or decrease in association with hypothesized changes in thalamic volumes in our TBI with SB group. We then examined our morphological findings in relation to symptoms of impulsivity and suicide.The Institutional Review Boards at the University of Utah and the George E. Whalen Department of Veterans Affairs (VA) Medical Center approved this study. All subjects provided written informed consent prior to participation in this study. A total of 74 male subjects including, 40 Veterans with mild TBI and no SB (TBI-SB) (age = 34.6 ± 8.1 years), 19 Veterans with mild TBI and a history of SB (TB + SB) (age = 38.0 ± 7.8 years), and 15 HC (age = 36.5 ± 11.5 years) were recruited from the George E. Whalen VA Medical Center and the community via local advertisements and by word of mouth. Inclusion criteria for all participants in this analysis were: ages 18–55 years old. Demographic information on the diagnostic groups is shown in Table Comparison HC did not have a current major DSM-IV Axis I diagnosis, including substance use disorders (SUD) other than nicotine, based on clinical interviews. Exclusion criteria for all subjects included major sensorimotor handicaps ; estimated full scale IQ < 80; history of claustrophobia, autism, schizophrenia, anorexia nervosa or bulimia, active medical or neurological disease other than TBI that would impact neurobiology or brain function; history of electroconvulsive therapy; and metal fragments or implants that would be contraindicated in an MRI.All participants, including HC, completed the Structured Clinical Interview for DSM-IV Patient Version (SCID-I/P)  administMeasures of current clinical status were obtained prior to scanning and included the Hamilton Depression Rating Scale (HAM-D)  and the The Columbia Suicide Severity Rating Scale (C-SSRS) was administered to all participants to assess for the presence of past and current suicidal ideation and SB . In addi2 FOV, 160 slices, 1.0 mm slice thickness. A DT-MRI GRAPPA sequence was also obtained utilizing 64 directions, two diffusion weightings: b = 0, 1000 s/mm2, TE/TR = 88 ms/9 s; matrix = 128 × 128 on 256 × 256 on FOV; 2 × 2× 2 isotropic voxels, slice thickness = 2.0 mm with 0 gap, and 70 slices. The original imaging data were transferred from the scanner in the DICOM format and coded. All MRI scans were read by a neuroradiologist to rule out gross pathology.Structural imaging was performed at the Utah Center for Advanced Imaging Research (UCAIR) using a 3 T Siemens Trio scanner. Structural imaging data was acquired using a T1-weighted 3D MPRAGE GRAPPA sequence acquired sagittally using a 12-channel head coil with TE/TR/TI = 3.38 ms/2.0 s/1.1 s, 8 °flip, 256 × 256 acquisition matrix, 256 mmThalamic volumes for each participant were calculated within the FreeSurfer image analysis environment, which is documented and freely available for download onlineThe diffusion weighted imaging data was analyzed using FSL’s FDT  were created for the left and right ATR utilizing the JHU white matter tractography atlas , 62. Thet-tests and Chi-square tests were performed for demographic and clinical data. Left and right thalamic volumes extracted from FreeSurfer were evaluated first by Student’s t-tests and then further via Univariate analyses. Thalamic volumes were adjusted by total segmented brain volume (TSV) (volume/TSV × 1000) prior to any analyses. To determine whether or not a laterality effect was present within the thalamus an asymmetry index was calculated for the thalamus using the following formula: (right volume – left volume)/[(right volume + left volume)/2]. Pearson’s correlations were performed between thalamic volumes and mean FA values and clinical variables including suicide and impulsivity measures. Voxel-wise regressions were also performed via permutation testing for clinical variables and total ATR FA data. Cohen’s d was calculated for volumetric and mean FA data to determine effect sizes. Because we had specific a priori hypotheses regarding thalamic volumes and FA within the thalamic radiations, corrections for multiple comparisons were not performed.Statistical analyses were carried out with SPSS software (version 19) for Macintosh. Independent Student’s p < 0.001) (See Table p < 0.001) as was severity of anxiety symptoms, as evaluated by the HAM-A . Increased impulsivity personality traits, as assessed by the BIS total score were found in the TBI + SB group, relative to controls  a finding that seemed to be driven by the BIS attention subscale score  as other subscale scores failed to reach significance.There were no significant between-group differences in age, years of education, vocabulary measures, or social status as assessed by the Hollingshead ; howeverp = 0.01), social status (p = 0.03), and GAF scores (p < 0.001). Depression symptom scores were higher in the TBI + SB group relative to the combined HC and TBI-SB group (p < 0.01); however, no significant difference was found between groups on HAM-A scores. A significant increase in BIS total score was found in the TBI + SB group relative to the combined group (p = 0.02). Subscale BIS scores assessing impulsivity traits related to attention and planning were also significantly increased in the TBI + SB group relative to the combined group ; however, the subscale score assessing motor impulsivity was not significantly different between groups.There were no significant between-group differences in age or vocabulary measures; however, a significant difference was found between all three groups for years of education (p = 0.03) and GAF scores . Depression severity was higher in the TBI + SB group relative to the TBI-SB group ; however, no significant difference was found between groups on HAM-A scores.There were no significant between-group differences in age, social status, or vocabulary measures; however, a significant difference was found between groups for years of education ; MDD, n = 23 (57.5%); TBI + SB: PTSD, n = 14 (73.7%); MDD, n = 14 (73.7%), χ2 = 0.26 and 0.26 respectively), or both PTSD and MDD (TBI-SB: n = 16 (40.0%); TBI + SB: n = 12 (63.2%), χ2 = 0.10). In the TBI-SB group, 10 subjects had neither a PTSD nor MDD diagnosis, 7 had PTSD only, 7 had MDD only, and 16 subjects had comorbid PTSD and MDD diagnoses. In the TBI+SB group, 3 subjects had neither a PTSD nor MDD diagnosis, 2 had PTSD only, 2 with MDD only, while 12 subjects had comorbid PTSD and MDD. In the TBI + SB only group, the number of suicide behaviors was 2.05 ± 1.61, number of suicide attempts was 0.95 ± 1.03, and intensity of ideation was 17.00 ± 5.26.No significant difference in the overall number of TBI events was found between groups. Furthermore, there were no statistically significant differences between groups in the number of subjects diagnosed with lifetime PTSD or MDD , benzodiazepines (χ2 = 0.001), hypnotics/non-benzodiazepines (χ2 = 0.02), and stimulants (χ2 = 0.04) use compared to the TB-SB group.Of the 19 TBI + SB subjects, 13 were taking an antidepressant (68.4%), 4 were on a mood stabilizer (21.1%), 7 were taking a benzodiazepine (36.8%), 3 were on an atypical antipsychotic (15.8%), 12 were taking a hypnotic/non-benzodiazepine (63.2%), and 2 participants were prescribed a stimulant (10.5%). Out of 40 TBI-SB subjects, 14 were taking an antidepressant (35.0%), 4 were on a mood stabilizer (10.0%), 2 were taking a benzodiazepine (5.0%), 4 were on an atypical antipsychotic (10.0%), 12 were taking a hypnotic/non-benzodiazepine (30.0%), and none were prescribed a stimulant. Chi-squared analyses indicated the TBI + SB group had higher frequency of antidepressants . Depression scores reduced between group significance in the left thalamus between HC and TBI +SB to a trend level (p = 0.07), however, the remainder of the findings still maintained significance .Separate general linear model analyses were conducted to examine the impact of depression and anxiety, separately, using left and right adjusted thalamic volumes as the dependent variables, the appropriate groups as a fixed factor, and HAM-A and HAM-D as covariates. Anxiety scores did not significantly impact our original findings between groups  = −2.10, p = 0.04], and left  volumes were found to be larger in the TBI + SB group. Left and right thalamic volumes were also found to be significantly larger in the TBI + SB group compared to the HC plus the TBI-SB group . When comparing TBI + SB to TBI-SB, again those with a lifetime history of SB had increased volumes in the right thalamus  and demonstrated a trend in the left thalamus  (See Table p = 0.53) or right (p = 0.62) adjusted thalamic volumes. No significant differences in the asymmetry index for the thalamus was found between HC versus TBI + SB, HC versus TBI-SB, TBI-SB versus TBI + SB, or HC versus TBI-SB and TBI + SB.Thalamic volumes, adjusted for whole brain volume, for TBI + SB was compared to HC, and both right  and total ATR . No significant between group differences were found between HC and the combined TBI-SB and TBI + SB group for left (p = 0.12), right (p = 0.45), or total (p = 0.56) ATR FA values. No statistically significant differences were found between TBI + SB and either HC or the combined HC and TBI-SB group  . Adjusted thalamic volumes did not significantly correlate with any TBI measures. Furthermore, in the TBI + SB group, no measure of suicide or impulsivity was found to significantly correlate with adjusted thalamic volumes.Correlations with clinical and TBI measures were performed on bilateral thalamic adjusted volumes for the TBI + SB group. In the TBI + SB group, the right hemisphere thalamus