Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development.Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish ( Motor neuron disorders (MNDs) are characterized by progressive loss of motor neurons of the spinal cord (‘lower motor neurons’) or motor neurons of the brain (‘upper motor neurons’), or both, leading to atrophy and/or spasticity of the associated musculature. Spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP) are the most common MNDs, and we summarise their aetiologies and genetics. We then present a critical overview of the methods available to model genetic mutations in zebrafish as well as their limitations. In the main body of the article, we highlight zebrafish models for each of these MNDs and discuss recent advances in developing drug screens, with a special focus on ALS.SMA is a neurodegenerative disease presented clinically by progressive degeneration of lower motor neurons in the anterior horn of the spinal cord, resulting in hypotonia, muscle atrophy, paralysis and, in severe cases, death . It is aALS is a late-onset neurodegenerative disorder that affects both upper and lower motor neurons and is the most common MND. Although the majority of ALS cases are sporadic, up to 10% are inherited, with mutations in over two-dozen genes accounting for the majority of familial causes . The recHSP is the collective term for a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by progressive spasticity and weakness in the lower limbs due to loss of upper motor neurons . The cliAlthough MNDs have quite different aetiologies, they are all typified by progressive paralysis resulting in severe disability. The pathogenesis of MNDs remains to be fully understood and there is a crucial need for the development of novel therapeutic strategies for their treatment. A thorough understanding of disease pathophysiology is necessary to direct rationale design of therapeutic strategies. As mentioned above, MNDs are characterized by the degeneration of upper and/or lower motor neurons and this is due to a dysfunction of several different cellular processes. Despite the apparent heterogeneity in these MNDs , several pathogenic pathways are common among at least two of these three diseases  stress as well as mitochondrial dysfunction have been reported as important pathological mechanisms in MNDs. For example, the inclusions found in ALS are thought to be associated with misfolding of mutant proteins and an inability of the unfolded protein response to dispose of them . MoreoveDanio rerio) has received favourable attention from clinicians owing to its advantages as a model system for the study of disease, because the zebrafish models often closely resemble the relevant human disease  rescue by the homologous human mRNA. Furthermore, the technique is not appropriate for studying late-onset-disease-related phenotypes such as in many MNDs. Nonetheless, transient knockdowns can be useful for drug testing pertinent phenotypes at earlier embryonic and larval stages, and their advantage for high-throughput screening lies in this area . RecentlTol2 transposon system , or rescue of gene-knockdown or -knockout phenotypes (to test for loss of function). As with morpholinos, the transient expression of injected mRNA is not useful for late-onset phenotypes but can be quite useful for drug screening in embryos and larvae. Stable transgenesis for the study of gain-of-function mutations is a popular approach in zebrafish using the efficient n system . Howeverpatterns . Severalpatterns . New andloxP  gene  gene . Clinicatein SMN . The sevy number , with noSMN is a ubiquitously expressed nuclear and cytoplasmic protein. It is part of a multiprotein complex localised to the nuclear Gem (for Gemini of coiled bodies) bodies and is necessary for the assembly of small nuclear ribonucleoproteins (snRNPs), a central element of the spliceosome. These Gem bodies are lost in SMA, thus indicating altered splicing as a possible cause for the disease . Additiosmn to levels similar to those observed in humans with SMA results in truncated and under-branched ventral root axons and impairments in pathfinding to target musculature  also confirmed that the human SMN2 gene is spliced in zebrafish and that increasing expression from this transgene can increase survival  causes frontotemporal lobar degeneration, rescued the shortened motor neuron phenotype upon ebrafish . Plastinebrafish , rescuedebrafish . As mentebrafish  and rescebrafish  were obsurexin 2  and withurexin 2 .Drosophila and mouse models of SMA, dysregulation of ubiquitylation pathways caused β-catenin accumulation, and pharmacological inhibition of β-catenin by quercetin robustly ameliorated neuromuscular pathology  was the first gene linked to ALS . Mutations in SOD1 are responsible for ~20% of fALS and ~1–7% of sALS  gene encodes the protein TDP-43, which was first linked to ALS as the major component of the cytoplasmic, ubiquitin-positive inclusions found in the majority of ALS cases, i.e. without mutations  were identified in individuals with ALS  was identified in a large percentage of fALS cases (~40%), and was also observed in ~7% of cases of sALS  GGGGCC hexanucleotide repeat expansion in the non-coding first intron of chromosome 9 open reading frame 72  (sod1 (G93R and G85R) heat-shock reporter lines   have beeer lines . These mG85RSOD1 mice have revealed hyperexcitability in motor neuron populations in the spinal cord  onto motor neurons occur at a lower frequency than in motor neurons of wild type and zebrafish expressing human wild-type SOD1  presymptomatic nal cord . An advaype SOD1 . Althougype SOD1 .SOD1, models for the more recently (since 2008) identified ALS genes TARDBP, FUS and C9ORF72 have also been developed in the zebrafish. For these genes, the role of loss of function, toxic gain of function or perhaps their combination is still under evaluation in the field. Consequently, models for both genotypes have been developed, although mostly by transient approaches because knockouts and stable transgenic lines have taken longer to develop. Mutations in the TARDBP gene were identified in individuals with both familial and sporadic forms of ALS , but importantly not wild-type TARDBP at the same level of expression (which is a problem in other models) , reduced quantal transmission, orphaned presynaptic and postsynaptic structures at the NMJ, and motor neuron death following exposure to the glutamatergic agonist N-methyl-D-aspartate (NMDA)  see , resulteant mRNA . Given tant mRNA . In a foe (NMDA) .TARDBP utilized transient genetic models of zebrafish orthologues. Recent efforts to generate tardbp knockout lines in zebrafish have highlighted an unexpected subtlety associated with the use of this model organism. Unlike mammals, zebrafish possess a paralogue of tardbp [Tar DNA binding protein-like (tardbpl)], which lacks the C-terminal glycine-rich region, presumably owing to a chromosome duplication event deep in its ancestral evolution (tardbpl has been shown to recover loss of tardbp following gene disruption of the latter with ZFNs (tardbp was isolated by TILLING (tardbp and tardbpl (the latter either by gene disruption by ZFNs or by knockdown) resulted in a severe phenotype and early lethality, although the phenotype of double heterozygotes has not been reported and might more closely resemble the ALS genotype (if due to haploinsufficiency) and perhaps the  knockdown phenotype. This strong tardbp/tardbpl phenotype is highly reminiscent of Drosophila knockout models of the related tbph gene, which display early lethality in the second instar stage, and Tardbp knockout mice, which fail to implant during embryogenesis  resulted in disrupted nuclear import  and in particular we observed that several neuroleptics protect against development of the motor phenotype. The FDA-approved drug that emerged from this screen offers an exciting starting point for further ALS research because one can work back to identify the target(s) of positive hits and investigate their mode of action to better understand the mechanism of disease pathogenesis. In addition to ‘repurposing’ drugs identified as hits, it is possible to screen novel structural analogues to develop more selective therapeutics. Thus, eventually, it should be possible to identify better drug targets and develop more potent disease modifiers.We have capitalized on the strength of our SPG4, encoding spastin, a ubiquitously expressed, mainly cytosolic protein that has microtubule-severing properties (SPG3A (atlastin-1) account for ~10% of autosomal dominant HSP and are the second most common cause of HSP as well as the most common form of early-onset HSP; this form is usually clinically described as uncomplicated (SPG11 (spatacsin) mutations occur for ~21% of autosomal recessive cases of HSP, and all except one of the 100 mutations identified in this gene to date lead to truncation of the protein, indicating a possible loss of function  have also been reported. They account for ~4% of HSP and are inherited in an autosomal dominant manner , SPG39 (PNPLA6), SPG53 (VPS37A), SPG46 (GBA2) and SPG43 (C19ORF12)  . A recen19ORF12) . The HSP19ORF12) .SPG4) caused dramatic defects in spinal motor axon outgrowth, abnormal motility and perturbation of axonal microtubule dynamics (SPG3A) severely impaired spinal motor axon architecture and fish motility (spastin and atlastin are (major) causes of HSP, rather than loss of function, caution is necessary when interpreting these knockdown models in zebrafish. Motor axons in fish injected with strumpellin (SPG8) morpholino were also shorter and had abnormal branching and impaired motility, which were rescued upon co-injection of wild-type human SPG8 mRNA but not mRNA with HSP-related variants, validating the latter as loss-of-function mutations (spatacsin (SPG11) or spastizin (SPG15) severely compromised branching of spinal cord motor neurons at the NMJ and caused locomotor impairment (slc33A1 [encoding Solute carrier family 33 (acetyl-CoA transporter), member 1; SPG42) in zebrafish embryos also caused a tail curvature phenotype and abnormal axon outgrowth from the spinal cord  led to abnormal locomotion and outgrowth of spinal motor axons (pnpla6 (SPG39) knockdown resulted in developmental abnormalities and motor neuron defects, including axon truncation and branching  knockdown zebrafish larvae , although all are studies of transient knockdown because no genetic models of HSP have yet been generated. Most HSP genes cause disease in an autosomal recessive fashion, thus implying loss-of-function and dominant-negative molecular mechanisms, which can be readily modelled at a genetic level in zebrafish. However, because zebrafish lack upper motor neurons, the phenotypes reflect at best a general effect rather than one of a specific nature. A number of reports have convincingly shown that the loss of function of HSP-associated