Late-life depression (LLD) is a common disorder associated with emotional distress, cognitive impairment and somatic complains. Structural abnormalities have been suggested as one of the main neurobiological correlates in LLD. However the relationship between these structural abnormalities and altered functional brain networks in LLD remains poorly understood. 15 healthy elderly comparison subjects from the community and 10 unmedicated and symptomatic subjects with geriatric depression were selected for this study. For each subject, 87 regions of interest (ROI) were generated from whole brain anatomical parcellation of resting state fMRI data. Whole-brain ROI-wise correlations were calculated and compared between groups. Group differences were assessed using an analysis of covariance after controlling for age, sex and education with multiple comparison correction using the false discovery rate. Structural connectivity was assessed by tract-based spatial statistics (TBSS). LLD subjects had significantly decreased connectivity between the right accumbens area (rA) and the right medial orbitofrontal cortex (rmOFC) as well as between the right rostral anterior cingulate cortex (rrACC) and bilateral superior frontal gyrus (bsSFG). Altered connectivity of rrACC with the bsSFG was significantly correlated with depression severity in depressed subjects. TBSS analysis showed a 20% reduction in fractional anisotropy (FA) in the right Forceps Minor (rFM) in depressed subjects. rFM FA values were positively correlated with rA-rmOFC and rrACC-bsFG functional connectivity values in our total study sample. Coordinated structural and functional impairment in circuits involved in emotion regulation and reward pathways play an important role in the pathophysiology of LLD. As a common psychiatric disorder in older adults, late-life depression (LLD) has a negative impact on the lives of the elderly by adversely affecting cognitive, emotional and somatic aspects of their mental and physical health Among the abnormalities associated with LLD, structural alterations, including gray matter volume and shape alterations, and white matter micro- and macrostructural changes in frontolimbic circuitry which have received a great deal of attention and have been widely shown in several structural imaging studies in LLD Neuroimaging studies of LLD have been primarily focused on structural brain alterations and an increasing effort has been made in understanding the mechanism of LLD at the functional network level. Classically, the function of a given network is tested by measuring the level of activity in terms of oxygen or glucose utilization in connected brain regions during the performance of a specific task. Task-based functional magnetic resonance imaging (fMRI) studies in LLD have revealed abnormal activity in several key regions of frontostriatal-limbic circuitry during cognitive and emotion tasks Another method for exploring the dynamics of brain networks is measuring the correlation and synchronicity of activity between brain regions at rest using functional connectivity (FC) analysis The relationship between aforementioned structural changes in LLD and FC alterations remains poorly understood. In healthy individuals, a strong relationship has been demonstrated between anatomical and resting state functional connectivity To our knowledge, no study has explored whole brain rs-fMRI functional connectivity changes in unmedicated and symptomatic patients with LLD. Furthermore, there have been no studies to date that have investigated the relationship between whole brain white matter tract integrity and FC values in patients with LLD compared to healthy control subjects. The purpose of our study was to examine FC alterations using rs-fMRI in association with white matter integrity measured by DTI in unmedicated patients with LLD. We hypothesized that abnormal changes in brain network FC and pathologic alterations in white matter integrity can occur in concert in LLD and relate to symptom severity in unmedicated patients suffering from geriatric depression. We also hypothesized that there will be a significant correlation between structural connectivity assessed by FA and functional connectivity assessed by rs-fMRI in unmedicated elderly with LLD. To test our hypotheses, a data-driven method was applied for FC analysis of rs-fMRI data acquired from unmedicated and symptomatic patients with LLD and a group of healthy comparison subjects. Changes in integrity of whole brain white matter tracts were also tested by applying an automated tract-based spatial statistics (TBSS) method to analyze diffusion tensor imaging (DTI) data We studied 25 subjects 60 years of age and older. Of these, 10 were unmedicated subjects with unipolar major depression (LLD) and 15 were nondepressed comparison subjects (HC). All study subjects were recruited from the local community through advertisements in flyers, newspapers, and radio. The inclusion criteria for all subjects were 60 years of age and older, antidepressant-naive or free of antidepressant use for at least two weeks and no history of unstable cardiac or neurological diseases. Six LLD subjects were treatment-naïve. For the remaining four, there have been varying degrees of antidepressant exposure. One subject started citalopram and bupropion 6–7 years ago and discontinued the medication 6 months prior to study entry. Another subject had been on venlafaxine for 4 years stopped 6 years before study entry. One subject had been on sertraline for unknown duration prior to study entry and the last subject stopped fluoxetine 9 years prior to study entry. The exclusion criteria included: schizophrenia, bipolar or any psychotic disorders; history of anxiety disorder outside of major depressive episodes; history of head trauma or loss of consciousness; history of substance abuse; contraindications to MRI such as metal implants; Mini Mental Status Exam (MMSE) Score ≤ 24. This study was approved by the University of Illinois-Chicago Institutional Review Board, and written informed consent was obtained from each participant in accordance with the Declaration of Helsinki.All eligible subjects were assessed by a trained research assistant with the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition1-weighted images were acquired with a MPRAGE (Magnetization Prepared Rapid Acquisition Gradient Echo) sequence . Resting-state data were acquired with the following parameters: Single-shot gradient-echo EPI sequence, TR/TE  =  2000/30 ms, Flip angle  =  80 degree, EPI factor  =  47, FOV  =  23 × 23 × 15 cm3, in-plane resolution  =  3×3 mm2, slice thickness/gap  =  5/0 mm, slice number  =  30, SENSE reduction factor  =  1.8, NEX  =  200, total scan time  =  6∶52. Subjects were instructed to keep their eyes close and “not think of anything in particular”. DTI images were acquired using single-shot spin-echo echo-planar imaging (EPI) sequence . Sixty seven contiguous axial slices aligned to the anterior commissure–posterior commissure (AC-PC) line were collected in 32 gradient directions with b = 700 s/mm2 and one acquisition without diffusion sensitization (B0 image). Parallel imaging technique was utilized with factor at 2.5 to reduce scanning time to approximately 4 minutes.Brain MRI data were acquired on a Philips Achieva 3.0T scanner  using an 8-channel SENSE (Sensitivity Encoding) head coil. Participants were positioned comfortably on the scanner bed and fitted with soft ear plugs; foam pads were used to minimize head movement. Participants were instructed to remain still throughout the scan. High resolution three-dimensional Thttp://www.nitrc.org/projects/conn;http://www.nitrc.org/projects/artifact_detect) was used to measure motion artifacts in all subjects. There was no significant difference in composite motion between groups , nonetheless we controlled for any motion artifacts using realignment parameters detected by ART. The principal components of the white matter and CSF signal were regressed out of the signal using the CompCor method Functional connectivity was measured using the resting-state fMRI toolbox, CONN v.1.2 Demographic, clinical, and network variables were analyzed for between-group differences using an independent sample t-test for continuous variables and chi-squared test for categorical variables. Levene's test was used to test the equality of variance. FC and FA group differences were analyzed using univariate analysis of covariance with age, sex, and education as covariates. Multiple comparison correction for FC measures at the connection level was conducted using the false discovery rate (FDR), implement in CONN 2 = .11,df = 1, p  = .74), MMSE  or education . As expected, depressed subjects scored significantly higher on both measures of depression severity  . The mean age of onset for LLD subjects was 39.6±19.9 years of age. 2 of the 10 subjects had late-onset depression (at 60 years of age or older). There were no significant differences between the two groups in gender  .2 = .53) as well as between the right Accumbens area (rA) and the right medial orbitofrontal cortex (rmOFC) (2 = .47). Our results revealed a significant negative correlation between rA-rmOFC connectivity and depression severity across the whole sample  but not among depressed subjects . In the whole sample and in the depressed group, a significant negative correlation was also detected between rrACC-bsFG connectivity and GDS score .Compared to the healthy comparison group, depressed subjects had significantly lower connectivity between the right rostral anterior cingulate cortex (rrACC) and bilateral superior frontal gyrus (bsFG)  in depressed subjects relative to healthy subjects  . We founTo the best of our knowledge, this is the first study investigating whole-brain rs-fMRI FC changes and DTI in unmedicated and symptomatic patients with late-life depression. Our data-driven rs-fMRI FC analysis revealed significant decrease in connectivity between the right rostral ACC and bilateral superior frontal gyrus as well as between the right Accumbens area and the right mOFC. TBSS analysis revealed in LLD subjects a significant decrease in the FA value of the right FM hat correlated with observed alterations in FC.We found reduced functional connectivity between the right rACC and bilateral sFG in LLD. Based on the results of several functional brain imaging studies in healthy subjects and patients with mood and anxiety disorders, both the sFG and rACC participate in a complex frontolimbic circuit suggested as the neural correlate of emotion regulation. Coactivation of the right sFG and the right rACC was detected during a task that required emotional self-regulation in normal participants The subgenual anterior cingulate cortex (sgACC) is located in the ventral part of rACC. A set of neuropathological, structural and functional brain imaging and animal lesion studies have suggested a strong involvement of the sgACC in depression and mania The NAcc and the OFC comprise part of the “reward network” involved in reward processing and hedonic experience. Accumulating findings from neuroimaging, neuropsychology and neurophysiology studies link the OFC to sensory integration, reward value processing, decision making and subjective pleasantness The OFC and medial prefrontal cortex sends direct afferent projections to the NAcc In a recent structural/functional connectivity study of adults with MDD, Kwaasteniet et al, showed a negative correlation between uncinate fasciculus integrity and subgenual ACC functional connectivity with the bilateral hippocampus in subjects with MDD compared to a healthy control group. They also found a positive correlation between this reported negative structure-function relationship and depression severity in depressed patients Interestingly, our main