was significantly correlated with HAM-A score (r(19) = 0.47, p = 0.04]. No other suicide, TBI, or other clinical measures were found to correlate with mean FA values. Voxel-wise regressions were also performed via permutation testing for clinical variables and total ATR FA data. Significant positive regressions were found for bilateral ATR and HAM-A, BIS total, and BIS planning (p ≤ 0.01) and trends were found for bilateral ATR and HAM-D and BIS attention (p ≤ 0.05)  or were not the appropriate measure to detect trait (most severe intensity of ideation) behaviors. However, several other DT-MRI studies have proven effective in observing the relationship between behavioral traits and structural connectivity, particularly impulsivity and suicidality, within the brain. For example, in a study of patients with attention-deficit/hyperactivity disorder, DTI measures of frontal FA were reported to correlate with impulsivity . In addiThere are a number of factors that must be considered when interpreting these study findings. First, the classification of TBI subjects is an area of ongoing debate and the application of different classification criteria can impact study results. Our categorization was based on subject self-report using a structured interview (OSU-TBI) , a methoThus far, reports on the effects of medication on DTI measures appear inconsistent, although some studies suggest that FA measures may be impacted by medication. For example, white matter FA has been found to be inversely related with mood stabilizer load in individuals with BD , antipsyIn summary, this is one of the first studies to evaluate thalamic structural and microstructural brain changes as measured by FA using DTI and morphometry using MRI in a cohort of Veterans with TBI + SB compared to Veterans with TBI-SB and HC. The present results suggest that Veterans with TBI + SB had enlarged thalamic volumes and increased FA in bilateral ATR as compared to both TBI-SB and HC. Furthermore, the increased FA in bilateral ATR regions was positively associated with self-reported measures of impulsivity. These findings are consistent with previous evidence indicating that suicide may be associated with behavioral disinhibition and frontal-thalamic-limbic dysfunction and suggest a neurobiologic mechanism that may increase vulnerability to suicide. One potential application of these findings may be identifying individuals at-risk for suicide. Further evaluation of key fronto-thalamic-limbic circuits via multimodal imaging combined with candidate genes for SB such as the SERT gene is warranted to ascertain whether or not individuals can be identified as “at-risk” for future suicide. In addition, examining the functional connectivity of the prefrontal-thalamic-limbic circuitry utilizing both resting state and task-based fMRI data in this population would also have the potential to expand current knowledge into the neurobiological underpinnings that may increase risk for suicide. Finally, the thalamus is a region of high susceptibility to diffuse axonal injury in TBI and therefore further study in a high risk Veteran population is needed.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Obesity is closely associated with various cardiovascular diseases (CVDs). Adipose tissue inflammation and perturbation of adipokine secretion may contribute to the pathogenesis of CVD. This study aimed to evaluate whether the 2 most abundant adipokines, adipocyte‐fatty acid binding protein (A‐FABP) and adiponectin, are independent risk factors predisposing to CVD.P<0.001), but similar adiponectin levels (P=0.881) compared with the non‐CVD group. In Cox regression analysis including both biomarkers, the adjusted HR for A‐FABP and CRP for subjects above the optimal cutoff values were 1.57  and 1.60 , respectively, after adjustment for traditional risk factors. The category‐free net reclassification index, but not the c‐statistic, showed improvement in predictive performance by the addition of A‐FABP to the traditional risk factor model (P=0.017).We investigated prospectively the 12‐year development of CVD in relation to the baseline levels of A‐FABP and adiponectin in a population‐based community cohort comprising 1847 Chinese subjects recruited from the Hong Kong Cardiovascular Risk Factors Prevalence Study 2 (CRISPS 2) cohort without previous CVD. Baseline serum levels of A‐FABP, adiponectin, and C‐reactive protein (CRP), an established biomarker predictive of CVD, were measured. In all, 182 (9.9%) of the 1847 Chinese subjects developed CVD during a median follow‐up of 9.4 years. The CVD group had more traditional risk factors, higher baseline levels of A‐FABP and CRP (both Circulating A‐FABP level predicts the development of CVD after adjustment for traditional risk factors in a community‐based cohort. Its clinical use for CVD prediction warrants further validation. A‐FABP and adiponectin are the 2 most abundant adipokines produced by adipocytes. Recent studies suggest that adipose tissue inflammation, characterized by the infiltration of inflammatory cells and perturbation of adipokine secretion, may play a central role in the pathogenesis of obesity‐related pathologies.2 A‐FABP is a cytoplasmic lipid chaperon and plays an important role in mediating intracellular fatty acid trafficking. In addition, A‐FABP may potentiate vascular inflammation and participate in the pathogenesis of CVD.4 In animal studies, A‐FABP deficiency resulted in a significant reduction of aortic atherosclerotic lesions in apolipoprotein‐E (apoE)–deficient mice, a mouse model with accelerated atherosclerosis, and a higher survival rate when they were fed on a high‐fat atherogenic diet.6 In contrast, the administration of recombinant adiponectin has been shown to protect against obesity‐related disorders, such as insulin resistance in obese diabetic (ob/ob) mice, hypertension in severely obese mice, dyslipidemia in mice with protease inhibitor–induced metabolic disorder, and atherosclerosis in apoE‐deficient mice.10 Furthermore, adiponectin‐knockout mice are more susceptible to ischemia‐induced myocardial infarction when compared with their wild‐type littermates.11 In line with these animal studies, hypoadiponectinemia has been demonstrated to be an independent risk factor for insulin resistance, hypertension, and myocardial infarction in humans.14 On the other hand, elevated circulating A‐FABP levels are closely associated with obesity, the number of components of metabolic syndrome, increased carotid intima‐media thickness, and ischemic stroke.17 In addition, we have shown in prospective studies that A‐FABP can predict the development of the metabolic syndrome and type 2 diabetes.19 A recent study also revealed that A‐FABP levels in atherosclerotic plaques were associated with an unstable plaque phenotype and were predictive of the occurrence of adverse CVD among subjects who underwent carotid endarterectomy.20 However, prospective studies linking circulating A‐FABP levels with cardiovascular outcomes in the general population are still lacking, and controversial results have been obtained from prospective studies addressing the relationship between serum adiponectin levels and incident CVD.22 To evaluate whether the circulating levels of these 2 most abundant adipokines are independent risk factors that predict CVD in humans, we investigated prospectively the 12‐year development of incident CVD in relation to the baseline levels in a population‐based community cohort comprising 1847 Chinese subjects recruited from the Hong Kong Cardiovascular Risk Factor Prevalence Study (CRISPS).23Obesity is closely associated with various metabolic and cardiovascular diseases (CVDs), including insulin resistance, type 2 diabetes, hypertension, coronary heart disease, and stroke. Adipose tissue, apart from its traditional role as energy storage, acts as an endocrine organ through the production of adipokines, and thereby participates in the regulation of insulin sensitivity, lipid metabolism, and cardiovascular homeostasis.24 Subjects attended all assessments after an overnight fast. At each assessment, demographic data, including age, sex, occupation, smoking, alcohol consumption, and physical activity, were ascertained. Detailed medical, drug, and family histories, including history of CVD, were obtained using a detailed questionnaire. Anthropometric parameters, including body weight, height, body mass index (BMI), waist and hip circumferences, and blood pressure were measured as previously reported.25 All subjects except those on antidiabetic drugs underwent a 75‐g oral glucose tolerance test. Blood was also drawn for fasting lipid and insulin measurements. At CRISPS‐2, plasma and serum samples were stored in aliquots at −70°C for the measurement of A‐FABP, adiponectin, and C‐reactive protein (CRP). As only 644 of these stored samples were available from CRISPS‐1, assessments of the relationship between these biochemical parameters and incident CVD were done using CRISPS‐2 as baseline, and after excluding those subjects with known CVD. Incident CVD events since CRISPS‐2 were evaluated during 2000–2011 (median follow‐up of 9.4 years). Measurements of the biochemical parameters including glucose, insulin, and lipid levels were described previously.26 A‐FABP was measured using an ELISA .18 Adiponectin was measured with an in‐house sandwich ELISA established in our laboratory.27 CRP was measured with a high‐sensitivity, particle‐enhanced immunoturbidimetric assay .The Hong Kong CRISPS cohort is a population‐based prospective study with comprehensive cardiovascular