genes lead to major motor neuron defects, usually with impaired locomotion, consistent with the roles of spastin and other less common HSP genes that are involved in axonal transport . For insdynamics . Loss ofmotility . Howeverutations . Knockdopairment . Knockdonal cord . Knockdoor axons . pnpla6 ranching . A loss h larvae . In a nuAlsin (ALS2) is a gene that is frequently mutated in juvenile HSP and ALS through an autosomal recessive mode of action, which leads, in most cases, to protein truncation. Similarly to spastin and strumpellin models, knockdown of alsin causes shortening of motor axons and a loss of neurons in the spinal cord  had morpholino knockdown phenotypes in zebrafish  and usp8 , yielded knockdown defects in touch-induced and spontaneous motor behaviour as well as motor axon morphology that are relevant to HSP, establishing the power of this combination of human genomics and zebrafish knockdowns for the identification and validation of disease-related variants, respectively. It is worth noting, however, that genetic knockout has not yet been reported for any HSP gene and will be necessary to help advance studies of this disorder using zebrafish models.Finally, a recent exome sequencing study identified 18 new putative HSP genes, four of which  (dystrophin-null)  as a potential lead compound for Dravet syndrome because it effectively inhibited spontaneous convulsive behaviours and electrographic seizures in zebrafish  mutants . In anotPROTO-2)  and an FPROTO-2)  that proin-null) . Interesin-null) . BecauseGal4/UAS approach (Epha4 as a gene that, when knocked down, could rescue the phenotype caused by expression of mutant SOD1 or TARDBP. Upon co-knockdown of SMN1, Epha4 also increased survival of mouse and rat models of ALS (EPHA4 expression in individuals with ALS inversely correlated with disease onset and survival, and loss-of-function mutations were associated with long survival. These results, starting with zebrafish, then validated in mammalian models and ultimately correlated with patient aetiology, nicely indicate the power of the zebrafish model for providing translational insights into MND.To date, over a hundred papers have shown that various pathological aspects of MNDs caused by expression of mutated genes can be modelled in the zebrafish. This model is now poised at the beginning of a new age of genome manipulation and the new editing approaches will undoubtedly improve the ability to generate more and better MND models in zebrafish. These models can then be used to study the molecular basis of disease and they will certainly provide novel and useful insights into MNDs. The ability to simultaneously target multiple genes and in different combinations in a single embryo, such as using combinations of morpholinos and mRNAs  and by iapproach , represes of ALS . Furtherin vivo in a vertebrate and design high-throughput screens, enabling novel hypotheses to be subsequently tested in mammalian models or in clinical studies, is a major strength of the zebrafish model. Although, to date, there is not a single ‘positive hit’ published from a zebrafish-based drug screen for neurological disorders that has entered a Phase 1 clinical trial, the protective compounds discovered in the recent studies described above are very promising leads for future investigation. The prospect of using zebrafish for drug discovery opens new avenues for discovering treatments for MNDs.The opportunity to apply discovery-driven approaches


Using a strict subject selection procedure, we tested in High and Low Hypnotizable subjects (HHs and LHs) whether treatments of hypoalgesia and hyperalgesia, as compared to a relaxation-control, differentially affected subjective pain ratings and somatosensory event-related potentials (SERPs) during painful electric stimulation. Treatments were administered in waking and hypnosis conditions. LHs showed little differentiation in pain and distress ratings between hypoalgesia and hyperalgesia treatments, whereas HHs showed a greater spread in the instructed direction. HHs had larger prefrontal N140 and P200 waves of the SERPs during hypnotic hyperalgesia as compared to relaxation-control treatment. Importantly, HHs showed significant smaller frontocentral N140 and frontotemporal P200 waves during hypnotic hypoalgesia. LHs did not show significant differences for these SERP waves among treatments in both waking and hypnosis conditions. Source localization (sLORETA) method revealed significant activations of the bilateral primary somatosensory (BA3), middle frontal gyrus (BA6) and anterior cingulate cortices (BA24). Activity of these contralateral regions significantly correlated with subjective numerical pain scores for control treatment in waking condition. Moreover, multivariate regression analyses distinguished the contralateral BA3 as the only region reflecting a stable pattern of pain coding changes across all treatments in waking and hypnosis conditions. More direct testing showed that hypnosis reduced the strength of the association of pain modulation and brain activity changes at BA3. sLORETA in HHs revealed, for the N140 wave, that during hypnotic hyperalgesia, there was an increased activity within medial, supramarginal and superior frontal gyri, and cingulated gyrus (BA32), while for the P200 wave, activity was increased in the superior (BA22), middle (BA37), inferior temporal (BA19) gyri and superior parietal lobule (BA7). Hypnotic hypoalgesia in HHs, for N140 wave, showed reduced activity within medial and superior frontal gyri , paraippocampal gyrus (BA34), and postcentral gyrus (BA1), while for the P200, activity was reduced within middle and superior frontal gyri (BA9 and BA10), anterior cingulate (BA33), cuneus (BA19) and sub-lobar insula (BA13). These findings demonstrate that hypnotic suggestions can exert a top-down modulatory effect on attention/preconscious brain processes involved in pain perception. Human pain is a multi-faceted protective experience involving the activity of sensory-discriminative, affective-emotional, attention-cognitive and behavioral systems –6. MelzaUsing a variety of stimulation methods, painful events have been shown to produce an early contralateral response in the primary somatosensory cortex (S1) reflecting the sensory-discriminative aspect of pain –29, follIn the EEG based search for the acknowledged multiple brain generators involved in pain perception, the superiority of standardized low-resolution brain electromagnetic tomography (sLORETA) ,60 over A secondary aim of the present study was to identify the major cortical regions sensitive to individual differences in hypnotizability and hypoalgesia and hyperalgesia treatments by using sLORETA source localization ,60 of N1Participant upon arrival to the EEG lab were first informed about the use of hypnosis, and all of them were informed that, during EEG recording, they would receive painful electric stimulations in waking and hypnosis condition. All of them gave their written informed consent for participation.The research was conducted according to the ethical standards of the American Psychological Association (APA), and according to the principles expressed in the Declaration of Helsinki. The study was approved by the Ethics Committee of the Department of Psychology, La Sapienza University of Rome, Italy (2008).From a group of 79 right-handed women  naïve volunteers, 10 high hypnotizable and 10 low hypnotizable subjects were recruited through university courses by advertisements. Handedness was measured by the Italian version of the Edinburgh Inventory Questionnaire . SubjectAll participants went through three experimental sessions, 1 week apart. These sessions were identical except that EEG was recorded only in the third session and is the basis of this present report. The purpose of the first session was to administer the HGSHS:A to naïve participants in groups of 15–20, and to make participants familiar with the experimental procedures. In the second session the SHSS:C was administered singularly to each participant. Selected subjects for hypnotic susceptibility (10 HHs and 10 LHs) participated in the third session wherein painful stimuli were delivered. Stimuli were unipolar electric shocks (2 ms) applied to the palmar surfaces of the distal and medial phalanges of the middle finger of the right hand. A constant current stimulator  served to generate these stimuli. Two silver-silver-chloride cup electrodes (1 cm in diameter) were filled with an electro-conductive hypo allergic cream and impedance was kept below 30 kΩ. For each participant we determined first sensory threshold and then pain threshold just before of EEG recordings. Throughout the experiment, electric pulses were applied with a frequency of 1.150 Hz and duration of 2 ms. Pain thresholds were determined for the right middle finger using the method of limits. This stimulation approach was derived from a previous study reported by Ray and collaborators , since o"… Although you will continue to experience normal sensation, your experience will seem more unpleasant,. as if your right middle finger is hold in a vise that is enhancing the grip,. more painful and uncomfortable,. now the vise is enhancing the grip at its maximum level. It is more disturbing than you might have expected…". For hypoalgesia treatment, the subject was suggested to produce an obstructive hallucination of stimulus perception on the right middle finger: "… When you feel the unpleasant stimulus on your right middle finger, you may experience how much less intense the sensation is than you might have expected it to be, it can be as if your hand is covered by a plastic glove which makes the unpleasant stimulation more attenuated,…you can experience that all sensations in the finger will be attenuated…. For control treatment, subject was suggested to feel feet, legs, arms, and hands deeply relaxed, the body more tired, sleepy, heavy, the breathing freely and deeply: "… You are relaxed. relaxed. deeply relaxed. When you think to be relaxed, your muscles will relax. You are going to reach a state of deeper, whole relaxation… May be you are feeling that a pleasant warm tingle is spreading throughout your body and you are getting more and more tired and sleepy. sleepy… drowsy… and sleepy. You feel numb or heavy in your legs and feet,… in your hands, arms and shoulder. All your body is getting heavy,. heavy, and you are so heavy that you feel sinking into the chair… Your breathing is slow and regular,. slow and regular. You can feel your breath flowing slowly and deeply,. slowly and deeply". Hyperalgesia, hypoalgesia, and control tasks were performed with eyes-closed in both waking and hypnosis conditions. Following each suggestion, 120 pain stimuli were presented over a 132 sec period resulting in 120 trials of EEG data. After each recording session, subjective pain experiences were rated. At the end