TBSS finding of LLD-related alteration in the rFM is consistent with our FC results. The forceps minor is the anterior extension of corpus callosum in the frontal lobes connecting the lateral and medial regions of frontal lobes and extends to the striatum and limbic areas. It is very likely that the disruption in integrity of this fiber bundle compromises the axonal tracts mediating both bsFG-rACC and OFC-NAcc anatomical connections. This possible white matter structural abnormality can reasonably be suggested as an underlying etiological mechanism for observed FC changes in our depressed subjects and explains the significant correlation between structural and functional connectivity values reported in the present study. However, prospective longitudinal studies are needed to determine causality.2 values. Approximately half the variance in FC values for the rrACC – bilateral SFG and rA– rmOFC was explained by group differences. Another limitation is the use of anatomical ROIs instead of functional ROIs. The use of anatomical ROIs can result in the combination of multiple functionally distinct regions and thus confound our results. Notwithstanding this caveat, there are several methodological strengths to consider. In addition, rather than examine a selected set of predefined brain regions of interest, we applied a comprehensive data-driven connectome method for broadly exploring whole brain FC changes in LLD. Compared to a priori hypothesis-based methods, our approach for data analysis was not biased by the choice of seed selection and therefore has higher sensitivity without compromising specificity. Another strength of the present study is our subject selection which involved unmedicated and symptomatic depressed participants. Several studies have shown significant effect of psychotropic medications including SSRIs on brain networks dynamics and FC in depression a priori region of interest based on the FC findings. However, the uncorrected threshold for significance was set at p = .0005. Future studies expanding the sample size would strengthen the validity of our findings. Another limitation is the relatively older age of our comparison subjects compared to the depressed patients. To address this issue, we carefully corrected all significant group differences for age. Furthermore, it should be noted that the older age in the comparison group would likely have a mitigating effect on our results.A limitation of the present study is the small sample size. Despite the small sample size, the results demonstrated a very robust effect size based on the partial ηBased on the findings of the present study, we propose two main circuit-based mechanisms for LLD at the network functional level: impairment in top-down frontolimbic emotion regulation circuitry and frontostriatal reward networks reflected respectively in the associated decrease in bsFG-rrACC and rA-rmOFC connectivity. We found those FC changes serve as significant biological markers for symptom severity in unmedicated and symptomatic subjects with LLD. Our DTI structural imaging results detected a pathologic white matter integrity measure alteration in the rFM that significantly correlated with above-mentioned FC differences and depression severity. This correlation suggests an interesting relationship between structural and functional connectivity in LLD. The association of structure-function alterations with specific cognitive, affective and somatic depressive symptoms will be an important direction for this line of study in the future.


Undifferentiated stem cells  were either maintained in their pluripotent state or released into differentiation . Glucose utilization was assayed through 2-deoxyglucose uptake measurement, and clock gene and glucose transporter expression was assayed every 4 hours for 2 days in ESCs and dESCs by quantitative PCR (qPCR) in the same cell lysates. Undifferentiated stem cells expressed a self-sustained rhythm in glucose uptake that was not coincident with rhythmic expression of clock genes. This physiological rhythm was paralleled by glucose transporter mRNA expression. Upon differentiation, circadian patterns of some but not all clock genes were expressed, and the amplitude of the glucose utilization rhythm was enhanced in dESCs. These data provide the earliest evidence of a functional circadian clock, in addition to further challenging the idea that rhythmic transcription of clock genes are necessary for rhythmic physiological output and suggest a role for a clock-controlled physiology in the earliest stages of development.The appearance, progression, and potential role for circadian rhythms during early development have previously focused mainly on the suprachiasmatic nucleus (SCN) and peri- and postnatal expression of canonical clock genes. More recently, gene expression studies in embryonic stem cells have shown that some clock genes are expressed in undifferentiated cells; however rhythmicity was only established when cells are directed toward a neural fate. These studies also concluded that a functional clock is not present in ESCs, based solely on their gene expression. The  Zeitgebers, such as light or timed feeding Circadian rhythms and the cellular/physiological processes they control have been established as a highly conserved mechanism by which organisms are able to anticipate daily changes in the environment and to temporally coordinate complex processes mClock and mBmal1 heterodimerize to activate transcription of negative elements Periods  and Cryptochromes (mCry1 and 2) via binding to E-box elements within the promoter regions of those negative elements. PER and CRY proteins heterodimerize and reenter the nucleus to inhibit the interaction of positive elements CLOCK and BMAL1. Thus, the PER/CRY inhibition itself acts to repress these genes’ own transcription, which is eventually relieved by Casein Kinase-1 Epsilon-mediated proteosomal degradation mBmal1 promoter region and act to inhibit and activate mBmal1 transcription, respectively In mammals, circadian rhythmicity is maintained via a transcriptional/translational feedback loop in which positive elements At the systems level, the master pacemaker that confers time of day information throughout the organism resides in the suprachiasmatic nucleus (SCN). This paired structure - which is located in the anterior hypothalamus and situated just above the optic chiasm – consists of 20,000 neurons and receives photic input from the retina. Lesion of the SCN abolishes physiological and behavioral rhythms, demonstrating its role in conferring time of day information to coordinated behaviors and physiological processes Previously, investigations into the development of the mammalian circadian system have concentrated primarily on the development of the SCN pacemaker mPer1 expression in utero using live fluorescence imaging. Although this was a proof of concept study, the authors clearly showed mPer1::luciferase expression as early as day 7. Earlier, Johnson et al. Several studies have investigated clock gene expression earlier in mammalian embryogenesis. Saxena et al mBmal1 promoter or clock-controlled gene mDbp promoter. The results from this study showed that undifferentiated cells were not rhythmic with respect to mBmal1 or mDbp, but cells that were directed towards a neural fate were rhythmic after synchronization with forskolin. Kowalska et al. Recently, two independent groups have investigated circadian rhythms in embryonic stem cells. Yagita et al. mReverb-a and mRor-a.Here we present evidence that rhythmicity in ESCs precedes the development of clock gene rhythms. Primary ESCs were cultured either in the presence or absence of the differentiation inhibitor Leukemia Inhibitory Factor (LIF). 2-DG uptake was assayed as a physiologically relevant output of the clock and a comprehensive profile of transcripts from both positive and negative limbs of the molecular clock was analyzed for rhythmicity over 2 days in culture, as well as the “stabilizing loop” consisting of 6 cells/well into 6-well culture plates (BD Bioscience) for experiments. Cells were allowed to grow for 12 hours after final passage before sampling began, at which point time is referred to as 0 hours on each graph.The ES cells were derived from blastocysts of SV129 mice for molecular studies and C57BL/6 mice for the real-time analyses. Females  were superovulated by i.p. injection of pregnant mare serum gonadotropin  followed by human chorionic gonadotropin  prior to mating with males of the same respective genotype). ES colonies were initially expanded from a 96-well format to 24-well plates, and finally to 6-well plates while being maintained on mitomycin C-inactivated STO feeder cells. For the experiments, ES cells were passaged nine times into feeder-free cultures, resulting in pure ESC cultures that were maintained as pluripotent ESCs or allowed to differentiate by the removal of LIF, which was done one passage prior to experimentation (passage 8). The culture environment consisted of a standard water-jacketed incubator held at 37°C and 5% CO2 and controlled humidity. Standard ES media (ES-DMEM), Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum, sodium bicarbonate (2.2 g/L), MEM Non-Essential Amino Acids (0.1 mM), L-glutamine (2 mM), B-mercaptoethanol (0.1 mM), penicillin (50 U/ml)/streptomycin (50 µg/ml) antibiotic (1 mM), and LIF  was used for maintenance of ES cell pluripotency. Differentiation medium was identical to ES-DMEM but without LIF. Cultures were maintained in 100 mm, gelatin-coated culture dishes, fed daily, passaged every 2 days and replated at 1×1014C-2-deoxyglucose  for one hour by complete removal and replacement of the medium. The medium was then removed and the cells rinsed twice with Dulbecco’s PBS (Invitrogen). Cells were harvested in 1 ml Trizol reagent (Invitrogen) to extract cellular RNA and soluble protein. 200 µl of cell lysate was placed in 5 ml of scintillant and each sample counted in duplicate on a Beckman scintillation counter. Disintegrations per minute (DPM) were converted to molar quantities as per Sokoloff’s method At the onset of each timepoint, one 6-well plate from each cell type was incubated with mCyclophilin D.DNase-treated cDNA from total RNA was generated using Superscript II reverse transcriptase (Invitrogen). Relative quantification of clock genes and stem cell marker genes was accomplished using SYBR chemistry-based qPCR on either ABI Prism 7500 Fast or StepOne Plus system . Both machines are capable of generating identical thermal profiles and this was confirmed by testing identical samples on both machines. 