risk assessment in Hong Kong Chinese. The study first commenced as a cross‐sectional survey in 1995–1996, conducted to assess the prevalence of cardiovascular risk factors in the Hong Kong population. In all, 2895 subjects (1412 men and 1483 women), aged 25 to 74, were recruited from the general population through random selections of telephone numbers. After baseline assessment at the Queen Mary Hospital, subjects were invited for prospective follow‐up to assess their development of major cardiovascular risk factors, including type 2 diabetes, hypertension, and dyslipidemia, with subsequent assessments carried out in 2000–2004 .28 Hypertension was defined as having a sitting blood pressure ≥140/90 mm Hg or on regular antihypertensive drugs, and dyslipidemia was defined as having ≥1 of the following criteria: (1) triglycerides (TG) ≥1.7 mmol/L; (2) HDL‐cholesterol <1.04 mmol/L in men and <1.29 mmol/L in women; (3) LDL‐cholesterol ≥3.4 mmol/L; and (4) already on lipid‐lowering drugs. Diagnoses of CVD events, based on the ICD‐9 , which included, among others, acute myocardial infarction, angina pectoris, stroke, and heart failure, were verified from the Hospital Authority database or its private practitioners in 2011, including the dates of the events and the discharge diagnosis. For subjects who had died, causes and dates of death were ascertained from the Hong Kong Death Registry. The medical diagnoses were reviewed by 2 physicians independently; disagreements between them were resolved by a third. The concordance between the 2 physicians was 0.98. This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster, and all participants gave written informed consent.Diabetes was defined as being on antidiabetic drugs or fulfilling the diagnostic criteria for diabetes according to the World Health Organization 1998 diagnostic classification.29 Optimal cutoff points were derived from the Youden index criterion (maximum of [sensitivity+specificity−1]). Cox proportional hazards regression (Cox regression) was used to estimate the hazard ratios (HRs) and 95% CIs for incident CVD after adjustment of the traditional risk factors including age, sex, BMI, smoking status, diabetes, hypertension, and dyslipidemia. Assumption of proportional hazard was validated for all covariates  and, variable by variable, using scaled Schoenfeld residuals. According to these tests, all Cox regression models did not violate the assumption (P>0.05). In addition, a propensity score–based approach was undertaken to control for baseline traditional risk factors in the logistic regression model. Subjects were then stratified into quintiles according to their predicted propensity score. The Mantel–Haenszel odds ratio for biomarkers was estimated. The predictive performances of various models were assessed by receiver operating characteristic (ROC), category‐free net reclassification improvement (NRI), and integrated discrimination improvement (IDI). Comparison between 2 areas under the ROC curves (AUCs) was examined using the DeLong method.30 Interrater reliability was determined by Cohen's kappa. Two‐sided P values <0.05 were considered statistically significant.All analyses were performed with SPSS Statistics 19  and R‐programming language, version 2.14.2. Results were presented as mean±SD or median with interquartile range (IQR) as appropriate. For data that were not normally distributed, natural logarithmic transformation was applied before analyses. Variables were compared between groups by 1‐way ANOVA for continuous data and chi‐square or Fisher's exact test for categorical data as appropriate. Partial correlation was used to examine the associations between biochemical parameters and various anthropometric measurements. The optimal cutoff values for A‐FABP and CRP were derived using Youden's j criterion.P<0.001). They had higher BMI, waist circumference (WC), systolic and diastolic blood pressure , fasting glucose (FG) levels, and greater insulin resistance as measured by homeostasis model assessment–insulin resistance , compared with those who did not develop CVD events (non‐CVD group) (P<0.001), but similar LDL‐cholesterol levels (P=0.081). Of the biomarkers, the CVD group had higher baseline CRP (P<0.001) and A‐FABP (sex‐adjusted P<0.001), but similar adiponectin levels (sex‐adjusted P=0.881) compared with the non‐CVD group . Subjects with incident CVD (CVD group) were older, more likely to be male, and more likely to be hypertensive (57.7% versus 22.5%), diabetic (28.6% versus 13.5%), dyslipidemic (77.5% versus 62.0%), and a current/former smoker (42.3% versus 24.2%) at baseline  . They alVD group .P<0.001 except for FG, P=0.002; CRP: all P<0.001 except for LDL‐cholesterol, P=0.001) and inverse correlations with HDL‐cholesterol (both P<0.001). In contrast, adiponectin was negatively correlated with these risk factors , and positively correlated with HDL‐cholesterol (P<0.001). Among the biomarkers, A‐FABP showed an inverse correlation with adiponectin , but a direct correlation with CRP , whereas adiponectin was negatively correlated with CRP , with all P values being age‐ and sex‐adjusted  and 1.30 , respectively, after adjustment for traditional risk factors , whereas smoking (current/former smoker) was significant only in model 2 (P<0.05). There was no sex interaction between A‐FABP and incident CVD (P=0.157). In addition, both NRI  and IDI  were statistically significant, demonstrating improvement in the predictive performance by the addition of A‐FABP to the traditional risk factor model, although a similar finding could not be shown using the minimal differences of area under the curve (P=0.837)  .P=0.006; and 1.60 [1.12 to 2.27]; P=0.01 for A‐FABP and CRP, respectively). The cumulative survival curves for incident CVD, based on the Cox proportional hazards model and stratified by the optimal cutoff values of A‐FABP, are shown in Based on the highest Youden's j, the optimal cutoff value for A‐FABP was 26.2 μg/L for men and 30.2 μg/L for women and for CRP was 1.0 mg/L. Using the optimal cutoff values in the Cox regression analysis, A‐FABP, even after adjustment for CRP and traditional risk factors, predicted incident CVD  Study, demonstrated that CRP enhanced global coronary risk, as assessed by the Framingham Risk Score. In their cohort, 191 (5.6%) of 3435 subjects developed events during an average follow‐up of 6.6 years. Subjects with CRP levels ≥3.0 mg/L had an adjusted hazard ratio of 2.21 , when compared with subjects with levels <1.0 mg/L. Our study revealed an adjusted hazard ratio of 1.60  when comparing subjects with high or low CRP levels, using a cutoff value of 1.0 mg/L. It should be noted that our study population had a low cardiovascular risk, with the exclusion of known CVD subjects, and only 182 (9.9%) of 1847 subjects  developed CVD over a median follow‐up of 9.4 years. Consistent with the low cardiovascular risk, our cohort had relatively low CRP levels (CVD/non‐CVD group: 1.28 mg/L [0.63 to 2.42 mg/L]/0.68 mg/L [0.33 to 1.47 mg/L]). Hence, our data suggest that CRP level is also useful in predicting CVD even among low‐risk subjects, when used in conjunction with traditional risk factors.CRP, produced mostly by hepatocytes in response to cytokines such as IL‐6 and TNF‐α, is the most extensively studied inflammatory marker in the literature. Koenig et al33 Although A‐FABP was originally identified as a cytoplasmic protein, we and others have shown that it is abundantly present in human serum and is increased with age and obesity.34 Apart from its correlations with various CVD risk factors, elevated circulating levels of A‐FABP have been associated with several markers of CVD, such as carotid intima‐media thickness, coronary atherosclerotic burden as measured by intravascular ultrasound, and the number of stenotic coronary arteries.36 In addition to its circulating levels, A‐FABP levels in carotid atherosclerotic plaques have been demonstrated to be associated with an unstable plaque phenotype.20 After a mean follow‐up of 2.4 years, subjects with elevated A‐FABP plaque levels showed a 2‐fold increased risk of reaching the primary outcome, defined as the composite of vascular death, vascular event, and surgical or percutaneous vascular intervention. We have previously demonstrated that high serum A‐FABP levels are independently associated with diabetic nephropathy and diabetic macrovascular diseases,37 and a recent study showed that serum A‐FABP predicts CV events in end‐stage renal disease.38 Furthermore, Tuncman et al39 demonstrated that the presence of a genetic variant at the A‐FABP promoter region that results in decreased A‐FABP expression was associated with reduced risk for CVD. Whereas the above studies are supportive of a role of A‐FABP in the pathogenesis of CVD in humans, this is the first outcome study demonstrating that elevated circulating A‐FABP levels can predict incident CVD on long‐term follow‐up of a community‐based cohort of relatively low CVD risk. Our results also show that the predictive power of A‐FABP can be attenuated after adjustment for CRP, which is consistent with the current knowledge of a close pathogenic link between A‐FABP and subclinical systemic chronic inflammation.40 In a murine macrophage cell line, expression of A‐FABP was increased on toll‐like receptor activation, whereas an orally active A‐FABP inhibitor significantly reduced the expression of several inflammatory cytokines, including TNF‐α and IL‐1β in macrophages, and decreased the atherosclerotic lesion area in mouse models.41 Nevertheless, our analysis could only