of pain treatments, the subject was waked up from hypnosis. Both waking and hypnosis conditions and the order of treatments within each condition were counterbalanced across subjects in order to avoid possible order effects or habituation. Between waking and hypnosis conditions, a resting period of 10 min was given. Each treatment condition lasted about 3 min (.8 min for suggestion instruction and 2.2 min of painful stimulation). At the end of each treatment, subjects were asked to rate both pain intensity and distress intensity, respectively as measures of the sensory-discriminative and affective-motivational components of pain, by using two separate 10 point numerical rating scales (NRS)  using Neuroscan Acquire setting. The ground electrode was located 10 mm anterior to Fz. The EEG was later processed by using Brain Vision Analyzer system (Brain Product). During a pre-processing stage, each signal was first applied a digital 50 Hz notch filter and then a bandpass filter (.5–70 Hz). The EEG was then reconstructed into discrete, single-trial epochs. For each stimulus, an EEG epoch length of 900-ms was used with a 150-ms pre-stimulus baseline and a 750-ms time window following the onset of the electric pulse. Epochs were rejected from averaging if amplitude exceeded ±75 μV, and eye blinks were corrected using Gratton’s et al.’s procedure . AdditioIn line with previous reports –49, two A simple ANOVA with hypnotizability, serving as between group variables, was used to assess individual differences on subjective threshold measures.To test the impact of hypnotizability on responses to hypoalgesia and hyperalgesia suggestions during waking and hypnosis condition, pain and distress rating measures were submitted to analysis of variance  with Hypnotizability (HHs vs LHs) as between subjects factor and Condition  and Suggestion  as within subjects factor.For each N140 and P200 peak amplitude measures, a repeated measures ANOVAs was carried out according to the following design: 2 Condition  x 3 Treatment  x 3 Head Level  x 5 Location . Contrasts F values were also obtained when necessary. To prevent the risk of falsely significant results, as may happen using repeated measures ANOVAs if the sphericity assumption has been violated , the Huyhttp://www.uzh.ch/keyinst/NewLORETA/LORETA01.htm). This was done since sLORETA software has been found useful for the analysis of different time segments of ERPs [3 rather than 7 mm3 offered by the previous LORETA version, and localization inference is based on standardized values of the current density estimates [SERP responses were exported for further analysis using sLORETA software provided by the KEY Institute for Brain-Mind Research . At the cluster level, all voxels showing a statistically significant difference were detected. For descriptive issues, the Talairach coordinates (TCs) of local maxima for the statistical comparison have been reported in the text. It is important to note that this localization is not a complete listing of all significantly different cortical areas, but a listing of the local maxima of these differences. Electrophysiological data analyses have been banked in our lab archive and are available upon request.For source reconstruction, the subtractions of SERP traces between painful stimuli of hyperalgesia, hypoalgesia, and control treatments, as well as between high hypnotizable and low hypnotizable subjects, were assessed using sLORETA within time intervals of 130–160 ms, 200–250 ms respectively for the N140, and P200 waves. sLORETA enables the computation of statistical maps from EEG data that indicate the locations of the underlying source processes with low error . Statist2), differences in activity between each treatment of noxious stimulation and a resting-baseline stimulation-free, in waking and hypnosis conditions, were statistically assessed using nonparametric permutation test with 5000 randomizations accounting for multiple voxel-by-voxel comparisons implemented in sLORETA software. Maps were statistically analyzed using the sLORETA paired Student t test (threshold p<.05). All voxels showing a statistically significant amplitude increase in response to noxious stimuli, compared with resting baseline, were detected. Clusters of significant activation were defined as ROIs and included the middle frontal gyrus (BA6), postcentral gyrus (BA3), and anterior cingulate (BA24) within a time window of 190–270 ms. For further activation magnitude analysis, each ROI was characterized by a single-voxel spatial local maximum. To accept a spatial local maximum, the thresholds of the t values in the particular voxel had to be greater compared with all surrounding voxels. All voxel locations are given according to the Talairach coordinate. For each treatment and condition, current density waveforms of each ROI were obtained at 512 Hz in a time window of 450 ms from stimulus onset, yielding high-resolution temporal curves. For further activation magnitude analysis, each ROI was characterized by a single-voxel local spatial maximum. Subsequently, areas under the curves (AUCs) in a neighborhood of the maximum were calculated using a 25 ms time window and then correlated with subjective pain and distress ratings obtained during each treatment in both waking and hypnosis conditions. Current density scores of ROIs showing significant Bonferroni's corrected correlations with subjective numerical pain and distress scores were used in multiple regression analyses as predictors of pain/distress scores. These analyses were mainly performed to highlight brain responses coding for pain/distress and also served to evaluate if the brain pattern of pain/distress coding may change depending on the experimental treatment and condition. We first entered as predictors in the regression model (Step-1) the BAs density scores that were significantly correlated with pain (or distress) scores (Based on estimated electric current density (μA/mm) scores . SubsequTo examine, in a more direct way, how pain modulation relates to brain activity, we performed separate multivariate regression analyses for each treatment and condition including difference scores of current density as predictors and pain rating difference scores as a dependent variable. Difference scores were calculated by subtracting scores obtained for hypoalgesia from those for control treatment , scores obtained for control from those for hyperalgesia (Hyperlgesia vs Control) and, to optimize leverage in the analysis , scores obtained for hypoalgesia were subtracted from those obtained for hyperalgesia treatment . In addition, hypnotizability was entered in the model as a potential mediator of the relation between pain modulation and brain activity.Since recent findings indicate that highly hypnotizable individuals are distributed across two classes of response patterns, one suggesting an inward attention subtype and the other a dissociative subtype , there iAs indicated by the statistics in The ANOVA on distress rating scores showed a main effect for Condition and for Treatment . The firAs shown by the statistics in The ANOVA on P200 amplitudes disclosed two significant main effects, one for Head Level and the other for Location . The firSource estimation analysis revealed significant activation of BA3, BA6, and BA24  of perceived pain, while this relation was found to be more robust during hyperalgesia treatment wherein the inclusion of hypnotizability level was highly significant in this relation. Regression analyses using distress rating as a dependent variable were carried out for the hypnotic hypoalgesia alone, since we failed to find significant correlations for control and hyperalgesia treatments in hypnosis . This anTo test in a more direct way the relation between pain modulation and cortical activity, we performed further multivariate regression analyses for each treatment and condition using difference scores of current density as predictors of numerical pain difference scores see  section.A general finding of these regression analyses performed for a waking condition was that, for each treatment, the individual change in current density at BA3 contralateral to the stimulation side, was a stable predictor of the change in pain rating, suggesting a stable pattern of pain coding changes in the brain across all contrasted treatments in waking condition  obtained for each treatments and t-test maps for HHs are reported in sLORETA source localizations of P200 wave (210–230 ms) obtained for each treatments and t-test maps for HHs are reported in In this paper we describe a SERP study examining the effects of hypnotic susceptibility and hypnotic suggestions on electro-cortical responses and on sensory-discriminative (pain rating) and affective-motivational (distress rating) components of pain induced by noxious electric stimulation. These measures were obtained in waking and hypnosis condition under a relaxation-control and suggestions to either increase  or decrease pain sensation . In this context, we did an attempt to validate previous pain-ERP findings to noxious electric stimulation during hypnosis . We askeWe found little differentiation between pain and distress scores across experimental treatments, indicating that suggestions for pain control influenced in a similar fashion both perceptual and affective expressions of pain. LH group showed little differentiation between hypoalgesia and hyperalgesia treatments in waking and hypnosis conditions, with the exception that they disclosed significant pain and distress reduction during hypoalgesia and hyperalgesia compared to control treatment in waking condition. In contrast, HHs showed more pronounced rating changes in the instructed direction, an effect that was amplified during hypnosis . We thinScalp distribution of N140 amplitude showed a pronounced lateralization to the left hemisphere, contralateral to the side of noxious stimulation . This fiResearch has demonstrated that both N140 and P200 waves are enhanced in amplitude to stimuli coming by the attended hand and reduced to stimuli coming from the unattended hand , 97, 99,In addition, since the P2 wave is believed to reflect the painfulness of the stimuli ,94, we tHowever, it must be stressed that the obtained findings are limited by the fact that ERPs of the present study were elicited using transcutaneous electric stimulation. Even if the stimuli produced a percept qualified as painful, it can be expected that the elicited ERPs were mainly related to the activation of non-nociceptive A-β afferents and the lemniscal pathway rather than to the activation of A-δ and C fibers . This crIn line with general models of selective attention  and theisLORETA source analysis of ERP response to noxious electric stimulation revealed clusters of significant activation as ROIs, including the BA3, BA6, and BA24 cortical regions in the left hemisphere contralateral to the