5 cells per plate and allowed to attach and grow for 12–15 hours. At the onset of the experiment, media was replaced with ES media with the following modifications: Sodium bicarbonate concentration reduced to 0.35 g/L and 10 mM HEPES added to compensate for atmospheric conditions. The dishes were sealed with glass coverslips and vacuum grease and placed in a Lumicycle luminometer . One group of cells was maintained in the undifferentiated state via LIF administration, while another group of cells, passaged from the same initial culture, did not receive LIF and, therefore, underwent differentiation. Bioluminescence recording and detrended plotting of the counts from the cells was accomplished using a Lumicycle photomultiplier detection system and Lumicycle software, respectively .ESCs of C57Bl/6/SV129 hybrid mice expressing the PER2::LUCIFERASE fusion protein  were used to monitor the real-time expression of mPER2 throughout differentiation. For these studies, feeder-free ESCs or dESCs (passage 9) were plated onto gelatin-coated 35 mm dishes (n = 10 for both ESCs and dESCs)  in ES media at 1×10bona fide circadian rhythms were as follows: a) both days show statistically significant rhythms using Circwave analysis and b) the acrophases of day 1 and day 2 occur within 23–25 hours of each other. Data were also tested using ANOVA for non-rhythmic expression profiles to determine significant changes over time. ANOVA was performed using Sigmastat software . Where performed, amplitudes were calculated by subtracting absolute peak levels from previous or following trough levels - as previously determined using ANOVA - and dividing by 2.Time course data were subjected to cosinor analysis based on linear harmonic regression  where each 24-hour period was tested separately. The software assumes a 24 hour period and applies harmonics to the basic sinusoidal function. These attributes allowed us to use the software to determine whether the data were rhythmic for each day of the 2 day sampling as well as providing centers of gravity to determine acrophase for each day. The criteria for determining mOct4 expression was high during the first day of culture and decreased during the second, suggesting that some of the cells began differentiating on the second day  expression might also be rhythmic. qPCR analysis of 6 different mGlut members revealed only two that were detectable: mGlut1 and mGlut8. Of these two, mGlut8 was rhythmic in both ESCs and dESCs  and elsewhere in vitroin vivo have no effect on metabolism These data cannot be explained by possible effects of 2-DG on metabolism itself. While high mM concentrations 2-DG inhibits cellular metabolism Thus, although undifferentiated ESCs do not possess a functioning canonical molecular clock, a circadian rhythm of glucose utilization persists in these cells. This rhythm is coincident with rhythmic expression of one glucose transporter gene, suggesting rhythmic transcriptional control of glucose utilization. Whether this rhythm is initiated intrinsically or in response to glucose availability is unknown. In addition, acute differentiation by withdrawal of LIF increases the amplitude of glucose utilization rhythms and initiates rhythms of the expression of canonical clock genes, suggesting a potential role for the cytokine in initiating the transcriptional-translational feedback loop.Table S1Primers used for qPCR analysis.(DOCX)Click here for additional data file.


Neurons may compete against one another for integration into a memory trace. Specifically, neurons in the lateral nucleus of the amygdala with relatively higher levels of cAMP Responsive Element Binding Protein (CREB) seem to be preferentially allocated to a fear memory trace, while neurons with relatively decreased CREB function seem to be excluded from a fear memory trace. CREB is a ubiquitous transcription factor that modulates many diverse cellular processes, raising the question as to which of these CREB-mediated processes underlie memory allocation. CREB is implicated in modulating dendritic spine number and morphology. As dendritic spines are intimately involved in memory formation, we investigated whether manipulations of CREB function alter spine number or morphology of neurons at the time of fear conditioning. We used viral vectors to manipulate CREB function in the lateral amygdala (LA) principal neurons in mice maintained in their homecages. At the time that fear conditioning normally occurs, we observed that neurons with high levels of CREB had more dendritic spines, while neurons with low CREB function had relatively fewer spines compared to control neurons. These results suggest that the modulation of spine density provides a potential mechanism for preferential allocation of a subset of neurons to the memory trace. Aplysia  in a similar small percentage of LA neurons did not affect auditory fear memory, likely because the neurons expressing CREBS133A were largely excluded from the memory trace. Furthermore, post-training ablation  is an activity regulated transcription factor that modulates the transcription of genes with cAMP responsive elements (CRE) located in their promoter regions. Early research in CREB is a ubiquitous transcription factor implicated in many diverse cellular processes in addition to memory formation, including regulation of proliferation, survival, apoptosis, differentiation, metabolism, glucose homeostasis, spine density, and morphology  and the National Institutes of Health (NIH) and approved by the Animal Care Committee at the Hospital for Sick Children.Adult male F1 hybrid (C57 BL/6NTac × 129S6/SvEvTac) mice were used for all experiments. This genetic background has been used extensively in behavioral studies and are well characterized  vectors were used to manipulate CREB function in individual LA principal neurons. Wild-type or dominant negative CREBMice were pretreated with atropine sulfate , anesthetized with chloral hydrate  and placed in a stereotaxic frame. Skin was retracted and holes were drilled in the skull above the LA  according to  Plexiglas and metal chamber  located in a soundproof room. After 2 min, a tone  that co-terminated with a footshock  was presented. Mice remained in the chamber for an additional 30 s and then returned to the homecage. Testing for auditory fear memory occurred 24 h later by placing mice in a novel context  and 2 min later, presenting the tone previously paired with footshock for 3 min. The percentage of time mice spent freezing  before and during the tone was measured using an automated system (Actimetrics) and was used as our index of memory. Immediately after testing, mice were deeply anesthetized and perfused.To visualize the number and morphology of dendritic spines in the neurons we infected, we took advantage of the GFP expressed by all viral vectors. We amplified the GFP signal using an antibody directed against GFP. 72 h after surgery, mice were deeply anesthetized using chloral hydrate  and transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde (PFA). Brains were post-fixed overnight in 4% PFA and transferred to 30% sucrose for cryoprotection. Coronal (50 μm) sections were prepared and immunohistochemistry for GFP was performed. Free-floating sections were incubated in blocking solution  for 1 h and labeled with anti-GFP rabbit polyclonal antibody  overnight at 4°C. Following PBS washes, sections were incubated with goat anti-rabbit Alexa 488  for 2 h at room temperature. Sections were washed with PBS, mounted on gelatin-coated slides and coverslipped using Vectashield Hardmount with DAPI (Vector Laboratories).GFP-positive LA neurons  were first identified using a 10x objective . Infected neurons were included in the subsequent spine analysis if (i) cell body was not damaged; (ii) dendritic projections remained within the LA; (iii) neurons could clearly be identified without interference from neighboring infected cells; (iv) neurons had first, second, and third order branches. Fourth order branches were not included in the analysis as they often appeared truncated in our 50 μm sections. Selected GFP-positive neurons were imaged using a 100x oil-immersion objective. Z series were obtained by imaging serial confocal planes at 0.25 μm intervals. Dendrites and spines were traced manually from the image stacks using Neurolucida software and analyzed with Neurolucida Explorer (MBF version 9).Image analysis was performed by two researchers unaware of the treatment condition of the mouse. Dendrites were traced. The first dendritic process emanating from the cell body was defined as the primary (first order) branch. Subsequent branches that bifurcated from the first branch order were designated as second order branches, and so forth. Truncated branches or those that did not remain within the image window were excluded from subsequent analysis.Dendritic spines were defined as small protrusions connected to the dendritic shaft . Newman-Keuls post-hoc tests were performed where appropriate. To protect against potential type 1 errors resulting from multiple comparisons of 5 different measures of neuronal morphology , we also performed a Bonferroni correction (corrected α = 0.01). All significant main effects remained significant after correction.Data were analyzed with 1 or 2-Way analyses of variance (ANOVAs) using Statistica (Statsoft) software. For the auditory fear conditioning data, we analyzed the percentage of time spent freezing to before (2 min) and during (3 min) the tone. For dendritic and spine morphological analysis, data were first averaged by branch order per cell, then by animal and finally by vector group  into the LA of adult mice 3 d before fear conditioning  × Time (5 min) ANOVA showing significant main effects of Vector  and Time  but no Vector × Time interaction . A subsequent One-Way ANOVA performed on freezing during the entire CS presentation showed a significant effect of Vector , as mice microinjected with CREB vector froze significantly more than mice with GFP (p = 0.006) or CREBS133A vector (p = 0.004), which did not differ from each other (p = 0.83) (Newman-Keuls post-hoc) . We next examined a possible mechanism underlying this preferential recruitment to the memory trace.We first confirmed the effects of manipulating CREB function in a small portion (~8–10%) of LA neurons on the formation of tone fear memory by microinjecting HSV vectors encoding GFP, CREB or dominant-negative CREB  Figure . Importaat the time of training than neurons infected with CREBS133A or Control GFP vector. We microinjected a separate co-hort of mice with GFP, CREB, or CREBS133A vector as above but did not train these mice. Instead, 72 h following surgery (at a time when they would have received auditory fear conditioning) we removed their brains and examined spine density  by Branch order (3) repeated-measures ANOVA showed significant main effects of Vector  and Branch order  but no significant interaction between Vector × Branch order . Post-hoc Newman-Keuls analysis on the significant main effects revealed that neurons with CREB vector had significantly greater spine density across branch orders compared to neurons infected with GFP (p = 0.02) or CREBS133A (p = 0.0002) vectors  -ergic interneurons  = 2.8, p = 0.09] or interaction of Vector × Branch order , but a significant main effect of Branch order . Therefore, dendritic length increased with increasing branch order, but this was not changed by CREB manipulation  = 1.3, p = 0.29], Branch order  or Vector × Branch order interaction . Therefore, manipulations of CREB function changed dendritic spine density without changing dendritic morphology.Importantly, dendritic length per branch Figure  or total Figures . We also Figures . An ANOVVector  or Vector × Branch order interaction , but a significant main effect of Branch order }. We next examined whether CREB manipulation influenced spine head diameter  = 0.7, p = 0.50] or Vector × Branch order interaction  but a significant main effect of Branch order }. Therefore, although CREB manipulations produced changes in dendritic spine density, these were not accompanied by changes in dendrite or overall spine morphology.Alterations in spine morphology have been correlated with changes in spine function  showed impaired in LTM for both context and tone fear memories   information directly from auditory cortex and thalamus (LeDoux et al., Here, we observed that changes in CREB function alone were sufficient to change dendritic spine density, and that neurons with increased CREB function showed higher dendritic spine density. Furthermore we observed that neurons with higher CREB function were preferentially allocated to the memory trace. Because synapses and spines play a key role in neuronal information processing, changes in dendritic spine density or morphology of a neuron may affect synaptic function and local circuit organization. Along with other factors, such as changes in neuronal excitability (Zhou 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.