demonstrate the close correlations among A‐FABP, CRP, and incident CVD but provide no evidence for any causal relationships.A‐FABP, also known as FABP4 and adipocyte fatty acid binding protein (aP2), is one of the most abundant proteins in mature adipocytes and is also expressed in macrophages.42 recently reported the association of an increase in serum A‐FABP level with fatal CVD outcomes over 10 years in subjects with known coronary heart disease. It is noteworthy that their cohort had a 2‐fold higher incidence rate than ours (24.00 versus 10.89 per 1000 person‐years). Taken together, these findings strongly support the role of A‐FABP in mediating CVD in humans, although our study design precluded the investigation of its direct causal relationship. To determine this causal relationship, long‐term interventional studies involving therapeutic agents that reduce A‐FABP expression or action such as A‐FABP inhibitors are warranted.41Our study was limited by the low incident CVD event rate in our cohort, which precluded sex‐specific subgroup analyses of A‐FABP and adiponectin in predicting CVD. The test for sex interaction was also underpowered because of the low event rate among the female participants. We included only community‐based Chinese subjects without previous CVD, and therefore our findings could not be extrapolated to other ethnic groups or those subjects with higher CVD risk. Interestingly, during the revision of this article, we found that von Eynatten et alIn conclusion, we have demonstrated that an elevated circulating level of A‐FABP above the optimal cutoff value can potentially serve as a biomarker for the prediction of CVD, as an adjunct to traditional risk factors and established biomarkers such as CRP. These findings support the role of A‐FABP as a possible mediator of obesity‐related CVD, possibly by coupling lipid abnormality to subclinical inflammation.


Intraocular pressure (IOP) may vary according to the change of ocular conditions. In this study, we want to assess the effect and mechanism of pupil dilation on IOP in normal subjects.We prospectively evaluated 32 eyes of 32 patients  with normal open angles under diurnal IOP. IOP was measured every two hours from 9 AM to 11 PM for one day to establish baseline values and was measured again for one day to assess the differences after dilation. To induce dilation, we administered 2.5% phenylephrine and 1% tropicamide every 5 minutes from 8:30 AM to 8:45 AM and for every two hours from 11 AM to 9 PM to keep the pupil dilated. Diurnal IOP, biometry, Visante OCT, and laser flare photometry were measured before and after dilation.p = 0.002). IOP elevation remained significant until about four hours after dilation. Thereafter, IOP decreased slowly and eventually reached pre-dilation level (p > 0.05). Flare values decreased, and the anterior chamber angle became wider after mydriasis.We observed a significant increase in IOP after dilation, 1.85 ± 2.01 mmHg (Dilation of the pupil significantly and incidentally elevated IOP in normal subjects. Further related studies are warranted to characterize the mechanism of the increased IOP after dilation. Like many biological parameters, IOP is a dynamic parameter and varies throughout the course of 24 hours, possibly following circadian rhythms. The mean range of diurnal IOP variation is approximately 2 to 6 mmHg in the normal population and 5 to 18 mmHg in glaucoma patients,4. As suMydriatics are regularly used to dilate pupils in patients presenting to ophthalmology clinics for assessment and follow-up of a wide variety of ophthalmic conditions. An increase in IOP has been observed after dilation with topical application of both parasympatholytic and sympathomimetic mydriatics-7. HowevThis prospective study was performed to investigate the effect and mechanism of dilation on IOP in normal subjects.This study adhered to the tenets of the Declaration of Helsinki and was approved by the institutional review board of Kangbuk Samsung Hospital in Seoul, Korea. We examined 32 eyes of 32 patients  who provided informed consent. All subjects were patients scheduled for a bilateral cataract operation who underwent a full ophthalmic examination including visual acuity, Goldmann applanation tonometry, gonioscopy, slit lamp evaluation, fundus biomicroscopy, auto refractometry , and pachymetry . All eyes presented as normal (except cataract) with an open angle by Goldmann three-mirror gonioscopy. Exclusion criteria included the following: high IOP (>20 mmHg) on the visit before dilation; preoperative ocular medication that could influence IOP level; pre-existing ocular pathology such as glaucoma, uveitis, or high myopia; and previous ocular surgery.IOP was measured in both eyes of each patient by experienced personnel using a Goldmann applanation tonometer every two hours from 9 AM to 11 PM to establish baseline values. On another day (1 ~ 3 months after the baseline test), we induced mydriasis by administering one drop of Mydrin-P  in the conjunctival sac every 5 minutes from 8:30 AM to 8:45 AM and every two hours from 11 AM to 9 PM to maintain a dilated state. IOP was measured every two hours from 9 AM to 11 PM.The following variables were assessed before and after dilation: diurnal IOP, anterior segment examination, axial length (AL), anterior chamber depth (ACD), central corneal thickness (CCT), anterior chamber flare with laser flare photometer , and anterior chamber angle with Visante OCT . AL and ACD were measured with an IOL Master . CCT was measured with a hand-held ultrasonic pachymeter, and the average of three readings was recorded.p-value of less than 0.05 was considered statistically significant.The Visante OCT was used to perform anterior chamber angle width measurements every two hours before mydriasis and after instillation of mydriatics from 9 AM to 9 PM. The average of three consecutive readings of the mean angle value at 3 and 9 o’clock was included in analysis. Laser flare photometry was performed once before mydriasis and every two hours after instillation of mydriatics, from 9 AM to 5 PM. The laser flare photometer quantifies anterior chamber protein (flare) and particles (cells) by measuring light scattering of a helium-neon laser beam projected into the anterior chamber. A singlp = 0.005). The maximum IOP also significantly increased from a mean pre-dilation level of 13.10 mmHg ± 2.91 to a post-dilation level of 14.96 ± 3.25 mmHg (p < 0.001). However, there was no significant difference between the minimum IOP before (10.50 ± 2.74 mmHg) and after (10.50 ± 2.35 mmHg) dilation (p = 0.978).The mean pre-dilation IOP was 11.48 ± 2.85 mmHg. The mean post-dilation IOP was 12.36 ± 2.58 mmHg. This change was statistically significant (p = 0.002). Diurnal IOP was elevated in 22 eyes (68.9%), decreased in two, and unchanged in eight. We noted a non-significant IOP increase at 9 AM, 30 minutes after dilation. IOP was significantly increased at 11 AM and 1 PM (Tablep > 0.05) from pre-dilation levels.Regarding diurnal IOP variation, the mean pre-dilation value was 2.60 ± 1.14 mmHg, and the mean post-dilation value was 4.45 ± 2.01 mmHg. The difference in the mean change of diurnal IOP variation was 1.85 ± 2.01 mmHg, and this change was statistically significant (p < 0.001) . Mean pupil diameter increased from 2.975 ± 0.498 mm to 6.725 ± 0.717 mm 2 hours after dilation and 6.793 ± 0.616 mm after 8 hours, but these changes were not statistically significant (p > 0.05). There was no significant variation of pupil size in either time interval (p > 0.05).The flare value decreased after dilation and remained constant Figure. The wid) Figure. After dp-values: 0.232-0.966). Changes in the mean CCT and axial length measurements before and after dilation were not statistically significant.IOP measurements after dilation were not related to the mean pre-dilation IOP, AL, or diurnal IOP value according to the results of multivariate analysis  sustained increases in IOP. Only one patient experienced a rise in IOP to a level greater than 21 mmHg after dilation. The patient’s pressure dropped after an additional two hours with no medical intervention. This patient exhibited the highest pre-dilation IOP level of all of our patients but did not have any other distinctive findings.Tropicamide is an anticholinergic drug, and phenylephrine is an alpha-1-adrenergic agent. These agents are commonly used together to achieve mydriasis for fundus examination-11. It hThe mechanism responsible for IOP elevation after dilation is unclear. Mydriatic agents can cause increases in IOP that may be related to decreasing aqueous outflow resulting from decreased traction on the trabecular meshwork due to ciliary muscle paralysis,15. UndeIris pigment liberation into the anterior chamber and subsequent obstruction of the trabecular meshwork has also been noted as a possible mechanism responsible for the increase in IOP-20. Krisp < 0.01). The flare value decreased just after dilation and remained decreased while the pupil was dilated. It is possible that, just after mydriasis, the flare may be increased by iris pigment liberation or by protein, but crowding in the angle and subsequent interruption through the outflow facility of the trabecular meshwork can explain the elevation of intraocular pressure. Jewelewicz et al. reported similar results in pigment dispersion syndrome cases[In our study, we investigated aqueous flare and anterior chamber angles before and after mydriasis to help determine the aetiology of increased IOP. We demonstrated that anterior chamber angles widened with dilation, which