stimulated side. These clusters of significant activations were temporally localized within a time window of 190–270 ms, clearly within the latency variability range of the P200 wave. In terms of ROIs, these findings parallel previous reports of the most commonly activated cortical pain-related regions reported in imaging studies, namely the primary somatosensory cortex, the frontal lobe, and anterior cingulate cortex –114. IntOur multiple regression findings, testing the association between subjective pain/distress ratings and activity in the ROIs, disclosed that current density measure at BA3, contralateral to the stimulated side, was the only brain response yielding a stable coding for the pain felt across treatments in both waking and hypnosis conditions Tables . These fTwo contrasting conceptual positions have emerged in the literature of pain research. One position stated that functional imaging data may contain a genuine and objective signature of the painful experience , 124 so Our sLoreta analysis of N140 wave showed that, during hypnosis, hypoalgesia, compared to a control treatment, had significantly reduced activity in both hemispheres within postcentral gyrus (BA1), medial and superior frontal gyri , and paraippocampal gyrus , anterior cingulate (BA32), middle temporal and supramarginal gyrus . These findings are also in line with PM notion suggesting these cortical regions as part of the conceptualized second-order and third order pain matrix network .In terms of the P200 wave we found during hypnosis that in HH participants the hypoalgesia treatment produced a reduced activity at middle (BA9) and superior frontal (BA10) gyri, anterior cingulate (BA33), cuneus (BA19) and sub-lobar insula (BA13). In contrast, hyperalgesia treatment produced an increased activity at superior temporal , medium temporal (BA37), inferior temporal/middle occipital gyri (BA19) and at superior parietal lobule/precuneus BA7; . These oIn the whole, the above mentioned findings suggest that reduced and enhanced activity in these higher-order contextual matrices may influence nociception via top-down projections by changing the sensory gain at the source, which is, in cortical receiving areas , thalamuOur most striking source activation findings fit well with the suggested cortical regions outlined in the re-conceptualization of pain matrix  and suggThe present findings describe hypnotic modulation of brain activation patterns associated with nociceptive processing. Correlation analyses distinguished BA3 activity, contralateral to the stimulation side, as the only one reflecting a stable pattern of pain coding changes across treatments in waking and hypnosis conditions. A more direct regression analysis testing also showed that hypnosis reduced the strength of the association of pain modulation and brain activity changes at BA3. The study convincingly demonstrates that hypnotic hypoalgesia is associated with reduced activity of the N140 and P200 SERP components, whereas hypnotic hyperalgesia is associated with increased activity of these components. Our source findings are among the first to clearly distinguish separate regions of the first, second, and third order pain matrices  as sensi


Mechanistic data have revealed a key role for selenium (Se) and selenoproteins in biological pathways known to be altered in multifactorial diseases, such as cellular maintenance, response to oxidative stress and correct protein folding. Although epidemiological studies indicate that low Se intake is linked to increased risk for various chronic diseases, supplementation trials have given confusing outcomes, suggesting that additional genetic factors could affect the relationship between Se and health. Genetic data support this hypothesis, as risk for several chronic diseases, in particular cancer, was linked to a number of single nucleotide polymorphisms (SNP) altering Se metabolism, selenoprotein synthesis or activity. Interactions between SNPs in selenoprotein genes, SNPs in related molecular pathways and biomarkers of Se status were found to further modulate the genetic risk carried by the SNPs. Taken together, nutritional genomics approaches uncovered the potential implication of some selenoproteins as well as the influence of complex interactions between genetic variants and Se status in the aetiology of several chronic diseases. This review discusses the results from these genetic associations in the context of selenoprotein functions and epidemiological investigations and emphasises the need to assess in future studies the combined contribution of Se status, environmental stress, and multiple or individual SNPs to disease risk. The applications of these technologies to nutrition research led to the development of nutritional genomics which studies both the effect of an individual’s genetic makeup on the response to nutrients (nutrigenetics) and the modulation of gene expression by nutritional compounds (nutrigenomics). It is now recognized that the study of reciprocal interactions between genes and nutrients holds the key to our understanding of mechanisms underlying diseases, to the discovery of novel biomarkers of disease risk and to the development of novel food policies and therapeutic approaches adapted to the individual’s needs. As a result, there is a possibility for A nutrient that particularly benefited from the development of nutritional genomics approaches to understand its relationship with human health and disease is the micronutrient Selenium (Se). There are three main reasons for this: (i) the risk-benefit window for Se intake is very narrow, leading to confusing results from epidemiological and intervention studies and highlighting the importance of inter-individual variations in response to Se intake; (ii) the geographical distribution of Se in the soil varies greatly with regions poor in Se such as Europe, New Zealand and some parts of China ,2,3,4 anThis review discusses the relationship between diseases and genetic variations in selenoprotein genes with an emphasis on the function of selenoproteins and factors that governs Se homeostasis.The identification of genetic associations between single nucleotide polymorphisms (SNPs) in selenoproteins and Se-related genes has shed some light on conflictual results from epidemiological studies investigating the relationship between of Se and disease risk, and on the understanding of the role of selenoproteins in tissue function. In particular, functional studies have revealed that SNPs in Se-related genes have the potential so affect (a) Se homeostasis and selenoprotein synthesis, (b) antioxidant defenses and redox control, and (c) endoplasmic reticulum (ER) signaling and degradation of misfolded proteins .The table summarizes the protein name, function, subcellular and histological localization, the gene symbol and chromosomal location. Proteins are grouped according to their main function.(SEPSECS) and selenophosphate synthetase 1 (SEPHS1)) and Se transport and delivery (SEPP1) ; P = 0.04, Pinteraction = 0.03) or high-grade PCA  per 10 μg/L increase in serum Se concentration [Additional evidence coming from genetic association studies support an interaction between Se status, genetic variants in selenoprotein genes and risks of PCA or aggressive PCA . In partulations ,61. In t = 0.05)  and for  = 0.05) . Moreoventration  and genontration .SEP15  significantly associated with PCA-related mortality. In addition, an inverse relationship between high levels of Se and PCA mortality was observed among men who did not have the increased risk genotype for rs561104, revealing the PCA mortality was modulated by the interaction between rs561104 and Se [SEPP1 locus, the authors ssubsequently identified two genetic variations in the SEPP1 gene that influence prostate cancer incidence [SEPP1) and plasma Se levels was observed, with a reduced risk of PCA for individuals with high Se status carrying the minor C allele but not for TT men. On the other hand, the T allele was associated with an increased mortality in smokers or obese individuals [TXNRD1  and GPX4 (rs2074452) were shown to be modestly associated with risk of mortality by prostate cancer [In a nested case-control study within the Physicians Health Study in the US, Penney and collaborators identified that three variants in 4 and Se . Using tncidence . In thisividuals . Supporte cancer .SELK (rs9880056), TXNRD1 (rs7310505) and TXNRD2 (rs9605030 and rs9605031) genes [TXNRD1-2 and SEP15 and prostate cancer incidence was recently supported by evidence that an association between genetic variants in these genes and risk of high-grade prostate cancer and prostate cancer recurrence [SEPP1) and rs17650792 and rs1800668 (GPX1) modulate risk for advanced PCA stages [Finally, in a case-control study nested within the EPIC-Heidelberg population, a pathway-wide analysis, using 384 tagSNPs, was developed to simultaneously genotype all SNPs in 72 genes. This alternative approach revealed that risk for high-grade or advanced stage of PCA was significantly modified by interactions between plasma markers of Se status  with SNPs in 1) genes . The lincurrence . The infA stages .Colorectal cancer (CRC) is the second most frequent cause of death by cancer in Europe and the third most common cancer worldwide . SeveralSEPP1 and TXNRD1 genes but not with SNPS in GPX1, GPX2, GPX3, and GPX4 [In addition, genetic association studies carried out in different populations have provided evidence that the effect of Se status on CRC or colorectal adenoma risks could be modulated by genetic variations in selenoprotein genes . In a caand GPX4 .GPX4 gene, a SNP affecting Sec incorporation and selenoprotein hierarchy [SEPP1, SOD2 and TXNRD2 genes were found to further modified the disease risk in the Czech population [GPX4 knock-down in Caco-2 gut epithelial cells results in the disruption of mitochondrial function [SEPP1) and rs713041 suggests that the reduced binding affinity for the selenoprotein synthesis machinery carried by the T allele for rs713041 could be amplified in carriers of a variant in SEPP1 associated with reduced Se bioavailability [GPX4 expression will be decreased and the availability of the synthesis machinery for the translation of selenoproteins lower in the hierarchy will be increased [CRC risk was shown to be modulated by genotype for rs713041 in the ierarchy ,17. In aierarchy .On the cierarchy  and no aierarchy . No datapulation , suggestpulation  and sincfunction , this sulability ,12,16. ASELS and rs7579 inducing a G/A change in the 3′UTR of the SEPP1 mRNA [SEP15) induces a base change in the SECIS element of SEP15 mRNA resulting in an alteration Sec insertion, via a mechanism comparable to rs713041 in GPX4. Sep15 like SelS is involved in in the control of protein folding in the ER and genetic interactions were identified between rs7579 and rs3877899 in the SEPP1 gene and rs5859 in