Donor-specific blood transfusion (DST) can lead to significant prolongation of allograft survival in experimental animal models and sometimes human recipients of solid organs. The mechanisms responsible for the beneficial effect on graft survival have been a topic of research and debate for decades and are not yet fully elucidated. Once we discover how the details of the mechanisms involved are linked, we could be within reach of a procedure making it possible to establish donor-specific tolerance with minimal or no immunosuppressive medication. Today, it is well established that CD4+Foxp3+ regulatory T cells (Tregs) are indispensable for maintaining immunological self-tolerance. A large number of animal studies have also shown that Tregs are essential for establishing and maintaining transplantation tolerance. In this paper, we present a hypothesis of one H2-haplotype-matched DST-induced transplantation tolerance (in mice). The formulated hypothesis is based on a re-interpretation of data from an immunogenetic experiment published by Niimi and colleagues in 2000. It is of importance that the naïve recipient mice in this study were never immunosuppressed and were therefore fully immune competent during the course of tolerance induction. Based on the immunological status of the recipients, we suggest that one H2-haplotype-matched self-specific Tregs derived from the transfusion blood can be activated and multiply in the host by binding to antigen-presenting cells presenting allopeptides in their major histocompatibility complex (MHC) class II (MHC-II). We also suggest that the endothelial and epithelial cells within the solid organ allograft upregulate the expression of MHC-II and attract the expanded Treg population to suppress inflammation within the graft. We further suggest that this biological process, here termed MHC-II recruitment, is a vital survival mechanism for organs  when attacked by an immune system. Today, transplantation tolerance in humans who receive an organ from a genetically disparate donor can very rarely be achieved without life-long administration of immunosuppressive therapy. While the current, non-specific immunosuppressive drugs are very good at inducing short-term graft survival, the impact on long-term engraftment has remained unchanged because most of the patients lose their transplants due to chronic rejection. Furthermore, due to general suppression of the immune system, the patients are exposed to a higher risk of infections and developing cancer. Therefore, the ultimate goal is to find a way to induce permanent donor-specific tolerance , 2.Long-term alloantigen-specific transplantation tolerance of solid organs can easily be induced in rodents without continuous supply of immunosuppressants . AlthougA widely used strategy to induce long-term transplantation tolerance in experimental animal models is to introduce the organ recipient to alloantigens in the form of blood transfusion before transplantation . MultiplSharing of a HLA/H2 haplotype between the recipient and blood donor in both humans and experimental animal models is associated with prolongation of graft survival. The effect is further optimized when the haplotype shared blood is donor specific, i.e., taken from the organ donor [donor-specific blood transfusion (DST)] –15.The mechanisms responsible for DST-induced unresponsiveness are not fully understood. However, many experimental studies in animals indicate that perhaps the most powerful mechanism involved in prolongation of allograft survival is the suppressor function of Foxp3 CD4+CD25+ regulatory T cells (Tregs). Tregs are also found to be indispensable for maintaining peripheral self-tolerance. How DST may induce Tregs has been intensively studied for decades.One central issue is whether donor-derived leukocytes are actively involved in inducing transplantation tolerance, and if so, how and to what extent. In this paper, we re-interpret an immunogenetic experiment conducted 17 years ago by Niimi et al., and we present a hypothesis that suggests that actively involved donor blood-derived Tregs and the donor organ itself are crucial for the prolongation of the allograft survival achieved in their study.via the donor organ as “passenger cells” and pretransplanted donor blood. In the early 1990s, Thomas E. Starzl and his colleagues proposed a model of microchimerism and transplantation tolerance. According to this model, allograft acceptance is the effect of a balanced double immune reaction between donor and recipient cells, which is comparable to a mutual graft-versus-host/host-versus-graft reaction, and causes reciprocal clonal exhaustion. Although the reaction eventually attenuates, it remains at a low level and keeps a dangerous increase of either of the two reactions in check. The persistence of microchimeric cells is therefore considered to be a prerequisite for long-term allograft survival in this model   . RecogniUnfortunately, this model does not explain allotolerance in mice because mice T-cells do not produce MHC-II. Therefore, a different model is required to explain similar phenomena that occur in mice.The discovery by Sakaguchi et al. in 1995 that a subset within the CD4+ T cell population that expresses CD25 are responsible for maintaining immunological self-tolerance in mice  triggerein vitro (iTregs) from the naïve CD4+ T cells population when encountering MHC/peptide complexes on dendritic cells in tolerogenic environments, such as in the presence of TGF-β and interleukine-2 (IL-2)  –34; i/p 2 (IL-2) . In heal2 (IL-2) . Tregs t2 (IL-2) , 37.foxp3-locus upstream of exon-1, also termed TSDR  Tregs show that they proliferate vigorously eostasis , 46, 47.eostasis , 48. It eostasis .In vitro studies have demonstrated that Tregs can regulate immune responses on multiple levels and via several different mechanisms. For example, they can