is possibly due to the posterior pull of the dilated iris-lens diaphragm, leading to a deep anterior chamber. The reduction in IOP after 3 pm may be explained by the widened anterior chamber angle and improvement of the aqueous outflow facility. A decreased resistance to aqueous outflow may be expected from deepening of the anterior chamber, which creates a larger surface area between the trabecular meshwork and the aqueous humour. Howeverme cases. The maxLaser flare photometry is an objective, quantitative method that enables accurate measurement with very high sensitivity and reproducibility. Guillen-Monterrubio et al. reported no significant differences in flare values measured by flare photometer between right and left eyes or between men and women.There are some limitations in this study. Ocular parameters, such as corneal thickness and shape,,25 anterAccording to the results of this study, pupil dilation caused an elevation of IOP. The elevation of IOP was significant until four to six hours after dilation. Afterwards, IOP decreased slowly until it reached pre-dilation level. Further related studies in glaucoma patients are warranted to characterize the mechanism of increased IOP after dilation in a diseased state.JMK; none, KHP; none, SYH; none, KSK; none, TWK; none, DMK; none, JC has received consultant fees and honoraria from Allergan.Literature screening and selection was performed by JMK and KSK. JMK, KHP, TWK, DMK and JC participated in the design of the study. Data collection was done by SYH and KSK, and SYH and KSK performed the statistical analysis. Preparation of the first draft of the manuscript was done by JMK. Critical revision was performed by KHP, SYH, KSK, TWK, DMK, and JC, and approval of the final version of the manuscript was performed by JMK, KHP, SYH, KSK, TWK, DMK, and JC.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/12/53/prepub


To examine which language function depends on early experience, the present study compared deaf native signers, deaf non-native signers and hearing German native speakers while processing German sentences. The participants watched simple written sentences while event-related potentials (ERPs) were recorded. At the end of each sentence they were asked to judge whether the sentence was correct or not. Two types of violations were introduced in the middle of the sentence: a semantically implausible noun or a violation of subject-verb number agreement.The results showed a similar ERP pattern after semantic violations (an N400 followed by a positivity) in all three groups. After syntactic violations, native German speakers and native signers of German sign language (DGS) with German as second language (L2) showed a left anterior negativity (LAN) followed by a P600, whereas no LAN but a negativity over the right hemisphere instead was found in deaf participants with a delayed onset of first language (L1) acquisition. The P600 of this group had a smaller amplitude and a different scalp distribution as compared to German native speakers.The results of the present study suggest that language deprivation in early childhood alters the cerebral organization of syntactic language processing mechanisms for L2. Semantic language processing instead was unaffected. The majority of deaf children have hearing parents, whose primary language is a spoken language. Children born severely or profoundly deaf do not have access to a spoken language and, thus, do not acquire a language from birth. In Germany, these children usually acquire DGS from school mates after entering primary school or even later ,2. Deaf In our ERP study, we investigated the effects of a delayed L1 acquisition in a violation paradigm. Semantic violations are known to be associated with a negative ERP, the so called ‘N400 effect’ that has mostly been observed over centro-parietal electrode sites, particularly for written languages . The N40L2 learners who are exposed to the L2 after puberty have shown lower performance and a higher variability in L2 processing compared to native speakers of the same language. A negative correlation between the age of onset of acquisition of an L2 and the achieved grammatical competence has been observed in many studies . ImpMany studies found that the semantic effect (N400) is robust to effects of age of acquisition (AoA) ,30, wherBy comparing the L2 processing of (1) deaf people who had acquired German Sign Language (DGS) as their L1 from their deaf parents (henceforth: ESL for Early Sign Language learners), and (2) deaf people who had not been exposed to sign language during the first years of life because they had hearing parents (henceforth: LSL for Late Sign Language learners) it is possible to study the effects of a delayed L1 acquisition compared to a timely L1 acquisition in sign language users when tested in their L2 German. German processing was assessed by analysing a group of (3) hearing German native speakers (henceforth: EGL for Early German Language learners). Both groups of deaf people, ESL and LSL, started to learn German at the time of primary school enrolment.In this regard, Mayberry and Lock  comparedThe effects of a delayed L1 acquisition on the neural processing mechanisms in a written L2 in sign language users have not been investigated yet. Using ERPs, we were able to assess the effects of a delayed L1-acquisition separately on semantic and syntactic aspects of language processing. Semantic ERPs are relatively robust to effects of AoA ,30, in cIn accord with the findings of Mayberry and Lock , the LSLWith regards to the ERP results of the group of EGL in accordance with previous findings in native speakers ,21,22,25For ESL a similar N400 effect in the semantic condition has been predicted. Previous findings have suggested that L2 learners who are neither highly proficient nor very familiar with the grammatical phenomenon do not show a LAN and/or a reduced P600 ,37,42. HAs previous studies give reason to expect differences between ESL and LSL , we predThe language proficiency test was run with all deaf participants. One-sided t-tests showed significantly higher performance in ESL than LSL in the following subtests of the ATBG: TGK (t21.766) = 2.219; p = 0.019), PPVT (t(24.794) = 1.865; p = 0.037), and GSV (t(22.666) = 1.822; p = 0.041). The groups did not significantly differ in the subtest ADST (t(24.703) = 0.552; p = 0.293). The subgroups of LSL and ESL who had reached the criterion of at least 60% correct sentence judgements in all conditions of the EEG experiment did not differ significantly in any of the four subtests of the ATBG . The results of the ATBG are shown in Table .766 = 2.As mentioned in the methods section, 8 out of 12 participants of the ESL group and 8 out of 15 participants of the LSL group performed at a level of at least 60% correct in all three conditions  in the EEG experiment. Here we report results of those participants who met the 60% criterion (high performing participants), since ERP violation effects are not expected for participants performing at chance level. The behavioural data is shown in Table The ANOVA for the three groups revealed main effects of Group  = 11.682; p < 0.001), and Condition  = 20.297; ϵ = 0.525; p < 0.001), as well as an interaction between Group and Condition  = 7.576; ϵ = 0.525; p < 0.001). The EGL differed significantly from LSL (t(8.096) = 3.632; p = 0.020) as well as from ESL (t(9.967) = 4.514; p = 0.003). The groups LSL and ESL did not differ (t(11.550) = 0.643; p = 0.533).In the correct condition, LSL performed significantly worse than EGL (t(8.760) = 4.378; p = 0.006) and ESL (t(9.775) = 3.271; p = 0.026).No group differences were observed in the semantic condition.In the syntactic condition, EGL performed at a higher level than LSL (t(9.591) = 3.105; p = 0.035) and ESL (t(9.856) = 3.909; p = 0.009).The results of the ANOVAs for each of the three groups of participants  are presented in the following sections. They are followed by between-group comparisons. Results for the semantic condition are always reported first, followed by the results for the syntactic condition. ERP effects of both the semantic and the syntactic condition were analyzed for the time epochs 300–500 ms and 600–800 ms. For the syntactic condition, the time interval of 300–500 ms was further divided into three sub-epochs of 66 ms each. The ANOVA model comprised the within-group factors Condition (CO), Hemisphere (HE), and Cluster (CL). For the between-groups ANOVAs the factor Group (GR) was added.In the time window of 300–500 ms, the ANOVA revealed a significant main effect of CO  = 46.717; p < 0.001) and an interaction of CO and CL  = 17.399; ϵ = 0.377; p < 0.001). The ERP difference between the incorrect and correct condition was negative for all clusters except L2 and L3 (p < 0.05)  = 22.307; p = 0.001). The interaction of CO and CL  = 16.294; ϵ = 0.302; p < 0.001) was significant as well. The ERP difference for incorrect and correct words was positive for all clusters except L1 and R2 (p < 0.05)  = 11.162; p = 0.007) was observed for the time window of 300–500 ms, indicating a stronger violation effect over the left than over the right hemisphere. The difference between the incorrect and the correct condition was significant for clusters L1, L2, L4, L5, and R4 (p < 0.05)  = 38.299; p < 0.001), interactions of CO and HE  = 8.541; p = 0.014), and of CO and CL  = 39.924; ϵ = 0.376; p < 0.001). The latter indicated a stronger ERP effect over the right hemisphere. The difference between the incorrect and correct condition was significant for clusters L2-7 and