relation to CRC risk [GPX4, these interactions could result in an altered SEP15 expression in condition of low Se bioavailability, and consequently could impact on protein folding regulation [In addition to rs713041, two other SNPs contributed to CRC risk in the Czech population: rs34713741 in the promoter region of PP1 mRNA . The incPP1 mRNA  was replPP1 mRNA  and gastPP1 mRNA . SelS isSEPP1 promoter in CRC cases [SEPP1 promoter region (TC)5/(TC)3 repeats and colon cancer risk [TNXRD2, Selenoprotein N, and Selenoprotein X genes were associated with survival after diagnosis with rectal cancer whereas SNPs in Selenoprotein N and Selenoprotein X were linked to colon cancer mortality and SNPS in TNXRD1-2 were shown to interact with both aspirin and cigarette smoking to alter colon and rectal cancer risk [The role of SePP in CRC carcinogenesis was further highlighted by the observation of a reduced expression of the protein in tumour samples compared with normal mucosa and the genomic instability of a polymorphic (TC)5/(TC)3 repeats in the RC cases ,86 and tcer risk . Moreovecer risk .SEPP1, affecting Se bioavailability, and SNPs in SEP15 and GPX4, affecting Sec insertion, underline the importance of the selenoprotein hierarchy in this tissue and the need for adequate Se supply to support selenoprotein synthesis to maintain a healthy colorectum.These studies emphasise the role of selenoproteins SePP, GPx4, TXNRD2, SelS and SeP15 in CRC and stress the importance of redox control and control of protein folding in colorectal function. Moreover, genetic interactions between variants in GPX1) has been linked to other cancers such as lung [GPX1 in high linkage disequilibrium, rs1050450 and rs18006688, was found to significantly modify the association between lead exposure and glioblastoma [GPX1 and NF-kappa-B p65 mRNA levels in whole blood and cartilage tissue samples of KBD patients [GPX4 mRNA level was dramatically lower in the blood of KBD patients and a significant haplotype A-T from the combination of two genetically linked SNPS in GPX4 (rs713041 and rs4807542) was shown to protect against KBD [Genetic variants in selenoproteins have been involved in other cancers and diseases. In particular, rs1050450  and rs1553153 in Sec tRNA synthase (SEPSECS) were significantly associated with Crohn’s Disease [In a Caucasian population from New Zealand, where the soil is poor in Se and Se intake is low , rs79013GPX1 and GPX4 loci were associated with Crohn’s disease and inflammatory bowel disease [TXNRD2 locus was associated with susceptibility to staphylococcus aureus infection [Moreover, GWAS studies revealed that  disease ,22 wherenfection  and glaunfection .SELS, known to influence pro-inflammatory cytokines levels [SELS was observed with type 1 diabetes, rheumatoid arthritis and the inflammatory bowel diseases, Crohn’s disease and ulcerative colitis [SELS gene were shown to affect markers of cardiovascular disease risk in European American families enriched for type 2 diabetes [Interestingly, several SNPS is the selenoprotein S gene were shown to affect risk of several chronic diseases. In particular, rs28665122 in the promoter of s levels , contribs levels . In addis levels . On the  colitis . Moreovediabetes  and episdiabetes .IDI2) gene has been linked with type 2 diabetes [SEPP1 gene  with fasting insulin and first phase insulin response was replicated in Hispanic cohorts [Finally, the coding SNP Thr92Ala (rs225014) in type 2 deiodinase . This observation reveals an extra level of complexity when considering the relationship between Se and health, as the effects of a genetic variant on disease risk not only depends on the function of the corresponding protein but as well on the biological pathway it is involved in  and on the hierarchical distribution of Sec between selenoproteins and Se bioavailability. In addition, several studies noted that these interactions were observed in certain ethnic groups or populations with different Se supply or genetic background. This suggests that, during evolution, the fixation of these SNPs in sub-group populations reflected probably the need for an adaptation to both changes in environmental Se levels and exposure to specific environmental stressors. Thus, a real appreciation of the effect of Se-gene interaction on cell responses and disease prevention will require, in the future, taking into account not only the effect of Se supply on multiple selenoproteins; it will also require considering the impact of changes in selenoprotein synthesis and activity on downstream molecular pathways and on the response to environmental stresses, as well as the contribution of these variants to disease risk within the genetic background of an individual. Mechanistic and epidemiological studies have shown that Se and selenoproteins play a key role in biological pathways, such as cellular maintenance, protein folding and oxidative stress response, known to be affected in multifactorial chronic diseases . As a repersonalised dietary recommendations of Se to individuals, depending on their genetic makeup, Se status and other risk factors.Another lesson from genetic epidemiological studies is that the role of selenoproteins and the consequences for downstream molecular targets may differ between tissues. These observations uncovered potential new clues to understanding the role of specific selenoproteins in certain tissues and molecular pathways and their implication in mechanisms underpinning diseases. The study of these mechanisms, using a functional genomics approach, has the potential to contribute to the identification of early disease susceptibility markers and to the design of novel disease prevention strategies, based on Finally, genetic associations provided strong evidence of a link between genetic variants in selenoproteins genes and risk for various chronic diseases but failed to demonstrate how these variants formally affect disease aetiology. Thus, in the future, more studies need to be designed to not only replicate these associations in multiple and larger cohorts but as well take into account measures of Se status, gene expression and functional approaches to formally establish and quantify the contribution of combined and individual SNPs to the disease risk. To clarify the effect of the SNPs, the design of these studies would have to include stratifications according to Se levels, genotype, disease stages and response to treatment.


Adolescence represents a crucial phase of synaptic maturation characterized by molecular changes in the developing brain that shape normal behavioral patterns. Epigenetic mechanisms play an important role in these neuromaturation processes. Perturbations of normal epigenetic programming during adolescence by ethanol can disrupt these molecular events, leading to synaptic remodeling and abnormal adult behaviors. Repeated exposure to binge levels of alcohol increases the risk for alcohol use disorder (AUD) and comorbid psychopathology including anxiety in adulthood. Recent studies in the field clearly suggest that adolescent alcohol exposure causes widespread and persistent changes in epigenetic, neurotrophic, and neuroimmune pathways in the brain. These changes are manifested by altered synaptic remodeling and neurogenesis in key brain regions leading to adult psychopathology such as anxiety and alcoholism. This review details the molecular mechanisms underlying adolescent alcohol exposure-induced changes in synaptic plasticity and the development of alcohol addiction-related phenotypes in adulthood. Alcohol exposure, whether acute or chronic in nature, produces profound morphological, structural, and molecular changes in the brain , ventral pallidum, and nucleus accumbens (NAc) are involved in the initial binge/intoxication stage of drug and alcohol use. Ethanol directly increases the firing rate of dopaminergic VTA neurons , bed nucleus of the stria terminalis (BNST), and a transitional zone of the NAc shell Alheid, , is propThe third stage of alcohol addiction involves preoccupation with drug taking/intoxication and “craving.” It should be noted that the concept of craving has not been successfully measured clinically and may not correlate with actual relapse Sinha, . This stAdolescence is marked by widespread changes in the developing brain that include the regions involved in alcohol addiction stated above. Brain white matter increases throughout adolescence as a function of ongoing myelination and gray matter reduction  and activity-regulated cytoskeletal-associated protein (Arc) and subsequently increased dendritic spine density  gene as a risk factor for AUD in discordant monozygotic twins  exposure leads to long-lasting increases in global HDAC activity, as well as specific increases in HDAC2 protein and mRNA, in the amygdala at adulthood  knockout animals  in the medial PFC that is reversed by local infusion of a DNMT inhibitor , termed Adult human alcoholic patients show characteristic structural brain abnormalities including presumed neurodegeneration in response to heavy and prolonged alcohol use , or the ability of synaptic activity to increase downstream signaling effects and ion flux during subsequent activation, at baseline compared to adult animals  in the hippocampus of adolescents vs. adult animals  in adolescents, but facilitate ePSCs in adult NAc slices in a mechanism dependent on presynaptic D1 receptors , high-mobility group box 1 (HMGB1), and the HMGB1 receptor TLR4 in the postmortem brains of human alcoholics  and interleukin 1 beta (IL-1β)  gene expression is increased in the cortex of adolescent rats during withdrawal to a greater extent than adults  persists into adulthood in the cortex  on DNA after phosphorylation and usually increases the transcription of downstream genes such as  Pandey, . As NF-κ Pandey, , these t Pandey, , possibl Pandey, .Bdnf expression in the NAc shell while also promoting alcohol drinking behavior  rats display innately heightened anxiety levels and excessive alcohol drinking behaviors, as well as lower levels of CREB and the functionally active phosphorylated form of CREB (pCREB) within the CeA and MeA when compared to non-preferring (NP) rats  infusion into CeA significantly decreases Arc expression, dendritic spines, and promotes anxiety-like and alcohol drinking behaviors in rats  occur across treatment paradigms including i.p. injection, i.g. gavage, vapor exposure, and voluntary consumption, and are therefore likely the result of ethanol exposure itself  contributed in the review of literature and writing of the article. The content of figures was conceived by SP and then prepared and edited by all authors.SP reports that a US patent application entitled “Histone acetyltransferase activators and histone deacetylase inhibitors in the treatment of alcoholism”  is currently pending. The other 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.