Muscle biopsies were taken from the exercising and non-exercising leg before and immediately after exercise and analyzed by microarray. One-legged cycling raised plasma lactate, free fatty acids, cortisol, noradrenalin, and adrenalin levels. Surprisingly, acute endurance exercise not only caused pronounced gene expression changes in exercising muscle but also in non-exercising muscle. In the exercising leg the three most highly induced genes were all part of the NR4A family. Remarkably, many genes induced in non-exercising muscle were PPAR targets or related to PPAR signalling, including PDK4, ANGPTL4 and SLC22A5. Pathway analysis confirmed this finding. In conclusion, our data indicate that acute endurance exercise elicits pronounced changes in gene expression in non-exercising muscle, which are likely mediated by changes in circulating factors such as free fatty acids. The study points to a major influence of exercise beyond the contracting muscle.Regular physical activity positively influences whole body energy metabolism and substrate handling in exercising muscle. While it is recognized that the effects of exercise extend beyond exercising muscle, it is unclear to what extent exercise impacts non-exercising muscles. Here we investigated the effects of an acute endurance exercise bouts on gene expression in exercising and non-exercising human muscle. To that end, 12 male subjects aged 44–56 performed one hour of one-legged cycling at 50% W Regular exercise training is generally recognized as a powerful preventive and therapeutic strategy for diseases such as type 2 diabetes, obesity and cardiovascular disease. At a systemic level, regular exercise training improves cardiac and lung function Immediately upon initiation of exercise, local demand for ATP, oxygen, glucose and fatty acids increases dramatically. These demands are accommodated by rapid changes in skeletal muscle activity of key enzymes and transporters involved in glucose and fatty acid oxidation via allosteric regulation and phosphorylation of rate-limiting enzymes. In addition, regulation at the mRNA level importantly contributes to the acute response and chronic adaptations to exercise. A large number of studies have shown that acute exercise induces genes involved in a variety of processes, including energy metabolism, hypertrophy and signalling 2max and Wmax (1 and 2 legged) values can be found in Twelve healthy middle-aged men  participated in the study. All subjects exercised less than 4 hours per week. Anthropometric parameters, VOAll subjects performed a single 60 minutes experimental endurance exercise bout, which was preceded by two preliminary exercise tests and two familiarization trials . During 2max; 2) and heart rate (HR) were measured . The tests were performed at the same ergometer as the endurance exercise bout and familiarization trails and were completed at least 14 days before the endurance exercise bout and 3 days apart to avoid a training effect. The day prior to both tests subjects were asked to refrain from alcohol and heavy exercise.Two graded cycling exercise tests to exhaustion were performed, the first with both legs, the second with one leg . The first two-legged exercise test was used to determine the subjects' maximum aerobic capacity VO2max; , while tAll participants were unfamiliar with one-legged exercise. Therefore the two familiarization trials were performed before the endurance exercise bout to make sure that subjects became familiar with one-legged cycling . The famSubject refrained from heavy exercise the last 4 days prior to the experimental day. The last day before the experimental exercise bout subjects received a standardized evening meal and refrained from alcohol. On the morning (8.15 h) of the experimental test subjects reported to the research facility, after an overnight fast (>10 hours). The experimental exercise bout consisted of 60 min one-legged cycling at 50% of the one-legged Wmax. Before (T0) and shortly after (T1) the exercise a venous blood sample was drawn and muscle biopsies were taken from both legs see . Two houBlood was collected in EDTA containing tubes. The samples were immediately centrifuged at 1000 g at 4°C for 10 minutes, after which plasma was stored in −80°C until further analysis. Blood samples were analysed for free fatty acids , Velp, NL), glucose, triglycerides, cortisol, lactate , catecholamines , and insulin .Percutaneous needle biopsies were taken before (T0) and shortly after (T1) exercise from the vastus lateralis muscle from both legs , using the Bergström technique with suction Total RNA was isolated from the skeletal muscle tissue by using Trizol reagent . Thereafter RNA was purified using the Qiagen RNeasy Micro kit  and RNA quality was checked using an Agilent 2100 bioanalyzer .Total RNA (100 ng) was labelled using an Ambion WT expression kit  and hybridized to human whole genome Genechip Human Gene 1.1 ST arrays coding 19.732 genes, . Sample labelling, hybridization to chips and image scanning was performed according manufacturer's instructions.Microarray analysis was performed using MADMAX pipeline for statistical analysis of microarray data Two subjects (4 and 8) were classified as outliers based on their aberrant response to exercise in the non-exercising leg. In subject 4 and 8, expression of 6 out of the 10 most highly upregulated genes was higher than the average plus two times the standard deviation. Accordingly, the analysis was repeated without these two subjects.All microarray data are MIAME compliant and have been submitted to the Gene Expression Omnibus (accession number GSE41769).http://www.broad.mit.edu/gsea/) was performed for both legs using MADMAX and genesets with a false discovery rate (FDR)<0.2 were considered significantly enriched. Possible transcription factors playing a role in the activation and inhibition of genes were identified using Ingenuity Pathway Analysis . The ClueGO plugin in Cytoscape was used for gaining insight into processes activated during exercise in both legs Geneset enrichment analysis  and can be found in Total RNA was reverse transcribed with a cDNA synthesis kit . Standard qPCR was performed using SensiMix real time PCR reagents  and a Bio-Rad CFX384 machine . Primer sequences were based on availability in the PRIMERBANK . Differences between the different time points for the plasma parameters  were determined using a repeated measure one-way ANOVA. Differences between T0 and T1 in qPCR were evaluated using a paired t-test, differences between the legs in qPCR were evaluated using a paired t-test for the changes between T0 and T1 in each leg. Data are mean ± SD and p<0.05 was considered statistically significant.Subjects performed one-legged exercise for one hour at 50% of their maximal workload with an average heart rate during the last five minutes of 132±18 beats per minute. The percentage heart rate reserve (%HRR) during the last 5 minutes was 65±12.5% . Plasma We first ruled out that there was a significant difference in baseline expression between the two legs. Only eight out of 19,732 genes were found to be significantly different between the two legs at baseline p<0.01; .Statistical comparison of the baseline and post-exercise samples revealed that in the exercising leg (E) one-legged exercise significantly changed expression of 938 genes (p<0.01), with the majority of genes being upregulated . The numTo gain more insight into the changes induced by exercise, genes were subsequently ranked according to mean fold-change in expression in the exercising leg and the changes in expression compared between the individual subjects . ExpressTo examine the impact of exercise on gene expression in parts of the body not directly influenced by exercise, we next focused our attention on genes upregulated in the non-exercising leg. Similarly to the exercising leg, genes were ranked according to mean fold-change in the non-exercising leg . The twoWe suspected that exercise-induced changes in gene expression were mediated by specific transcription factors. To identify these transcription factors and to identify pathways regulated by exercise, we performed Ingenuity pathway analysis (IPA) . IPA useThe only significant set of target genes in the non-exercising leg was the set controlled by PPARα, which is consistent with the marked upregulation of several existing and putative PPARα target genes . Since PTo further determine the biological processes activated during exercise, we used ClueGO and GSEA. ClueGO integrates GO categories and creates a functionally organized GO category networks based on the overlap between the different GO categories and the significance Here we used the one-legged exercise model to study the effect of acute endurance exercise on whole genome muscle gene expression and determine the relative importance of systemic versus local contraction-related stimuli. We found that acute exercise induced immediate and dramatic gene expression changes in the exercising leg, with the most dramatic inductions observed for the NR4A family. Strikingly, acute exercise also caused substantial gene expression changes in non-exercising leg. Overall, our data indicate that the molecular responses to acute exercise are not confined to the exercising muscle but also extend to resting muscle. The notion that exercise alters gene expression in non-exercising muscles is new. Our data provide a conceptual and molecular framework for the observation that immobilized muscle can experience favourable metabolic adaptations in response to repeated contractile activity in non-immobilized muscle In general, changes in gene expression in non-exercising muscle were less pronounced compared to the exercising muscle, both in magnitude of fold-changes and number of genes changed. A relatively small number of genes, many of which represent known target genes of the PPAR transcription factors including ANGPTL4, KLF10, SLC22A5, ZNF750, PDK4, was induced equally in exercising and non-exercising muscle. PPARs play a key role in regulation of lipid metabolism in a variety of tissue, including skeletal muscle We are the first to use microarray to assess the immediate effect of acute endurance exercise on human skeletal muscle gene expression. Prior studies focused on the recovery after exercise Many of the observed exercise-induced changes in gene expression are likely part of an acute stress response related to disturbances in homeostasis elicited by exercise. The most highly induced genes in the exercising leg were all members of the NR4A family, a subgroup of orphan receptors within the nuclear receptor superfamily. NR4A1 and NR4A3 have been reported to be