for R1-7 (p < 0.05)  = 6.883; ϵ = 0.351; p = 0.007), and of CO and HE  = 9.011; p = 0.020), and of CO, HE, and CL  = 5.416; ϵ = 0.278; p = 0.026), indicating a smaller ERP difference over the left than over the right hemisphere. Moreover, the interaction of CO, HE, and CL  = 7.486; ϵ = 0.506; p = 0.001) was significant   = 11.999; p = 0.010). The interaction of CO and CL  = 5.918; ϵ = 0.316; p = 0.016) was significant, as well. The t-tests revealed positive ERP differences between incorrect and correct conditions for clusters L3-7, and R3-7 (p < 0.05)  = 6.487; p = 0.038) was found for time epoch 300–366 ms, in which the violation effect was significant for cluster L1 and L2 (p < 0.05)  = 8.865; p = 0.021) and an interaction of CO and CL  = 4.889; ϵ = 0.291; p = 0.031) were observed. The positive difference between the incorrect condition and the correct condition was significant for clusters L3-7 and R3-7 (p < 0.05)  = 32.594; p < 0.001), and interactions of CO and CL  = 11.111; ϵ = 0.245; p = 0.004) and of CO, HE, and CL  = 5.239; ϵ = 0.330; p = 0.020) were significant in the time window of 300–500 ms after the onset of the critical word  = 28.762; p = 0.001) and an interaction of CO and CL  = 12.062; ϵ = 0.275; p = 0.002) reached significance. The difference between incorrect and correct conditions was positive  = 4.408; p = 0.074). This interaction was due to a more negative violation effect over the right hemisphere  = 7.635; p = 0.028), and the interaction of CO and CL  = 11.768; ϵ = 0.410; p < 0.001) were significant. Clusters L3, L5, L6, L7, and R5, R6, and R7 showed a more positive ERP in the incorrect condition than in the correct condition  = 5.368; p = 0.011 was significant in the ANOVA including all three groups.The comparison of EGL and LSL resulted in a significant GR, CO, and HE interaction  = 8.986; p = 0.008) that was due to a negative difference over the left hemisphere for EGL and a marginally significant difference (see above results for group LSL) over the right hemisphere for LSL  violation effects . Therefore, we used time epoch 366–433 ms for the EGL group, time epoch 300–366 ms for the ESL group, and time epoch 433–500 ms for the LSL group. The ANOVAs revealed significant differences between all three pair of groups  = 21.322; p < 0.001; GR*CO*HE*CL: F = 5.296; ϵ = 0.472; p = 0.003.EGL and ESL: GR*CO*HE: F = 5.671; p = 0.028.ESL and LSL: GR*CO*HE: F = 8.493; p = 0.011; GR*CO*HE*CL: F = 3.987; ϵ = 0.458; p = 0.017.These interactions show that LSL displayed a right lateralized topography whereas the syntactic violation effect was negative over the left hemisphere in EGL and ESL.In the time window of 600–800 ms, the ANOVA for all three groups did not reveal any significant effect involving the factors Group and Condition. However, the direct comparison of EGL and LSL resulted in a significant GR and CO interaction  = 5.906; p = 0.0258). A stronger violation effect was present in EGL compared to LSL. The ANOVAs of the other pairwise group comparisons - EGL and ESL, and ESL and LSL - did not reveal any significant effects  at the time of school enrolment (LSL) were compared to native signers who had learned written German at a comparable point in time but sign language from birth (ESL). Both groups were compared to a control group of hearing German native speakers (EGL).Only participants who performed above chance level were included in the analyses. All three groups showed an N400 effect to semantic violations that was followed by a broadly distributed positivity. These findings are in line with results of Neville et al. , who repIn the syntactic condition, EGL displayed a left temporally distributed negativity which has been considered as an index of early automatic processing . InteresBy contrast, we did not find a LAN like effect for the LSL. This finding is particularly interesting given the behavioural results. Both selected deaf groups did not differ in their performance neither in the syntactic condition of the EEG experiment nor in the corresponding subtests of the ATBG. These results suggest that even signers with a delayed L1 acquisition who have achieved a relatively high performance level do not show a cerebral organization of syntactic language aspects comparable to people who have grown up with a natural language.Across all participants of both deaf groups, LSL showed lower performances than ESL. This finding together with the ERP results supplement the observations of Mayberry and Lock , providiIt should be noted that seven out of fifteen participants from the LSL group and four out of twelve participants of the ESL group were not able to perform the EEG task above chance level. The drop-out rates for the two deaf groups did not differ significantly (chi-square-test: p = 0.76). Even among the best performing participants both groups of deaf signers performed worse compared to hearing German native speakers. This general disadvantage of the deaf might be partially due to overall effects of late acquisition, the available impoverished German language input , noun . Prepositional phrases consisted of either two  stall’) or three words . Each sentence was presented in three different conditions: (1) correct, (2) containing a semantic violation , or (3) containing a syntactic violation . Example sentences for each condition are listed in Table The critical word in an incorrect sentence had a counterpart of the same word class in its companion correct sentence at the same position. In order to reduce possible confounding influences of linear distance between subject and verb, phrase structure boundaries, working memory load, or decision and motor processes , we usedEighty different sentences were generated comprising two sets of 40 sentences each  was accomplished by the two groups of deaf participants in a different session. The test was presented on a computer monitor. The instructions were part of the test software. They were given in DGS and in written German.In the EEG experiment, the sentences were presented in random order with black letters against a grey background. The vertical visual angle was 1.53°. First, a centred fixation cross appeared for 600 ms, followed by the successive presentation of the words at a rate of 600 ms, followed by a grey screen also for 600 ms after which the presentation of two smileys, one happy and one sad, informed the participants to press one of two buttons with their index fingers, indicating whether the sentence was correct or incorrect. Half of the participants responded on the left side for a correct sentence and on the right side for an incorrect sentence, the other half in the reverse order. The participants had to press one of the buttons to go on with the experiment. Five blocks with 80 sentences each were presented. A short break was given between blocks. The experiment lasted for about 60 min.The electroencephalogram was recorded with 74 scalp electrodes, which were arranged into an elastic cap  according to the international 10/10 system. The electrode on the right earlobe was used as the recording reference. An averaged right/left earlobe reference was calculated offline. Electrode impedance was kept below 5 kΩ. The vertical electrooculogram (VEOG) was measured with two electrodes, one placed under each eye, and recorded against the reference electrode. Horizontal eye movements were monitored using electrodes F9 and F10.Three BrainAmp DC amplifiers with 32 channels each  were used. The recorded data was digitally stored with the BrainVision Recorder software , in which the analog EEG signal was sampled at 5000 Hz, filtered online with a bandpass of 0.1 to 250 Hz, and downsampled to 500 Hz for storing. Offline, the signal was filtered . Four adjacent electrodes were pooled into clusters, resulting in seven clusters on each hemisphere  and the within participant factor Condition  were predictor variables, the percentage of correct answers the dependent variable. Post-hoc t-tests were applied to compare each possible pair of Groups. The degrees of freedom from the t-tests were corrected using the Welch algorithm. Additionally, the p-values of these t-tests were Bonferroni corrected.For EEG data of the time periods 300–500 ms and 600–800 ms the mean amplitudes were analysed by repeated measurements ANOVAs, separately for semantics and syntax. For syntax the interval 300–500 ms was further divided into three sub segments 66 ms each. Repeated measurement factors Condition (CO: correct vs. incorrect), Hemisphere (HE: left vs. right), and Cluster (CL: one to seven) and the between participant factor Group  were included. Sums of Squares of Type II were calculated. To compensate for violations of the assumption of sphericity in multi-channel electroencephalographic data, the Greenhouse and Geisser correction was applied and the corresponding Greenhouse/Geisser Epsilons (ε) were reported for the F-tests. Statistically significant effects without the factor Condition are not reported. T-tests for the difference between the correct and the incorrect condition at each cluster were additionally applied to add information about the topographical distribution of the effect. The open source statistical programming language ‘R’ was used for statistical analyses. The data of EGL and ESL have been previously published in a different context .The authors declare that they have no competing interests.NS, MK, BHF, and BR designed the experiment. NS and MK run the ERP experiments. NS and BR analyzed the data. NS, US, MK, BHF, and BR wrote the paper. All authors read and approved the final manuscript.