The nature and significance of the molecular genetic aberrations in relation to the locations and function of oncogenes and tumor suppressor genes will be discussed. At last, a possible pathogenic role of embryonic duct remnants was observed in seven female fetal reproductive tract in endometriosis and may induce a discussion about the beginning of ovarian tumors and malignant proliferations.Endometriosis is a benign disease with high prevalence in women of reproductive age estimated between 10 and 15% and is associated with considerable morbidity. Its etiology and pathogenesis are controversial but it is believed to involve multiple genetic, environmental, immunological, angiogenic, and endocrine processes. Altered expressions of growth factors, cytokines, adhesion molecules, matrix metalloproteinases, and enzymes for estrogen synthesis and metabolism have been frequently observed in this condition. The possibility of genetic basis of endometriosis is demonstrated in studies of familial disease, in which the incidence of endometriosis is higher for first-degree relatives of probands as compared to controls. This review describes mainly the cellular, cytochemical, cytogenetic, and molecular genetic features of endometriotic lesions and cultured endometriotic cells. In attempts to identify candidate gene (s) involved in the pathogenesis of endometriosis, a tissue-based approaches including conventional cytogenetics (RHG-banding), loss of heterozygosity (LOH), and comparative genomic hybridization (CGH) were employed. In addition to the karyotypic anomalies, consistent chromosome instability was confirmed by CGH and fluorescence  Endometriosis is a common gynecological disorder accounting for 10–15% of pelvic pain and infertility in women of reproductive age. Despite its prevalence, relatively little is known about the underlying pathophysiological mechanisms. Numerous studies however suggest that it has a multidimensional etiology including, hereditary, hormonal, and immunological factors .in vitro . Thereafter, the glands are washed from the sieves and allowed to proliferate in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum and antibiotics. In separate experiments, stromal cells from the collagenase digested endometriotic tissues are allowed to adhere to the culture flasks and maintained in vitro following the same culture procedures. The primary cultures obtained from the peritoneal biopsies and from the chocolate cyst wall contain mostly mixed cell populations variable in their size and in their morphology. Within 1 week, small foci consisting of rather small spindle or polygonal cells appear among the loosely proliferating epithelial-like cells and gradually increase in size. After 3–4 weeks in culture, the cellular monolayer is successfully passaged in vitro at a density of 2 × 105 cells/ml.Briefly, the fragments obtained at surgery are rinsed, minced, and digested with collagenase (2 mg/ml) at 37°C in 5% COThe cultured stromal cells from both endometriotic implants and the endometrioma wall appear as adherent fibroblast-like cells, their doubling time being approximately 9 days. These stromal elements usually proliferate as elongated cells, densely packed, and reach confluence in about 10–14 days. An aspect on phase contrast microscopy of an endometriotic gland from a peritoneal implant in primary culture giving rise to growing epithelial-like cells and third passage of culture derived from an ovarian endometrioma is presented in Figure To determine if the cultured endometriotic cells retain specific epithelial markers, immuno-cytochemistry is routinely performed using a panel of specific antibodies. For immunostaining, the endometriotic cells cultured either in tissue culture chamber slides (Labtek) or prepared as cytocentrifuge smears are fixed in 4% paraformaldehyde or in cold acetone (10%), rinsed in PBS, and incubated with the primary antibody. Monoclonal and/or polyclonal antibodies are revealed using rabbit anti-mouse or goat anti-rabbit IgG peroxydase conjugated antibodies  according to standard methods. Control slides are incubated with normal mouse serum to replace the specific monoclonal or polyclonal antibodies.The results of the immuno-histochemical staining of both epithelial and stromal endometriotic cells with specific monoclonal and polyclonal antibodies are summarized in Table Among the epithelial markers, cytokeratin expression remained one of the most specific characteristics of the cultured cells. The anti-cytokeratin antibody detecting cytokeratin 8, 18, and 19 produce cytoplasmic immunolabeling of the majority of epithelial endometriotic cells from various passages Figure . These cConcerning the differentiation capacity, experiments were performed to determine whether the cultured epithelial and stromal endometriotic cells undergo changes after treatment with the estradiol metabolite 2-Ethoxyestradiol and medroxyprogesterone acetate (MPA). Phase contrast microscopy indicated that cells cultured in the absence of steroids remained growing until confluence. For cultures grown in medium supplemented with 1% of FCS, addition of with 2-Ethoxystradiol (1 μM) resulted in strong inhibition of cell growth and appearance of morphological changes, i.e., the cells became large with appearance of cytoplasmic projections Figure . In contMost genetic changes occur in somatic cells, but some occur as germ line defects that can result in an inherited predisposition to endometriotic development. In this context, convincing evidence in favor of a genetic basis for endometriosis is provided by several studies sowing that the prevalence of the disease is higher in the first-degree relatives of affected women than in controls thought to be representative of the general population –43.At the tissue level, cytogenetic, molecular genetic, and molecular cytogenetic techniques have been applied to endometriosis and have lead to the identification of consistent somatic genetic alterations. Altogether, these findings strongly suggest existence of genomic aberrations in endometriotic tissues probably related to distinct putative genes involved in development of the disease.in vitro against aneuploid cells. The evidence for the latter comes from a study where the FISH was performed on direct preparation revealing clonal aberrations and aneuploidy of several chromosomes  with painting probes for chromosomes 1, 6, 7, 8, 11, 12, 17, and 22.To investigate the chromosomal structure of the different types of endometriotic lesions, we performed conventional karyotype analysis of a series of seven endometriotic samples obtained from patients with advanced stage disease. In strictly defined culture conditions, increased percentage of aberrant metaphases showing aneuploidy, dicentric chromosomes, endomitoses, and chromosomal pulverization were detected in five of seven studied samples by RHG-banding Figure . These rFISH analysis with painting probe for chromosome 17, demonstrated presence of three copies of that chromosome in 17% of the analyzed metaphases in one ovarian endometrioma. In contrast, loss of chromosome 17 was detected in 2 cases of 7 studied. Loss of the q arm of chromosome 22 was detected in 3 cases (not shown). Similar data were previously reported by Shin et al.  in one cOnly a limited number of loss of heterozygosity (LOH) studies have been conducted using DNA from histologically homogeneous endometriotic tissues. Recent molecular allelotyping work has detected somatic genetic changes, distributed along several chromosomes including chromosome 9p, 11q and 22q in 11 of 40 (28%) examined endometriotic samples . When moIn general, FISH analysis has revealed more clonal aberrations than conventional cytogenetic analysis in a number of altered tissues. This approach has the advantage that culturing of endometriotic cells is unnecessary and eliminates the problems of tissues heterogeneity. Performed on interphase nuclei in severe/advanced stage endometriosis, higher frequency of monosomy for chromosome 16 and 17 and increased number of cells with trisomy 11 were recently reported , 49. TheComparative genomic hybridization (CGH) is a molecular cytogenetic method able to discover and map genomic regions for chromosomal gains and/or losses in a single experiment . RegionsBy means of CGH, we screened primary endometriotic lesions for chromosomal gains and/or losses in a series of patients with advanced stage disease. The study was performed on native, non-amplified DNA extracted from manually dissected endometriotic tissues Figure . RecurreThe average number of copy alterations in our series of endometriotic tissues was 3.1 per lesion, which is low compared to malignant tissues . The DNAThe most common regions of loss of genomic material were located on 1p involving at least the 1p32–36 (50%), 5p (33%), 6q (27%), 7p14-p22ter (22%), 16qter (22%), and 22q12.3-qter (50%) segments  .–Precancerous lesions .–Chronic inflammatory conditions.–Infectious diseases.–Diseases induced by viruses .It is defined as an alteration of the chromosome constitution occurring in various pathological conditions:–Chromosomal alterations in non-neoplastic precursor lesions and mutation of the P53 gene.–Errors in DNA replication detected by microsatellite instability (deficiency in DNA mismatch repair mechanism).The genomic instability appears in two different types:We have observed , 69 suchThe loss of either essential genes (TSGs) or even entire chromosomes, explain the high invasive potential of the endometriotic cells. Genomic alterations (rearrangements) initiated for instance by telomere dysfunction can be a primary event that facilitate endometriosis initiation and spread .The theory of transformation of the vestigial tissue of Müllerian or Wolfian origin and the celomic metaplasia theory can explain the origin of distinct entities: for instance endometriotic lesions as well as development of particular types of ovarian neoplasms. In the same context, a recent study has proposed the fetal origin of endometriosis that could develop on the basis of altered migration of primitive endometrial tissue during embryogenesis . These aIn a previous study, we evaluated the incidence and the anatomical location of displaced endometrial tissue in the reproductive tract in seven female fetuses. Serial sectioning of the reproductive organs was realized followed by immuno-histochemical analysis of tissue areas containing ectopic glands and embryonic cell rests Table  and 8.We have compared these results with those of two germ cells tumors, the young and adult forms using exclusively an immuno-histochemical analysis.It was observed that the anatomical and the phenotypic features of the misallocated tissue components recall some pathological characteristics of both benign and malignant gynecological conditions .The actual classification used around the world is only anatomic, with adjunction recently of pain and deep lesions . But, weIn addition, in this study, the proliferation is shown with the lesions having lost their chromosomal stability. All these lesions have genomic abnormalities. Thus, when endometriotic cells cultured from these lesions  are treated with progestins, an increase in the proliferation curve is observed  that is There are no correlations between the size of lesions and the severity of the disease Figure . ConsequAn immunohistochemical profile of the lesions (Endometriogram*) should be defined in a similar way as the procedure used to molecularly classify the breast cancer. Using this strategy, the medical treatment can be decided including the IVF technology in case of infertility.Thus, larger prospective randomized studies should be carried on in the future with the main basic concept to introduce the most apporpriate algorithm for therapeutic decision.Substantial progress has been made in the last few years to understand better the etiologies, pathogenesis, and means to treat endometriosis. This field of investigation is supposed to make greater advances in the near future. From the collected molecular data, it is evident that one or several genes may be disrupted by mutations or loss of DNA sequences in this condition. Loss of some protein functions may effect the regulation of several emerging down stream pathways, including the EGFR-axis, E-cadherin dynamics, transcriptional regulation, cell cycle regulation (proliferation/apoptosis), and cell adhesion. Divergences from mature endometrial epithelium to endometriotic epithelium must involve abnormal gene expression, which may directly cause or reinforce the alterations caused by changes of one or several genes undergoing germinal alterations. Within this context, the treatment strategies of endometriosis centered mainly toward ovarian function suppression and indirect limitation of cell growth and activity of endometriosis remain insufficient , 74.in vitro growth pressures. CGH and cDNA microarray studies has only been used to date on a limited number of primary endometriotic samples, yet has given us important informations (Future studies should concentrate on modifying and extending the use of the molecular biology techniques. Cytogenetic analysis with improved culture techniques of endometriotic tissue, capable of simulating the normal endometriotic environment, may help to prevent selective rmations , 75. GenFinally, the anatomical and the immuno-histochemical features of the ectopic organic structures identified in fetal female reproductive tract suggest that endometriotic as well as neoplastic disease in adult women may develop on the basis of misplaced endometrial glands and/or embryonic cell remnants.All authors were equally involved in the design of the study, data acquisition, interpretation and analysis. Luca Gianaroli and Jean Marc Bernard Ayoubi were involved in samples preparation and analysis. Jean Gogusev was involved in manuscript preparation and performed the image and statistical data analysis.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.