upregulated shortly after acute exercise and during recovery in rat Unlike in other subjects, in two subjects the overall magnitude of gene expression changes in the exercising and non-exercising leg were very similar. It is unclear how such an extreme response can occur in a resting leg that was not engaged in any concentric contractions. We cannot completely exclude these two subjects performed significant isometric contractions in the non-exercising leg, although subjects were instructed not to. Alternatively, a potential role for (involuntary) neural stimulation muscles may be envisioned. It is known that neural stimulation can induce gene expression changes via increased calcium concentrations in the skeletal muscle One-legged cycling is frequently used as a model to enable direct comparison between exercising and resting muscle. Earlier studies encountered problems with repeated biopsies, showing inflammatory gene expression changes induced by repeated biopsies In conclusion, exercise has profound effects on gene expression in human skeletal muscle, not only in exercising muscle but also in resting muscle. The latter effects are likely mediated by changes in circulating factors such as FFA and may explain why muscles not involved in the exercise movement may undergo favourable adaptations in response to exercise.Figure S1qPCR confirming microarray results of NR4A family, JUNB and FOS (N = 11): fold changes of both legs are displayed, which are calculated by dividing the post-exercise by the baseline sample for both legs. Before expression values were normalized by the housekeeping gene GAPDH. *  =  p<0.01. Depicted is mean ± SEM.(TIF)Click here for additional data file.Figure S2The 20 most highly induced genes in the non-exercising leg, including subjects 4 and 8: heatmap of the top 20 upregulated genes in the non-exercising (N = 9), left panel shows top 20 of the non-exercising leg, right the corresponding genes in the exercising leg. Green is a signal log ratio of −3, red a signal log ratio of 3. Values are displayed per subject to visualize inter-individual differences. FC  =  fold change, *  =  p<0.05, #  =  p<0.1 between exercising and non-exercising leg.(TIF)Click here for additional data file.Figure S3Acute endurance exercise induces several genesets in both legs related to immune response, skeletal muscle hypertrophy and stress. Venn diagram shows the overlap between the upregulated genesets in both legs. Next to the circles are the top 5 enriched genesets. Genesets depicted in bold are overlapping between exercising and non-exercising leg, whereas geneset with a normal font are unique for that leg. FDR  = 0.2, exercising leg N = 9, non-exercising leg N = 7.(TIF)Click here for additional data file.Figure S4Combined time course of gene expression of selected genes of Mahoney et al. and this study: signal log ratios of selected genes (based on presence in datasets and expression) are displayed directly after exercise (this study) and 3 and 48 hours after exercise .(TIF)Click here for additional data file.Table S1List of differentially expressed genes at baseline (T0) in exercising and non-exercising leg.(PDF)Click here for additional data file.Table S2Induced sets of transcription factor targets.(PDF)Click here for additional data file.


Seedlings of cowpea raised in HAuCl4, even at concentration as high as 1 mM, did not show any suppression in growth. Accordingly, Au3+, despite being a heavy metal, did not alter levels of stress markers  in cowpea. Interestingly, cowpea turned clear pale yellow HAuCl4 solutions colloidal purple during the course of seed germination and seedling growth. These purple colloidal suspensions showed Au-nanoparticle specific surface plasmon resonance band in absorption spectra. Transmission electron microscopic and powder X-ray diffraction investigations confirmed presence of crystalline Au-nanoparticles in these purple suspensions. Each germinating seed of cowpea released ∼35 nmoles of GAE of phenolics and since phenolics promote generation of Au-nanoparticles, which are less/non toxic compared to Au3+, it was contemplated that potential of cowpea to withstand Au3+ is linked to phenolics. Of the different components of germinating seed of cowpea tested, seed coat possessed immense power to generate Au-nanoparticles, as it was the key source of phenolics. To establish role of phenolics in generation of Au-nanoparticles (i) seed coat and (ii) the incubation medium in which phenolics were released by germinating seeds, were tested for their efficacy to generate Au-nanoparticles. Interestingly, incubation of either of these components with Au3+ triggered increase in generation of Au-nanoparticles with concomitant decrease in phenolics. Accordingly, with increase in concentration of Au3+, a proportionate increase in generation of Au-nanoparticles and decrease in phenolics was recorded. In summary, our findings clearly established that cowpea possessed potential to withstand Au3+-stress as the phenolics released by seed coat of germinating seeds possess potential to reduce toxic Au3+ to form non/less toxic Au-nanoparticles. Our investigations also pave a novel, simple, green and economically viable protocol for generation of Au-nanoparticles.While evaluating impact of Au nanoparticles on seed germination and early seedling growth of cowpea, HAuCl In general, gold comes into the environment from primary (i.e. ores) and secondary sources such as electronic scrap and waste electroplating solutions 6) bearing one or more hydroxyl groups The degree and mechanism of tolerance to heavy metals vary significantly amongst plant species 3+ on growth and development on a leguminous crop, cowpea (Vigna unguiculata). Our results showed for the first time that (i) cowpea posesses excellent potential to withstand Au3+-stress due to the presence of phenolics; and (ii) phenolics released during the course of seed germination and early seedling growth of cowpea play a vital role in detoxification of Au3+ by forming Au-nanoparticles.Owing to lack of any detailed studies, present investigations were initiated with an aim to evaluate impact of AuVigna unguiculata (L.) Walp., Fabaceae] were obtained from the local farmers of Haldwani .Seeds of cowpea [3+ on seed germination and early seedling growth of cowpea was evaluated using HAuCl4. After washing with 0.1% cetrimide and distilled water, seeds were surface sterilized with 0.1% mercuric chloride for 2 min, rinsed with sterile distilled water and inoculated in autoclaved bottles containing 75 g of uniform sized glass beads with 20 ml test solution under sterile conditions. These bottles were incubated at 25±2°C under a 16/8 h light/dark cycle at a light intensity of 60 µmol m−2 s−1. Growth of seedlings was measured in terms of length and fresh weight of root and shoot. Levels of malondialdehyde (MDA) and proline in root and shoot were measured in 4 d old seedlings.Impact of different concentrations  of Au−1 fresh weight.For determining levels of proline and malondialdehyde (MDA), root and shoot of seedlings were homogenized in 5% TCA and centrifuged at 15,000 xg for 15 min. Proline levels were measured according to Bates et al. Total phenolics were measured as per Ainsworth and Gillespie 4 for 24 h to test their potential to generate Au-nanoparticles. Seedlings devoid of cotyledons+seed coat were treated by immersing their roots in HAuCl4 solutions. Contol incubation medium (i.e. distilled water), in which seedlings of cowpea were raised for 4 d, was also tested for its efficacy to form Au-nanoparticles by incubating 500 µl of this medium with 10 ml HAuCl4.Different components namely seed coat and cotyledons were excised carefully from 4 d old cowpea seedlings raised in distilled water under sterile conditions. Seed coat, cotyledons and rest of the seedlings (i.e. seedlings devoid of cotyledons+seed coat) were treated with 10 ml of different concentrations of sterile HAuCl3+ solutions (i) in which seedlings of cowpea were raised; and (ii) incubated independently with various components of seedlings of cowpea and distilled water in which seedlings were raised as detailed above, were recorded from 190 to 1100 nm using Specord 200 Analytikjena UV-Vis spectrophotometer. For transmission electron microscopic (TEM) studies, 10 µl of colloidal suspension was drop-coated on 200 mesh copper grid with an ultrathin continuous carbon film and allowed to dry in a desiccator at room temperature. Grids were viewed in the transmission electron microscope (Technai G2 T30) at a voltage of 300 kV. The hardware associated with the machine also allowed (i) energy dispersive X-ray (EDX) analysis to measure the elemental composition; and (ii) selected area electron diffraction (SAED) analysis to determine crystalline/amorphous nature, of nanoparticles.UV-Vis spectra of Au3+ solutions were centrifuged. The pellet obtained was re-suspended in distilled water, drop coated on silica surface, dried in desiccator and used for collecting PXRD pattern using Rigaku Rotaflex RAD-B with copper target CuK(α)1 radiation with tube voltage 40 kV and 60 mA in 2 theta (θ) range of 30–80°.For powder X-ray diffraction (PXRD) studies, colloidal suspensions obtained by incubating different components with AuAll experiments were carried out independently at least seven times. Duncan's multiple range test was used to to determine the level of significance in physiological and biochemical data 4 was used as control. To our surprise, in spite of being a heavy metal ion, Au3+ was not associated with any significant alteration in seedling growth (measured in terms of length and fresh weight of root and shoot) of cowpea even when present at a concentration of 1 mM  is due to generation of Au-nanoparticles 3+  turned brown during the course of seed germination and early seedling growth. It is known that germinating legume seeds release phenolics 3+-stress tolerance to cowpea seedlings, four different components namely (i) control incubation medium (i.e. distilled water) in which seedlings were raised; (ii) seed coat; (iii) cotyledons; and (iv) seedlings devoid of cotyledons+seed coat, were tested for their efficacy to generate Au-nanoparticles by independently incubating them in different levels of Au3+. As anticipated, brown colored incubation medium possessed abundant potential to turn pale yellow Au3+ solutions purple indicating the generation of Au-nanoparticles (3+ solutions purple (To identify the key component(s) responsible for formation of Au-nanoparticles and imparting Auarticles . Of the s purple . Accordi3+ solutions. However, the size and morphology of nanoparticles varied depending on the component. Seed coat