Most of the tested phenols were not effective against the four bacterial strains when tested as single compounds at concentrations of up to 1000 μg mL−1. Hydroxytyrosol at 400 μg mL−1 caused complete growth inhibition of the four strains. Gallic acid was effective at 200, and 400 μg mL−1 against S. aureus, and S. pyogenes, respectively, but not against the gram negative bacteria. An OMW fraction called AntiSolvent was obtained after the addition of ethanol to the crude OMW. HPLC analysis of AntiSolvent fraction revealed that this fraction contains mainly hydroxytyrosol (10.3%), verbascoside (7.4%), and tyrosol (2.6%). The combinations of AntiSolvent/gallic acid were tested using the low minimal inhibitory concentrations which revealed that 50/100–100/100 μg mL−1 caused complete growth inhibition of the four strains. These results suggest that OMW specific fractions augmented with natural phenolic ingredients may be utilized as a source of bioactive compounds to control pathogenic bacteria.Polyphenols or phenolic compounds are groups of secondary metabolites widely distributed in plants and found in olive mill wastewater (OMW). Phenolic compounds as well as OMW extracts were evaluated  The OMW is rich with organic compounds (mainly phenols) which creates a number of acute environmental and ecological problems [The process of olive oil production is accompanied by generation of a considerable amount of olive mill wastewater (OMW). Up to 30 million mproblems , 2. So fproblems , 5, detoproblems , ozonatiproblems , as wellThe phenolic fraction of olive oil comprises only 2% of the total phenolic content of the olive fruits, with the remaining 98% being lost in olive mill waste (OMW) . A numbe p-coumaric and caffeic acids against Xylella fastidiosa strains (causes Pierce's disease in grapes) were 800 μM and 200 μM, respectively [p-coumaric acid [in vitro against both Gram-positive and Gram-negative bacteria responsible for respiratory and intestinal tract infections [Rhizoctonia solani and Fusarium solani [Phenols and polyphenols are diverse group of compounds which widely occur in a variety of plants including olives and are used in defensive functions in many plant species , 16 wherectively . Compounectively , oleuropectively , 4-hydroric acid . Olive pfections . In a rem solani . A large number of research papers have been published dealing with the chemical composition of olives and olive oil; however, only a few studies have focused on isolating and identifying compounds from the OMW . The isoThe aims of the current research were (a) to develop a simple and cost-efficient OMW extraction method yielding a highly active antimicrobial phenolic fraction effective against important human pathogenic bacteria, (b) to define the active constituents (and/or phenolic compounds) of such fraction (using pure compounds), and (c) to investigate the synergistic effects of known bioactive compounds and OMW fractions against human pathogenic bacteria.p-coumaric acid from Sigma-Aldrich Ltd, Israel, hydroxytyrosol, from TCI AMERICA, 3,4-dihydroxyphenylacetic acid from ACROS chemicals, and verbascoside from Apin Chemicals Ltd, UK.Phenolic and other standards used without further purification were ascorbic acid, tyrosol, protocatechuic acid, vanillic acid, caffeic acid, gallic acid, ferulic acid, and Streptococcus pyogenes (ATCC no. 19675) and Staphylococcus aureus (ATCC no. 25923) and the Gram-negative reference strains Escherichia coli (ATCC no. 25922) and Klebsiella pneumoniae (ATCC no. 700603). Bacterial strains were maintained on tryptic soy broth (TSB) containing 20% glycerol and stored at −80°C until use. Subcultures were freshly prepared before use by inoculation of a loop of stored culture into 5 mL TSB and incubation overnight at 37°C. The turbidity of the culture was adjusted with sterile saline solution to match 0.5 McFarland standards  (http://www.cdc.gov/ncidod/DBMD diseaseinfo/cholera/ch9.pdf).The tested bacterial strains included the Gram-positive reference strains The OMW was generated by the olive oil extraction using the three-phase known process. OMW for this study was obtained from a nearby olive mill press . The OMW was treated with 20% ethanol (v : v) and stored at 4°C until use. The total phenol (TP), COD, BOD, and pH values, of the collected OMW samples were determined according to the “Standard Methods for the Examination of Water and Wastewater, 20th Edition 1998.” The TP in OMW was determined according to the Folin-Ciocalteu method .The AntiSolvent was prepared in a very unique way in order to extract polyphenols from OMW. The AntiSolvent fraction was obtained by the addition of at least one polar organic solvent (acetone or ethanol) to the aqueous mixture. The polar solvent caused a precipitation and therefore forces out an organic fiber fraction from the solution which was identified mainly as cellulosic mixture (no phenolic chromophore was identified using the HPLC). The AntiSolvent used throughout these experiments was prepared as follows. One liter of OMW stored at 4°C with 20% ethanol was centrifuged (7000 rpm for 10 min) and then subjected to filtration using Wattman filter paper . The resμL. The main phenolic compounds in the extract were identified and quantified by comparison with relative retention times and UV spectra of pure standards .The presence and amount of the phenolic compounds in the AntiSolvent extract were studied using reversed-phase HPLC analysis with a binary gradient elution. The analysis was performed by reversed-phase HPLC using a Thermo Scientific Finnigan Surveyor system equipped with a PDA plus detector (220–340 nm). The chromatographic separation was achieved on a SYNERGI 4U POLAR-RP 80A 250 × 4.60 mm phenomenex. Its temperature was maintained at 30°C. The mobile phase was 0.1% acetic acid in water (A) versus 0.1% acetic acid in methanol (B) for a total running time of 40 min. The specific elution conditions were 0–5 min, 20% B; 5–10 min, 20–70% B; 10–21 min, 70–80% B; 21–30 min, 80% B; 30–32 min, 80–20% B; 32–40 min, 20% B. The flow rate was 1.0 mL/min, and the injection volume was 20 8 cfu mL−1. Each fraction/component or combination of compounds was examined for antibacterial activity in triplicate wells using 96-well plates, and the experiments were repeated at least twice. The plates were incubated at 37°C for 18 h. Subsequently, the plates were examined visually for bacterial growth inhibition. In each treatment, the inhibition was considered positive when there was no microbial growth in all the 3 wells of the triplicate. The antimicrobial activity of the different compounds and mixtures was tested against Gram-positive (S. pyogenes and S. aureus) and Gram-negative bacteria (E. coli and K. pneumoniae) in order to determine the MIC for the different combinations. The MIC was determined as the lowest combination of two compounds caused complete growth inhibition in the triplicate wells of each treatment.The inoculums were prepared by lifting 3–5 identical colonies from each agar plate with a sterile loop and transferred into a tube containing 5 mL of TSB and incubated overnight at 37°C. The turbidity of each bacterial suspension was adjusted to reach an optical comparison to that of a 0.5 McFarland standard, resulting in a suspension containing approximately 1 − 2 × 10−1, and the pH was 5.0. The AntiSolvent fraction was isolated without tedious extraction method for ease of isolation. The dark AntiSolvent liquid evaporated to give 10.0 grams of brown/black thick paste from 1 liter of OMW. The content of the paste was identified using HPLC method and constituted of hydroxytyrosol, 3, 4-dihydroxyphenylacetic acid, tyrosol, protocatechuic acid, verbascoside, vanillic acid, caffeic acid, ferulic acid, and p-coumaric acid in addition to other unidentified peaks   . E. coli and S. pyogenes at 1000 μg mL−1 as was visually observed  when gallic acid was supplemented up to 1000 μg mL−1  resulted in complete inhibition of the four strains. Also, it would require the combination of gallic acid/hydroxytyrosol (100/200 μg mL−1) to completely inhibit the growth of the same four bacterial isolates. It is interesting to note that ascorbic acid/hydroxytyrosol showed synergistic