The genetic cause of idiopathic congenital talipes equinovarus (ICTEV) is largely unknown. We performed a systematic review to describe the findings from 21 studies that have examined the genetic variants related to ICTEV, and to evaluate the quality of reporting. We found that ICTEV was positively associated with Hox family genes, collagen family genes, GLI3, N-acetylation genes, T-box family genes, apoptotic pathway genes, and muscle contractile family genes. Negative and controversial results were also discussed, and several genes associated with ICTEV were identified. Due to the limitation of the included studies, rare coding variants should be further investigated, sample size should be enlarged, and candidate genes should be replicated in larger ICTEV populations. Epigenetic study, pathways, chromosome capture, and detailed gene-environment interaction will also allow further elucidation of factors involved in ICTEV pathogenesis and may shed light on diagnosis and timely and accurate interventions. Idiopathic congenital talipes equinovarus (ICTEV), also called clubfoot, is a common orthopedic birth defect found in 1 of 1000 infants  screened the titles and abstracts of all the retrieved articles to determine whether they met the eligibility criteria, and appraised the methodological quality and evidence of each selected study. The second author (XFX) subsequently reviewed the accuracy and quality of the appraisal. Any disagreements were discussed between the first and the second authors until consensus was reached. The flow diagram describes the process used to select articles for this study, and the results of the literature search Fig. . The folWe identified 902 relevant studies from various resources Fig. ; the incFrom the studies gathered, we identified 21 that investigated genes or pathways which may contribute to the occurrence of ICTEV. Among them, some genes are positively associated with ICTEV, while others have shown no evidence of association. The genes with positive results include: (1) Hox family genes (HoxA and HoxD), (2) collagen family genes (COL9A1 and COL1A1), (3) GLI3, (4) N-acetylation genes (NAT2), (5) T-box family genes (TBX3 and TBX4), (6) apoptotic pathway genes , and (7) muscle contractile family genes (TNNC2 and TPM1). The genes with negative or controversial results are CAND2, WNT7a, MYH, and DTDST. Our critical review of these genes affecting ICTEV is summarized as follows:HoxD gene; transmission of disequilibrium was found at the 12th allele, indicating that HoxD may be a potential gene for ICTEV   collagen component of hyaline cartilage. Lack of Type IX collagen is associated with early onset of osteoarthritis, epiphyseal dysplasia, and intervertebral disc degeneration  gene expression. Mutations in the limb development related gene GLI3 have been associated with polydactyly  in the region of chromosome 12q24, where the TBX3 gene is located, was proven to have transmission disequilibrium in ICTEV patients, suggesting that TBX3 is a susceptible gene for ICTEV  are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis, and inflammation , a key protein complex in the regulation of striated muscle contraction, is composed of three subunits . The Tn-I subunit inhibits actomyosin ATPase. The Tn-T subunit binds tropomyosin and Tn-C. The Tn-C subunit binds calcium and overcomes the inhibitory action of the troponin complex on actin filaments. TNNC2 encodes Tn-C subunit and plays a key role in initiating muscle contraction in fast-twitching muscle fibers (Mckillop and Geeves TPM1 is a member of the tropomyosin family of highly conserved, widely distributed actin-binding proteins involved in the contractile system of striated and smooth muscles and the cytoskeleton of non-muscle cells. Tropomyosin functions in association with the troponin complex to regulate the calcium-dependent interaction of actin and myosin during muscle contraction. The associations of multiple SNPs in the TPM1 gene with ICETV suggest a potential role of genes that encode contractile proteins of skeletal myofibers on the etiology of ICTEV (Shyy et al. Besides the aforementioned candidate gene studies, a genome-wide association study was conducted in 396 isolated ICTEV patients and 1000 controls of European descent to identify novel genes for ICTEV (Zhang et al. Shyy et al.  studied In 2006, Sharp et al. found that children who carry the 677T variant of the MTHFR gene have a lower risk of ICTEV (Sharp et al. In this systematic review, positive associations between genetic variants and ICTEV were established in several studies. Certain genetic variants were found to have significant association with ICTEV. However, it must be noted that conflicting and negative results were also identified which do not necessarily undermine their contribution to the occurrence of ICTEV.ICTEV’s genetic study history is described in Fig. Several genes were identified, though none of them could solely explain the occurrence of ICTEV. Because the sample size of most association studies was small, most of the studies included are largely underpowered. In those included studies, rare coding variants were rarely investigated. Candidate genes were not replicated in larger ICTEV populations. These limitations should be addressed in future studies. In the future, genetic research on ICTEV could be focused on at least five aspects. First, high-throughput sequencing instead of GWA studies might be used to detect replicable candidate genes. The sample size should be calculated and candidate genes must be replicated in other studies. Second, epigenetic sequencing examining regulatory mechanisms for RNA could be studied. Third, novel genes like FOXN3 and SORCS1 identified by the GWAS may be further investigated. Studying genes and their interactions could reveal common pathways which are responsible for the occurrence of ICTEV. Their functions and interactions are worthy of clarification. Fourth, recent advances in chromosome conformation capture may show more structural changes on a chromosomal level (Imakaev et al.


Postprandial hyperlipemia produces long-term derangements in lipid/lipoprotein metabolism, vascular endothelial dysfunction, hypercoagulability, and sympathetic hyperactivity which are strongly linked to atherogenesis. The purpose of this review is to (1) provide a qualitative analysis of the available literature examining the dysregulation of postprandial lipid metabolism in the presence of obesity, (2) inspect the role of adiposity distribution and sex on postprandial lipid metabolism, and (3) examine the role of energy deficit (exercise- and/or energy restriction-mediated), isoenergetic low-carbohydrate diets, and omega-3 (n-3) fatty acid supplementation on postprandial lipid metabolism. We conclude from the literature that central adiposity primarily accounts for sex-related differences in postprandial lipemia and that aerobic exercise attenuates this response in obese or lean men and women to a similar extent through potentially unique mechanisms. In contrast, energy restriction produces only mild reductions in postprandial lipemia suggesting that exercise may be superior to energy restriction alone as a strategy for lowering postprandial lipemia. However, isoenergetic very low-carbohydrate diets and n-3 fatty acid supplementation reduce postprandial lipemia indicating that macronutrient manipulations reduce postprandial lipemia in the absence of energy restriction. Therefore, interactions between exercise/energy restriction and alterations in macronutrient content remain top priorities for the field to identify optimal behavioral treatments to reduce postprandial lipemia. Reductions in vocational physical activity and the availability and consumption of energy-dense foods are often cited as primary culprits of the rising incidence of obesity observed throughout the world –6. The iWhile the contribution of fasting TGs and HDL-C to the overall risk of CVD remains controversial , 13, accThe relationship between postprandial lipid metabolism and atherosclerosis is not surprising when one considers that the majority of individuals following a typical Western Diet consume 3 to 5 meals per day. Since each meal requires 6–12 hours to fully dissipate TGs in circulation, the implication is that most individuals spend over two-thirds of the day in a postprandial state with elevated TRLs  , 25. TheThe purpose of the current review is to (1) provide a qualitative analysis of the available literature examining the dysregulation of postprandial lipid metabolism in the presence of obesity, (2) inspect the role of adiposity distribution and sex on postprandial lipid metabolism, and (3) examine the role and mechanisms by which energy deficit produced via exercise and/or energy restriction, isoenergetic low-carbohydrate diets, and n-3 fatty acid supplementation improves postprandial lipid metabolism.Systematic abnormalities in fasting and postprandial blood lipid and lipoprotein metabolism have been well documented in obesity and other conditions which produce hyperinsulinemia/insulin resistance . InsulinSubsequent studies have attempted to address the precise role of adiposity distribution on postprandial lipemia. Overall, visceral adipose tissue distribution appears to be a better predictor of postprandial TG responses than body mass index (BMI) alone or gynoid distribution of adipose tissue –35. Sinc per se. Taken together, abnormal postprandial lipemia can be detected in the presence of excess visceral adiposity with comparable BMI and in the absence of fasting hypertriglyceridemia, providing further support that postprandial lipemia is a more sensitive predictor of metabolic and CVD risk than fasting parameters.In agreement with this interpretation, variation in postprandial TG responses between men and women was abrogated after controlling for visceral adiposity suggesting that the distribution of body fat is responsible for postprandial TG responses as opposed to sex differences , 37. FurCross-sectional and longitudinal studies show that regular aerobic exercise reduces postprandial lipemia in the presence or absence of weight loss –51. HoweAlthough the nutrient composition of test meals, populations studied, and exercise conditions/modalities employed are highly variable throughout the literature, several conclusions can be drawn that support the therapeutic benefits of consistent exercise participation as a means to improve metabolic health in the postprandial state. For instance, significant disparities exist between studies regarding the type of test meals employed to evaluate the impact of exercise on postprandial lipemia. The literature is split regarding the use of mixed high-fat meals and exclusively high-fat meals to explore these effects. Although the validity of high-fat diets exclusively as a test meal has come into question, what is important is that, in the majority of cases, a single session of aerobic exercise robustly lowers postprandial lipemia regardless of the composition of the test meal.It is important to point out that the amount of dietary fat in the test meal required to increase plasma TG concentrations appears to be dose-dependent . AbsolutAnother important aspect regarding test meal design and administration that should be considered is the fact that although the single test meal provides a powerful laboratory design, it may not reflect a real-world situation that occurs as additional meals are consumed. Farah and colleagues  addresseThe majority of investigations which have examined the effects of exercise on postprandial lipemia have been designed where a single session of aerobic exercise is conducted 11–20 hours prior to the test meal in a 2-day design. On day 1, participants perform a prescribed exercise modality and on day 2 they receive a test meal followed by temporal blood sampling for up to 8 hours , 53–64. 2max and that 90 minutes of walking at 31% of VO2max elicited little effect providing initial evidence that the effects of aerobic exercise on postprandial lipemia were due to either greater intensity or to greater energy expenditure at a higher intensity. In an attempt to answer this question, the authors conducted a follow-up study where they compared the effects of isoenergetic low- and moderate-intensity walking on postprandial lipemia  palmitate (5 mg/kg FFM) was administered in a liquid test meal. Lipids were isolated using a heptane/isopropanol extraction procedure and analyzed by LC/MS [More sophisticated approaches to apolipoprotein quantification and kinetics have been employed using stable isotopes to examine whether defects in secretion and/or catabolism of apolipoproteins are responsible for hypertriglyceridemia following a meal. For example, Wong and colleagues  used a bby LC/MS .Moderate-high intensity aerobic and resistance exercise produce consistent reductions in postprandial lipemia when performed 30 minutes to 20 hours prior to mixed or high-fat meals. Energy deficits created by moderate-intensity exercise appear to be responsible for a considerable proportion of the decrease in postprandial TGs with exercise. However, studies using resistance and HII exercise suggest that other factors may be involved. Future studies are needed to compare the effects of resistance exercise and HII in men and women. Furthermore, studies are required to determine the impact of weight loss (produced by energy restriction and/or increased energy expenditure) on the acute postprandial lipemia response to exercise. On a practical level, the minimum threshold of exercise energy expenditure to lower postprandial lipemia in sedentary and active populations is crucial. Additional studies using innovative approaches are also needed to more thoroughly evaluate the mechanisms by which exercise lowers postprandial TGs and if these mechanisms are different between obese and nonobese populations, sex, and race/ethnicity.Reductions in dietary carbohydrate without reducing energy intake also decrease postprandial lipemia. Future studies will be needed to determine the mechanisms by which low-carbohydrate (glucose and/or fructose) diets work. Studies using deuterated water could be used to evaluate de novo lipogenesis between tissues.