and incubation medium generated nanoparticles in range of 10–30 nm, while cotyledons and seedlings devoid of cotyledons+seed coat generated nanoparticles in the range of 10–25 and 5–10 nm, repectively . Potential of cowpea seedlings with seed coat to generate 5–6 fold higher level of Au-nanoparticles compared to the seedlings devoid of seed coat is evident from the intensity of Au-nanoparticle specific absorption peak of purple colloidal suspensions formed by them  that contained large quantity of phenolics to generate large proportion of Au-nanoparticles  were determined. As evident from 3+. This clearly established that phenolics played a critical role in reduction of Au3+ and generation of Au-nanoparticles. As stated earlier, phenolics possess potential to donate electrons to metal ions such as Au3+ and promote synthesis of Au-nanoparticles.To establish direct role of phenolics in generating Au-nanoparticles, the level of phenolics in Au3+-stress tolerance to cowpea. Earlier researchers had reported that seed coat plays important role in chemical protection from oxidative damage as it possesses phenolics which act as antioxidants 3+-stress tolerance by rapidly converting toxic ionic speciation state into less/non-toxic nanoparticle speciation state.In nutshell, our results convincingly demonstrated that seed coat, being key source of phenolics, is the most powerful component of developing seedling responsible for generation of Au-nanoparticles. In light of above elaborated experimental evidences, we believe that seed coat plays most vital role in imparting Au3+-stress during seed germination and early seedling growth. Our results established that (i) the potential of cowpea to withstand Au3+-stress is linked to phenolics; (ii) seed coat is the key source of phenolics during seed germination and early seedling growth and hence is important for imparting Au3+-stress tolerance; and (iii) phenolics impart Au3+-stress tolerance by converting toxic ionic speciation state of Au to less/non-toxic nanoparticle speciation state. Owing to the proven role of phenolics in imparting metal ion stresss tolerance, we believe that it is important to understand molecular and genetic basis of modulating the synthesis of phenolics for improving tolerance of agricultural/forestry plant species against metal ion stress. Our findings also furnish a novel, simple, green and economically viable protocol for using seed/seed coat of legume seeds for synthesis of metal nanoparticles.Our above findings demonstrated for the first time that cowpea has substantial potential to withstand Au


The reported findings suggest that these polyphenols target multiple signaling pathways involved in cell cycle modulation and apoptosis. Further work is required to understand its potential for health promotion and potential drug discovery for prostate cancer chemoprevention.The Mediterranean diet is rich in fruits and vegetables and has been associated with a variety of health benefits including cancer prevention. One aspect of the diet that has not received enough attention is Mediterranean herbs. Specifically, rosemary and its polyphenolic diterpenes (carnosic acid and carnosol) are known to possess anti-oxidant activity that may be beneficial for cancer control. Herein, we describe the  Rosmarinus officinalis), has long been associated with anti-inflammatory and anti-oxidant properties. A population-based study has even suggested that an overall risk reduction in cancer incidence is correlated with patients consuming herbs including rosemary  has approved the use of rosemary extracts for food preservation and has been adopted into the EU food additive legislation . The stu2+ mobilization, and inhibit the secretion of leukocyte elastase  value for PC3 cells was found to be 41.1 μM. Carnosic acid induced apoptosis as evidenced by DNA fragmentation, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Annexin V–PI flow cytometric analysis. At 80 μM, carnosic acid promoted apoptosis in PC3 cells with 56.1% apoptosis compared to 47.2% apoptosis in DU-145 cells /Akt signaling pathway which in turn suppresses IκB kinase/nuclear factor kappa B (IKK/NF-κB) pathway in PC3 prostate cancer cells anthracene (DMBA)-induced oral carcinogenesis  proteins-2 and -6  pathway. The expression of AMPK beta-1 regulatory subunit was found to increase by 365% in carnosol treated cells compared to control cells via a protein array. AMPK and its downstream molecule mammalian target of rapamycin (mTOR) have been considered as a therapeutic target for cancer treatments. Typically mTOR protein is upregulated in prostate cancer. Upon treatment of PC3 cells with carnosol we observed a dose-dependent decrease in phosphorylation of mTOR protein thereby leading to inhibition of prostate cancer in vitro  levels compared to untreated control animals (in vitro in prostate cancer cells (LNCaP and 22Rv1) as well as in vivo in nude mice  and their role in chemoprevention of prostate cancer. Epidemiological studies suggest a reduced risk of cancer in patients consuming rosemary. Herein, we have described the mechanism by which carnosic acid and carnosol inhibits prostate cancer. Essentially both diterpenes inhibit cancer by promoting apoptosis and inhibiting the critical PI3K/Akt signaling pathway which is an important regulator of tumor cell survival. These findings warrant further research to understand the potential of rosemary as a cancer chemopreventive agent in prostate cancer.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-transcriptional modifications of miRNAs with 3′ non-templated nucleotide additions (NTA) are a common phenomenon, and for a handful of miRNAs the additions have been demonstrated to modulate miRNA stability. However, it is unknown for the vast majority of miRNAs whether nucleotide additions are associated with changes in miRNA expression levels. We previously showed that miRNA 3′ additions are regulated by multiple nucleotidyl transferase enzymes. Here we examine the changes in abundance of miRNAs that exhibit altered 3′ NTA following the suppression of a panel of nucleotidyl transferases in cancer cell lines. Among the miRNAs examined, those with increased 3′ additions showed a significant decrease in abundance. More specifically, miRNAs that gained a 3′ uridine were associated with the greatest decrease in expression, consistent with a model in which 3′ uridylation influences miRNA stability. We also observed that suppression of one nucleotidyl transferase, TUT1, resulted in a global decrease in miRNA levels of approximately 40% as measured by qRT-PCR-based miRNA profiling. The mechanism of this global miRNA suppression appears to be indirect, as it occurred irrespective of changes in 3′ nucleotide addition. Also, expression of miRNA primary transcripts did not decrease following TUT1 knockdown, indicating that the mechanism is post-transcriptional. In conclusion, our results suggest that TUT1 affects miRNAs through both a direct effect on 3′ nucleotide additions to specific miRNAs and a separate, indirect effect on miRNA abundance more globally. Because microRNAs (miRNAs) are critical regulators of key processes such as development and differentiation, their expression levels must be precisely regulated. Recent progress has been made in understanding regulation of miRNA transcription and multiple stages of biogenesis  We and others have observed that mature miRNAs show a diversity of nucleotide additions at their 3′ end in metazoan miRNA high-throughput sequencing studies In humans, the role of 3′ additions is poorly understood, as only a handful of reports have examined the effects of A and U additions on the stability of a small number of miRNAs. The addition of a 3′ A to the miRNA miR-122 was associated with increased abundance of the miRNA In humans, many miRNAs feature 3′ non-templated additions of an adenosine (A) or uridine (U), which do not match the genomic or precursor sequences Figure S1A), although effective antibodies to determine the protein expression of the entire panel of nucleotidyl transferases were not available. Therefore, while the mechanism of compensatory 3′ additions remains unknown, our results indicate that increased 3′ additions are a frequent occurrence when the activity of specific nucleotidyl transferases is disrupted. Importantly, our suppression of nucleotidyl transferase enzymes also created a diversity of altered 3′ additions: Some 3′ additions were gained and others were lost, which enabled us to examine the association between each altered addition and total miRNA abundance.To investigate the association between miRNA 3′ additions and miRNA abundance, we developed a system to perturb 3′ nucleotide additions to endogenous miRNAs. We have shown that multiple nucleotidyl transferase enzymes regulate miRNA 3′ additions in an enzyme and miRNA specific manner Table S1). We found that miRNAs with increased addition following enzyme suppression showed a significant decrease in total abundance compared to miRNAs with unchanged  or decreased 3′ addition  . For three enzymes–PAPOLG, ZCCHC11, and TUT1–we again found that increased 3′ addition was associated with decreased miRNA abundance , indicating that this phenomenon may occur in an enzyme-specific manner.The relationship between 3′ additions and miRNA abundance has not been described on a global scale in humans. Because 3′ additions can modulate miRNA stability in other organisms, we hypothesized that changes in additions would affect the total abundance levels of miRNAs in humans. We examined the expression levels of miRNAs that displayed significant increases and decreases in 3′ additions following our suppression of nucleotidyl transferases. To quantitatively compare abundance levels, we used the NanoString nCounter platform, as this system does not involve sample amplification and provides a precise readout of total counts of a canonical miRNA sequence and its two abundant isomiRs present in a sample , with most miRNAs showing a 50% reduction compared to miRNAs with unchanged additions. Together, these results reveal a strong association between miRNA 3′ nucleotide modifications and abundance levels. While future in vivo and in vitro assays for miRNA turnover will be needed to directly address the differential stability of miRNA isomiRs, these experiments indicate that the acquisition of a 3′ U family addition is associated with decreased miRNA expression.Because changes in 3′ additions can involve the loss or gain of a diverse panel of nucleotides, we next investigated whether certain nucleotides are associated with characteristic changes in abundance. We classified each miRNA isomiR into an