activity against the four isolates and resulted in complete inhibition to S. pyogenes at the combination 100/50 μg mL−1. The combination of AntiSolvent/hydroxytyrosol in MIC of 50/50 μg mL−1 resulted in complete inhibition of the isolate S. pyogenes  and the Gram-negative bacteria (E. coli and K. pneumoniae) was found to be very low and required high concentration exceeding 1000 μg mL−1 per component to inhibit the growth of the four isolates (data not shown) except for hydroxytyrosol. However, an enriched AntiSolvent with combinations of specific phenolic compounds completely inhibited all four bacterial strains at different combinations with low concentration combinations of 50/50–200/400 μg mL−1. The MIC of the phenolic compounds extracted from olives (the phenolic amounts found in table olives ranged from 0.9 to 5 g/kg) was established against bacterial isolates responsible for human intestinal and respiratory tract infections such as Bacillus cereus , B. Subtilis , S. aureus , Pseudomonas aeruginosa , E. Coli , and Klebsiella pneumoniae  [μg mL−1 of AntiSolvent with hydroxytyrosol or with gallic acid caused complete inhibition to the four microbial strains. The growth inhibition of the different bacterial strains was tested using the broth dilution method which showed synergistic activity of the AntiSolvent fraction obtained from OMW in combination with hydroxytyrosol or with gallic acid . Also thobserved . Mixtureobserved . The bioμg mL−1) . These aμg mL−1 alone completely inhibited the growth of two isolates only, E. coli and S. pyogenes. Also hydroxytyrosol which is known as antimicrobial compound [μg mL−1 to cause full growth inhibition of the four strains. However, the combination of various phenolic compounds was effective against the four different bacterial strains because of the synegistic effect obtained using various components, that is, AntiSolvent/hydroxytyrosol, AntiSolvent/gallic, and gallic/hydroxytyrosol  showed more limited activity [The hydroxytyrosol alone was effective against all the 4 strains at 400 μg . Enrichmμg . In anotactivity . μg mL−1 and even less caused full growth inhibition of the four bacterial isolates (An interesting result was obtained by the combination of hydroxytyrosol/ascorbic acid (vitamin C) where 200/200 isolates . The expRecovery of phenols from OMW is a difficult analytical task for several reasons. Phenols are reactive chemical species, vulnerable to oxidation, conjugation, hydrolysis, polymerization, and complexation . This isThe most important conclusion drawn from our study is that simple, efficient, and cost-effective extraction of OMW yielding highly active antimicrobial extract can be done and the extract can be further augmented with additional natural compounds to achieve higher activity. These finding may lead to more attention to natural compounds as an alternative treatment of infectious diseases , 38. Suc


Capsicum is an important agricultural crop, not only because its economic importance, but also for the nutritional values of its pods, mainly due to the fact that they are an excellent source of antioxidant compounds, and also of specific constituents such as the pungent capsaicinoids localized in the placental tissue. This current study was designed to evaluate the antioxidant capacity and total phenolic contents from fruits tissues of two Capsicum chinense accessions, namely, Chak k'an-iik (orange) and MR8H (red), at contrasting maturation stages. Results showed that red immature placental tissue, with a Trolox equivalent antioxidant capacity (TEAC) value of 55.59 μmols TE g−1 FW, exhibited the strongest total antioxidant capacity using both the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the CUPRAC methods. Placental tissue also had the highest total phenolic content (27 g GAE 100 g−1 FW). The antioxidant capacity of Capsicum was directly related to the total amount of phenolic compounds detected. In particular, placentas had high levels of capsaicinoids, which might be the principal responsible for their strong antioxidant activities. In the past few years, there has been a renewed interest in studying a wide variety of food products that show beneficial effects on human health.  Capsicum spp.) are among the most consumed vegetables in the world. Mexico is considered an important center of domestication and diversification of chili peppers. Given the extension of the territory and variations in environmental conditions, the different regions have developed their own cultivars and varieties. For instance, although in the Yucatan Peninsula indigenous cultivars of C. annuum have arisen, Habanero peppers (C. chinense) are widely preferred and an extensive variety of cultivars is available  × 100, where ADPPH is the absorbance of the control reaction  and AS is the absorbance of the extract. Extract concentration inducing 50% inhibition (IC50) was calculated from the graph (slope = −0.0171) of radical scavenging activity percentage versus sample extract. Trolox  was used as a positive control [Free radical-scavenging activity was evaluated using a modified DPPH assay . DPPH wa control , 13.μL) or placentas (5–15 μL) extracts were mixed with 1 mL 0.4 mM DPPH dilution in Tris buffer , vigorously shaken and allowed to reach a steady state at room temperature. A standard curve (0 to 60 μM) was prepared using Trolox solutions. The decrease in absorbance at 520 nm 30 min after addition of a compound was used for calculating the TEAC. All determinations were performed in triplicate. Results were expressed as μmoles Trolox equivalent (TE) g−1 FW [The antioxidant capacity was assayed as the reaction of DPPH with Trolox, as described by Oomah et al. . Pericarcentas 5– μL extra) g−1 FW .−1) as a standard. Briefly, 0.02 mL of extract and 1.58 mL water were mixed with 0.1 mL of the Folin-Ciocalteu's reagent, followed by 0.3 mL 20% (w/v) sodium carbonate. After 2 h at room temperature, absorbance was measured at 765 nm. Total phenolic content was calculated as gallic acid equivalents (GAE) 100 g−1 FW.Total polyphenols in extracts were determined , using t2, 7.5 mM neocuproine alcoholic solution, and 1 M ammonium acetate buffer were mixed prior to addition of plant extracts and water to a final volume of 4.1 mL in a tube. Tubes were thoroughly mixed and let to stand for 30 min before absorbance at 450 nm was measured [Cupric reducing antioxidant power (CUPRAC) was determined as described in . Equal vmeasured , 20.Data were expressed as means ± SD. Statistical analysis was performed by using a one-way ANOVA statistical model and mean comparisons were made using Tukey's multiple-range test at a 5% level of probability.In order to obtain a complete prospect of the antioxidant potential of Habanero peppers, assays to evaluate radical scavenging and total reducing power were carried out.50 and TEAC. IC50 represents the dose of sample that reduces by half the absorbance of a DPPH reference solution; therefore, a low IC50 indicates a high antioxidant activity [50 values) was found in the immature, rather than mature pericarps of both C. chinense accessions  were found in pericarps from immature pods of both varieties. In contrast, the placental tissues presented between three- (orange) and eight- (red) fold higher phenolic contents than pericarps .Considering that high levels of phenolics were contained in tissues with also high antioxidant activity, it is proposed that an important component of this activity in the C. chinense Jacq. accessions from the Yucatan Peninsula have significant amounts of antioxidants, specifically in the placental tissue. The antioxidant properties of the Habanero pepper may be explained in terms of the phytochemicals that it contains, such as carotenoids, vitamins, phenolic compounds, and capsaicinoids [C. chinense pods may supply a substantial amount of the antioxidants needed to promote a better health and to prevent diseases and ailments. Our results can be the basis for the selection of suitable accessions for antimicrobial and anticarcinogenic trials of habanero extracts.In conclusion using different assays to determine a full prospect of the total antioxidant capacity, it was determined that the icinoids , 31–33.