Previous mitochondrial DNA analyses on ancient European remains have suggested that the current distribution of haplogroup H was modeled by the expansion of the Bell Beaker culture  out of Iberia during the Chalcolithic period. However, little is known on the genetic composition of contemporaneous Iberian populations that do not carry the archaeological tool kit defining this culture. Here we have retrieved mitochondrial DNA (mtDNA) sequences from 19 individuals from a Chalcolithic sample from El Mirador cave in Spain, dated to 4,760–4,200 years BP and we have analyzed the haplogroup composition in the context of modern and ancient populations. Regarding extant African, Asian and European populations, El Mirador shows affinities with Near Eastern groups. In different analyses with other ancient samples, El Mirador clusters with Middle and Late Neolithic populations from Germany, belonging to the Rössen, the Salzmünde and the Baalberge archaeological cultures but not with contemporaneous Bell Beakers. Our analyses support the existence of a common genetic signal between Western and Central Europe during the Middle and Late Neolithic and points to a heterogeneous genetic landscape among Chalcolithic groups. The evolutionary history of the human settlement of Europe has been shaped by a complex pattern of migrations, driven by the appearance of new socio-economical strategies, cultural innovations and ecological changes Agriculture first developed in the Fertile Crescent of West Asia around 12–11,000 years ago, being afterwards developed independently over the next few thousand years in other regions of the planet Archaeological, anthropological and paleoecological evidence supports the existence of a major demographic change that took place with the arrival of the Neolithic around 9,000 years ago and the disappearance of the hunting and gathering strategy in the European continent after only few thousands of years However, the scale and nature of the interactions between local Mesolithic hunter-gatherers and incoming farmers has been a subject of much debate, opinions being divided between those who argue in favour of a population expansion from the Near East into Europe , and those who think that the process of ‘neolithisation’ was mainly a spread of ideas that led to the acculturation of the indigenous population In recent years, several ancient DNA studies have contributed to this debate by directly analysing ancient human skeletal remains, mainly Neolithic, and comparing their mtDNA composition to those of modern populations from the same area With the next-generation sequencing (NGS) technologies it has been possible to retrieve also prehistoric European genomic data. The first of these studies was the 7x coverage genome of the exceptionally well preserved Tyrolean Ice man, Ötzi, dated to about 5,300 years BP While most of efforts have focused on the Mesolithic-Neolithic transition, more recent periods remain relatively unknown To further investigate the transition between Late Neolithic to the Chalcolithic period, we have analyzed the mtDNA in a recently excavated population from El Mirador cave in Atapuerca, Spain. This site is contemporaneous to the Bell Beaker culture (BBC) but does not carry the diagnostic items of this culture that include the distinctive bell-shaped ceramics and weapons. In fact, the archaeological sites with Bell Beaker remains are very scarce in the Meseta Central of the Iberian Peninsula. It has been suggested 2 was affected by clandestine excavators. Later, in 1999, the archaeological fieldwork was resumed, and it is still continuing The cave of El Mirador is located on the southern side of the Sierra de Atapuerca , at an altitude of 1,033 meters above sea level . The iniTwo different assemblages of human remains were recovered from this site.2 survey in an area unaffected by the clandestine workers. Six individuals dated to the Early Bronze Age  were found. These human remains had been cannibalized and abandoned as rubbish in the Early Bronze Age, being buried later in the Middle or Late Bronze Age The first human sample was recovered from a 6 mAfter this first survey, in 2009 the fieldwork focused on a small cavity near the wall of the cave, where a collective burial was found. There were some individuals in anatomical position, but the most superficial remains had been disturbed and mixed by the earlier actions of the clandestine excavators. The human remains are associated with pottery without decoration, antler deer tines and fluvial shells. The fieldwork on this burial site is still in progress; however, a minimum number of 22 individuals have been recovered. All of these human remains belong to the Chalcolithic period and have been dated to 4,760–4,200 years cal. BP.The Chalcolithic samples were left uncleaned until they were sampled for the genetic analysis, to prevent exogenous DNA contamination by handling. The samples analyzed are labelled Mirador 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 and 26  and are DNA was extracted from approximately 100 mg of dental root tips following the method described in detail in Lalueza-Fox et. al Extraction procedures were performed in an isolated pre-PCR area, adopting the standard precautions of ancient DNA studies Besides standard precautions in ancient DNA outlined, we followed several additional strategies to support the authenticity of our results. 1) we retrieved endogenous DNA sequences from an animal bone (wolf) from the same site, to ensure that the cave environment was favourable indeed to DNA preservation, 2) we retrieved the same mtDNA haplotype from two teeth belonging to the same individual, 3) we replicated mtDNA sequences of an individual that shares the same mtDNA haplotype with two of the molecular researchers in an independent laboratory (Florence) after sending an additional sample directly from the archaeological institution, and 4) we repeated the amplification and sequencing of potentially rare haplotypes.In Barcelona, the mtDNA hypervariable region 1 (HVR1) was amplified by polymerase chain reaction (PCR) in two overlapping fragments (L16055-H16218 and L16185-H16378 primers), following a two-steps protocol , in which primers are limited in the first PCR step to avoid the generation of unspecific primer-primer artifacts In both laboratories, PCR products were subsequently visualized under UV lights in a low-melting point agarose gel. The appropriate bands were excised from the gel, melted at 65° for 20 min, and purified with a silica-based method. Subsequently, the amplification products were cloned in bacteria . Resulting white colonies were picked up, amplified with M13 universal primers and sequenced in an ABI3730 capillary sequencer (Applied Biosystems).prcomp function using mtDNA haplogroup diversity  we have replicated at least twice most of the mtDNA fragments, as recommended in Hofreiter et al. From the analysis of 757 clone sequences , we haveDespite the recent retrieval of partial or complete Mesolithic and Neolithic genomes However, another recent study based on the analysis of 629,443 single nucleotide polymorphisms (SNPs) from 934 individuals belonging to 53 human populations has uncovered a previously unrecognized signature of Northern European genes into the Iberian Peninsula The mtDNA composition of El Mirador is quite unique, and different to that found in other contemporaneous Bell Beaker populations and to present-day Iberians  and 3. TThe clustering of the El Mirador sample with a Salzmünde culture  population from Germany and the roughly contemporaneous Treilles culture population from France in the PCA of ancient populations  as well To explore the genetic signal of these population affinities, we have estimated the correlation coefficient between haplogroups and PCA dimensions and performed a significance test . K, T2, In none of the analyses El Mirador sample shows close genetic affinities with a contemporaneous Bell Beaker population of 29 specimens gathered from three sites in Germany. The Bell Beaker mtDNA signal is characterized by high frequencies (around 50%) of H haplogroup that in El Mirador only reaches 26%. This heterogeneity in the genetic composition of geographically close populations adds further complexity to future reconstructions of these ancient expansions and correlates with the existence of contemporaneous groups with and without the typical Bell Beaker burial kit.The generation of complete genomes with the new sequencing technologies will likely contribute to provide a more detailed picture of the complex population movements and affinities along the European prehistory and would eventually explain the dynamics of pan-European Chalcolithic migrations.Figure S1mtDNA clone sequences of the El Mirador samples. Samples with potentially conflicting haplotypes or with substitutions that could be attributed to postmortem damage were repeatedly amplified to generate a consensus sequence. Some CRS clones attributable to background contamination have been suppressed for clarity.(PDF)Click here for additional data file.Figure S2Boxplot grouped by geography of El Mirador principal components and other prehistoric cultures. PC1 and PC2 show the differentiation between geographical distributions of ancient cultures. El Mirador population (in yellow) shows affinities with prehistoric Central European cultures despite its location in Spain.(TIFF)Click here for additional data file.Figure S3Boxplot grouped by chronology of El Mirador principal components and other prehistoric cultures. PC1 shows the differentiation between hunter-gatherer, Early/Middle Neolithic and Late Neolithic/EBA, whereas PC2 gives information for all the periods. El Mirador population (yellow) have traits of the Early/Middle Neolithic period in these components despite its Chalcolithic attribution.(TIFF)Click here for additional data file.Table S1Details and haplogroup frequencies of El Mirador and present-day populations used in principal component analysis. Relative haplogroup frequencies of El Mirador and previously published populations (XLSX)Click here for additional data file.Table S2Details and haplogroup frequencies of El Mirador and prehistoric cultures used in principal component analysis. Relative haplogroup frequencies of El Mirador and previously published cultures (XLSX)Click here for additional data file.Table S3Details and haplogroup frequencies of El Mirador, Central European metapopulation and prehistoric cultures used in Ward’s hierarchical clustering. Relative haplogroup frequencies of El Mirador and previously published populations (XLSX)Click here for additional data file.