addition category based on the first nucleotide of the 3′ addition , filtered for categories that included a minimum of at least 5 miRNAs, and compared the abundance changes associated with significant increase or decrease of each class of miRNA additions . The greatest magnitude of reduction in miRNA abundance was seen following TUT1 suppression, which resulted in a median relative miRNA expression value of 0.61 compared to negative control cells  (−30), although the median relative miRNA expression value of 0.71 in siPAPD4 cells was of a smaller magnitude than observed from TUT1 suppression. As an additional control, we performed Western blots for the TUT1 protein and found that RNAi effectively reduced expression of TUT1 at the protein level . Together, these results indicate that the loss of TUT1 has broad effects on miRNA expression levels that extend beyond its measurable effects on 3′ additions.To confirm and extend these results more broadly, we used Exiqon qRT-PCR arrays to globally profile the expression of over 300 miRNAs in HCT-116 cells following the suppression of our panel of eight nucleotidyl transferase enzymes. Exiqon miRNA qRT-PCR assays are based on a 3′ tailing reaction, and therefore allowed us to detect the total abundance of both canonical and isomiR miRNAs in a single reaction. In an independent series of transfections, we again found that suppression of the majority of the enzymes had very minimal effects on miRNA abundance . This effect was specific to TUT1 knockdown, as we did not observe a change in miRNA abundance following suppression of another nucleotidyl transferase serving as a negative control .We next tested whether this phenomenon extended to other cell lines. In A549 lung carcinoma cells, we suppressed TUT1 and used Exiqon miRNA qRT-PCR assays to quantify global changes in miRNA abundance. We again found that suppression of TUT1 resulted in significant decreases in miRNA expression levels . To determine whether this effect is specific to miRNAs, we also examined the expression of two small nucleolar RNAs assayed in parallel on the qRT-PCR miRNA array. We did not find significant changes in the expression of SNORD38B or SNORD49A following TUT1 suppression , rather there was a slight increase in the primary transcripts for most miRNAs, suggesting that TUT1 affects subsequent steps in miRNA processing and/or turnover. We considered whether TUT1 could indirectly regulate miRNAs by affecting the mRNA expression of miRNA processing enzymes, as TUT1 was previously found to regulate the stability of a subset of mRNA transcripts Dicer, Drosha, or the Drosha co-factor DGCR8 following TUT1 suppression . Thus, while the precise mechanism by which TUT1 maintains miRNA levels remains unknown, our results suggest that TUT1 acts at a post-transcriptional step. Recent work has shown that uridylation of specific precursor miRNAs by the ZCCHC11 enzyme can prevent miRNA maturation TUT1 could potentially affect miRNA levels via indirect or direct mechanisms. TUT1 has previously been described to act in the nucleus as a poly (A) and (U) polymerase for a variety of substrates Our work investigates the functional effects of miRNA 3′ additions by nucleotidyl transferases on a global scale. We found that the acquisition of 3′ additions–particularly of a 3′ U–is associated with decreased miRNA abundance. We also determined that the TUT1 nucleotidyl transferase globally regulates miRNA abundance and represents a newly identified post-transcriptional regulator of miRNA homeostasis in the cell. Future work will be needed to determine the mechanism by which TUT1 regulates miRNA abundance. Our results indicate that this is an indirect mechanism independent of regulation of miRNA 3′ NTA by TUT1, based on our observation that knockdown of TUT1 affected miRNA abundance broadly while only leading to changes in 3′ additions of a subset of the miRNAs we examined. However, we cannot exclude the possibility that TUT1 effects occur predominantly through direct nucleotide additions that we did not detect because they lead to rapid miRNA degradation. Because TUT1 suppression led to increased 3′ U addition on many miRNAs, our results support a model in which uridylation may serve as a signal for rapid miRNA decay in human miRNAs. This work extends a recent report that found an increase in 3′ uridylation during the decay of an exogenously expressed miRNA Cyclophilin B was used as the negative control (Dharmacon). TUT1 siRNAs described by Mellman et al. HCT-116 cells were obtained from Bert Vogelstein and maintained in McCoy’s media (Invitrogen) with 10% FBS . A549 cells were obtained from ATTC and maintained in DMEM (Invitrogen) with 10% FBS. Transfections with biological replicates were performed in 6-well dishes with 62,500 cells transfected with 66 nM siRNA using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. Dharmacon On-Target+ SMARTpool siRNAs were used for suppression of a panel of eight nucleotidyl transferases as previously described GUSB and GAPDH were used for normalization. For primary miRNA qRT-PCR profiling, Taqman assays were obtained from Applied Biosystems for 73 different primary miRNAs (assay numbers listed in Table S2). Primary miRNAs were assayed with two technical replicates according to the manufacturer’s instructions. For primary miRNA data analysis, we removed any miRNA with an undetermined cycle threshold (CT) or with CT >35 in any sample, which yielded 48 pri-miRNAs for subsequent analysis. Delta CT values were calculated using the average of three endogenous control genes run on each plate: GAPDH-FAM, ACTB-FAM, and GAPDH-VIC. Delta delta CT values to the siCyclophilin negative control cells were calculated, and relative expression values were determined.72 hours post transfection with siRNAs, cells were washed with PBS and lysed in 700 uL Qiazol (Invitrogen). RNA was isolated using the miRNeasy RNA isolation kit (Qiagen), quantified with a spectrophotometer (Bio-Tek), and normalized to equal concentration. Taqman assays from Applied Bioystems were used to measure the degree of nucleotidyl transferase suppression. The expression of the endogenous control genes T values were filtered to maintain only reliably detected miRNAs . Delta CT values were then calculated relative to cells treated with negative control siRNAs, and relative expression values were computed. Two-sided t-tests were used to compare the miRNA expression in enzyme suppressed versus control cells. The multtest package of R from Bioconductor Exiqon V2.0 Panel 1 Human miRNA qRT-PCR arrays in 384-well format were used to assess global changes in miRNA levels. Reverse transcription was performed using the Universal cDNA Synthesis kit (Exiqon) using equal amounts of RNA within each sample set. Real-time PCR was performed using a Viia7 instrument (Applied Biosystems), and all the data were imported into a single gene expression study and thresholded to 0.2 across all arrays to minimize plate to plate variation. Our approach to normalization was to first evaluate whether using the two endogenous normalizer control small RNAs  assayed on each qRT-PCR array led to a reduction in the standard deviation between biological replicates. If technical variation that can be normalized using endogenous controls is a significant component of the observed variation between samples, then we would expect endogenous control normalization to reduce the standard deviation between biological replicates. In the case of the HCT-116 cell experiments, endogenous control normalization did not decrease the variation in biological replicates, suggesting that technical variation was minimal compared to meaningful biological variation. Thus, for HCT-116 we have presented miRNA data not normalized to any additional RNAs. In the A549 cell experiments, we found that biological replicates showed decreased variation following snoRNA normalization, which suggested this normalization was an appropriate correction for technical variation . Therefore, we have presented the normalized data for experiments done with this cell line. Next, CFor protein quantification, HCT-116 cells were lysed in RIPA buffer 72 hours post transfection with siRNAs. Protein samples were resolved on Tris-Glycine gels (Invitrogen), and Western blots were performed using TUT1 antibody from Sigma (SAB2102610) on nitrocellulose membranes blocked with 5% non-fat milk.For NanoString quantification of 3′ additions, we performed additional analyses on data collected from previously described samples http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE26970.The NanoString data from this study are available at the NCBI Gene Expression Omnibus  qRT-PCR was used to evaluate potential compensatory changes in nucleotidyl transferase expression levels following knockdown of the TUT1 enzyme. (B) Boxplots display the changes in miRNA abundance following the suppression of each enzyme. MicroRNAs are categorized by whether they showed unchanged, increased, or decreased 3′ additions. MicroRNA abundance was significantly decreased in miRNAs with increased additions compared to unchanged additions following the suppression of PAPOLG and ZCCHC11 . (C) Western blot confirms the suppression of TUT1 following treatment of HCT-116 cells with TUT1 siRNAs from Dharmacon. Both lanes are from a single Western blot, with non-relevant lanes omitted and spaces indicating non-adjacent lanes.(TIF)Click here for additional data file.Figure S2TUT1 does not regulate themRNA expression of miRNA primary transcripts or miRNA processing enzymes. (A) The expression of 73 primary miRNA transcripts was measured with qRT-PCR following TUT1 suppression in HCT-116 cells. Scatterplots depict the delta CT values in the enzyme suppressed versus control cells. Suppression of TUT1, PAPD4, and ZCCHC11 all yielded slight increases in pri-miRNA abundance, indicating that changes in mature miRNA levels following TUT1 loss are not a result of upstream decreases in primary miRNA transcript abundance. (B) qRT-PCR was used to assess the expression of three miRNA processing components following knockdown of a panel of nucleotidyl transferases in HCT-116 cells. Although the expression levels of these genes are slightly variable following nucleotidyl transferase knockdown, suppression of TUT1 does not yield unique changes that account for the global decreases in miRNA abundance.(TIF)Click here for additional data file.Table S1Table of NanoString normalized miRNA counts and filtered miRNAs used in the analyses.(XLSX)Click here for additional data file.Table S2Table of miRNA primary transcript assays from Applied Biosystems.(XLS)Click here for additional data file.


Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts.A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way.We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data. Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids, the most widespread group of pigments found in nature. Carotenoids provide distinctive coloration to flowers and fruits, important for the visual attraction of animals for pollination and seed dispersal. Carotenoids in plants are also precursors for the synthesis of the hormone abscisic acid and other apocarotenoids with biological activity . They arIn most fruits, chromoplasts differentiate from chloroplasts at defined stages of the ripening process. Differentiated chromoplasts show high morphological diversity depending of the plant species and have been classified in different types according to their shape and suborganellar structures . The conLike the rest of isoprenoids, carotenoids are built from the 5-carbon precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plastids, IPP and DMAPP are synthesized from pyruvate and glyceraldehyde 3-phosphate (GAP) via the recently elucidated methylerythritol 4-phosphate (MEP) pathway . Carotenin vivo labeling of proteins using exogenously supplied 35S-methionine -glucose, [14C]-pyruvate or [14C]-malate. As shown in Figure 14C]-pyruvate and [14C]-malate were higher when chromoplasts were preloaded with sodium chloride and inorganic phosphate, respectively, in agreement with the involvement of known plastidial pyruvate and malate translocators -glucose, [14C]-pyruvate and [14C]-malate under the same conditions used for uptake assays and at different times samples were withdrawn and extracted with a mixture of hexane:acetone:methanol . The labeled compounds present in the organic fraction were analyzed by TLC and autoradiography. To our surprise, and as exemplified in Figure 14C]-pyruvate, only a small fraction of the radioactivity (less than 5%) was incorporated into lycopene (the major carotenoid present in tomato fruit) and β-carotene. In contrast, most of the radioactivity was found in more polar compounds, which after TLC analysis using appropriate mobile phases were identified as a mixture of neutral and polar lipids (data not shown). A detailed analysis of the labeled lipids is currently underway.To evaluate the relative contribution of the metabolites tested above as precursors for carotenoid biosynthesis, chromoplasts were incubated with [14C]-pyruvate supplemented with different concentrations of GAP. As shown in Figure The relatively high incorporation rate of the tested precursors into lipids has revealed that lipid biosynthesis is an active metabolic process in tomato fruit chromoplasts. However, the synthesis of fatty acids and complex lipids in chromoplasts is not an unprecedented observation as it was also reported in isolated daffodil petal chromoplasts . In cont14C]-mevalonic acid -pyruvate and [14C]-malate into lipids suggested that metabolic channeling to acetyl-CoA (the biosynthetic precursor of fatty acids) was a very efficient process in isolated chromoplasts. These compounds share in common that they can readily be converted into acetyl-CoA through the operation of well know metabolic steps. Whereas malate can be converted into pyruvate in a reaction catalyzed by malic enzyme, the conversion of pyruvate into acetyl-CoA can be catalyzed by the pyruvate dehydrogenase complex. The presence of these enzymes in tomato fruit chromoplasts has been predicted from proteomic data [14C]-pyruvate and [14C]-malate, and at different incubation times the [14C]-labeled metabolic intermediates were analyzed by HPLC. As shown in Figure 14C]-malate and [14C]-pyruvate were readily metabolized to [14C]-acetyl-CoA, thus confirming the presence of malic enzyme and pyruvate dehydrogenase activities in the isolated chromoplasts. The absence of [14C]-pyruvate in the incubations assays using [14C]-malate would suggest that the activity of pyruvate dehydrogenase activity is higher than that of malic enzyme in the chromoplast.The high incorporation rates of -glucose, [14C]-pyruvate and [14C]-malate have revealed that lipid biosynthesis is a very active process in tomato fruit chromoplast and that metabolic channeling of metabolic precursors to acetyl-CoA is a very efficient process. We have also shown that tomato fruit chromoplasts could bear endogenous mechanisms to produce ATP used for anabolic processes. Furthermore, the isolated chromoplast system used in this work may represent an appropriate platform to address the validation and characterization of novel functional processes predicted in tomato fruit chromoplasts from recent transcriptomic and proteomic data.We have set up a suitable system for metabolic and biochemical studies using isolated tomato fruit chromoplasts. The system is based on the development of a robust protocol for high-yield purification of intact and metabolically active chromoplasts from ripening fruits. Using targeted radiolabeled precursors we have been able to unravel novel aspects related with tomato fruit chromoplast metabolism. We have shown that a variety of metabolic precursors can be efficiently taken up by the isolated chromoplasts and metabolized through specific metabolic pathways. Labeling studies using -glucose, , -14C]-malate or [2-14C]-pyruvate, and incubated at 25°C. At the end of the specified incubation periods, chromoplast samples were loaded on top of a density gradient containing (from bottom to top) 100 μL of glycerol:methanol (2:1 [v/v]), 50 μL of silicone oil AR200 , 50 μL of buffer B containing 220 mM sorbitol and 110 mM sucrose, 50 μL of silicone oil AR200 in a long polyethylene microcentrifuge tube of 0.4 mL  and then centrifugated at 14,000 × g during 30 s. Tubes were frozen in liquid nitrogen and the lower part containing the chromoplast pellet cut and placed into a vial containing 2 mL of scintillation liquid (Ecoscint H). The incorporated radioactivity was counted in a liquid scintillation analyzer .Uptake assays were performed using the double silicone oil layer centrifugation method described by Gross et al.  and modi2 gas flow. Samples were dissolved in 100 μL of chloroform and used for counting the incorporated radioactivity or for analysis by TLC (SIL G-25 UV 254 plates). Different mobile phases were used for the TLC analysis: hexane:toluene  for highly hydrophobic compounds, like carotenoids, and chloroform:methanol:water  for less hydrophobic lipids. After drying, the TLC plates were placed in contact with Fujifilm screens for bioimaging analyzer BAS-IPMS 2325 and scanned with Bioimager (Bio-Rad). Lipids were visualized by brief exposure to iodine vapor.After incubation with the radiolabeled precursors as indicated above, chromoplast samples were extracted with 1 mL of hexane:acetone:methanol  by shacking for 15 min. Distilled water (100 μL) was then added and vigorously mixed using a vortex. After centrifugation for 3 min at 5,000 × g, the organic fraction was transferred into a new tube. The extraction process was repeated. The organic solvent used for the two extractions was mixed and dried under a NRadiolabeled metabolic intermediates were analyzed by HPLC using a Waters 600 HPLC system equipped with a Waters 2996 UV detector, a radioactivity detector and the Enpower software for data acquisition . After incubation with the radiolabeled precursors as indicated above for different time periods chromoplasts samples (100 μL) were mixed with 100 μL of distilled water and vortexed for 1 min. Lysed chromoplast samples were centrifuged for 5 min at 14,000 × g and the supernatants were used for HPLC analysis. Stock solutions (100 mg/L) of the standards were dissolved in Milli-Q water. All solutions were filtered through a 0.45 μm membrane filter, and finally stored in a refrigerator before use. According to the experimental requirements, citric acid, oxaloacetic acid, succinic acid, fumaric acid, malic acid, acetic acid and pyruvic acid stock solutions were mixed in volume ratio of 1:10:5:10:5:15:10, and then diluted to required concentrations to prepare a serial mix of standard solutions. The mobile phase consisted on 0.27 mL of sulfuric acid diluted to 1 L with Milli-Q water, then filtrated through a 0.45 μm membrane filter and degassed before use. Metabolites profiling was performed using the ionic exchange Aminex HPX-87H column  using 0.005 mol/L sulfuric acid as mobile phase at a flow rate of 0.5 mL/min. The column was maintained at 50°C. Injection volume was 10 μL for all runs. Target compounds were detected using a UV detector set at 210 nm and a radioactivity detector.HPLC: High-Performance Liquid Chromatography; TLC: Thin Layer Chromatography; MEP pathway: 2-C-methyl-D-erythritol 4-phosphate pathway; MVA pathway: mevalonate pathway; GAP: glyceraldehyde 3-phosphate; IPP: Isopentenyl diphosphate; DMAPP: dimethylallyl diphoshate.The authors declare that they have no competing interests.DMA and CRS performed the uptake assays and the metabolic analyses. RP and FHG participated in setting up the method for chromoplasts purification. IP and MB supervised experiments. AB supervised the experiments and prepared the manuscript. All authors read and approved the final manuscript.