inhibit activation and function of APCs and produce immunoregulatory cytokines, such as TGF-β and IL-10, which act directly on effector T cells by impeding their priming and effector function  are needed to induce indefinite transplantation tolerance  mice were transfused with one haplotype-matched or fully mismatched donor blood 7 days before transplantation. The recipient mice were then transplanted with a heart of different F1 mice with which they shared one haplotype  or none (groups III and VII). All combinations are illustrated in Figure  mice werTwo sets of experiments with different blood donors were conducted to ensure that the effects of pretransplant blood transfusion were not restricted to a specific strain combination. The results were similar.Optimal prolongation of graft survival was achieved in recipient mice which received one haplotype-matched DST (approximately 40% at day 100) (I). Graft survival at day 100 was also obtained in 10% of the recipients that shared one haplotype with the blood donor and the mismatched haplotype with the organ donor (IV). The survival curve in this group was quite similar to that in which the recipient and both donors shared the same haplotype but were mismatched for the other haplotype (II). However, in the latter test group, all hearts had been rejected by day 70. Recipients transfused with homozygous blood from a mismatched donor obtained moderate graft prolongation  (V). The graft was rejected immediately if the blood was derived from a haplotype-matched homozygous donor (VI). Prolongation of graft survival was also not achieved with fully mismatched DST (VII).In pilot studies with purified CD4+ T cells from mice after haplotype-matched blood transfusion, Niimi et al. found that these cells had the ability to downregulate donor-specific CTL responses in a specific way. This finding indicates that allospecific Tregs had been induced by the blood transfusion and that these Tregs were involved in prolongation of graft survival.According to Niimi et al., graft survival induced by the matched DST is probably due to priming of host T cells that recognize allopeptides presented by self(shared)-MHC indirectly on recipient APC and directly on donor APCs. However, since graft survival was also prolonged in combinations where the blood and organ donor shared only the mismatched haplotypes (IV and V), Niimi et al. suggest that presentation of allopeptides by recipient (self)-APCs is the essential pathway for tolerance induction.the organ did not have a recipient haplotype and was therefore not able to present the allopeptide.”Although indirect alloantigen recognition may have been essential to tolerance induction during the pretreatment regimen, the hypothetical-induced Tregs had to be reactivated by the transplanted organ for graft prolongation to occur. According to Niimi et al., this explains why the hearts in group III were all immediately rejected. In their own words: “… With regard to prolongation of graft survival in group II where the blood and organ donor share a recipient haplotype while being mismatched for the other, Niimi et al. suggest that the tolerance achieved is due to cross-reactivity or sharing of allopeptides between the mismatched haplotypes (haplotypes B and C).Several mechanisms may have contributed to the prolongation of graft survival obtained in the Niimi et al. study. It is, for example, well known that alloreactive T cells anergize or are deleted when reacting with donor-derived allopeptides or allo-MHC complexes in non-inflammatory transplantation settings . AlthougThe direct pathway of allorecognition is primarily associated with acute graft rejection . In expeI.via the indirect pathway of antigen recognition/presentation, i.e., recipient APCs, if (a) the donor and recipient have a sufficient match of major histocompatibility complex (MHC) classes I and II, for example, share one haplotype and (b) the recipient APCs present the donor-derived peptide that is recognized naturally by the donor-derived Treg and the indirect antigen-presenting profile (MHC/peptide profile) is functionally identical to the immunological self of the blood donor, i.e., that at least some self-peptide/MHC complexes are identical in the recipient and blood donor, the antigenic background on which that these blood donor-derived Tregs have been selected on.Whole live blood contains functional self-specific Tregs, which are transferred to the recipient by blood transfusion. These self-specific donor-Tregs can be activated  and proliferate All mice in the Niimi experiment were naive . Therefore, we also find it reasonable to assume that a substantial portion of the Tregs were self-specific tTregs, positively selected in the thymus upon encountering tissue-specific antigens before entering the periphery , 59. Becin vitro studies have shown that Foxp3 Tregs can physically out-compete non-Tregs in aggregating around APCs  responses directed against organs (either as a HVG-reaction or auto-reactive) cause the cells of the attacked organ  to produce or upregulate the production of MHC-II molecules. We have chosen to term the upregulation of MHC-II “MHC-II recruitment.” MHC-II recruitment allows for activated self-specific Tregs  to bind specifically to the organ and downregulate immune responses at the site of inflammation. This self-protecting mechanism will be activated if or when the transplanted organ is attacked by the host immune system.As indicated by the different outcomes of allograft survival in group I and, i.e., II and IV, the hypothetical multiplied self-specific donor tTregs appear to be restricted to carrying out their full regulatory potential of responses against a genetic identical organ donor. We further hypothesize thatvia rDCs before the donor organ is transplanted. The hypothetical induction of pTregs with specificity for a different MHC-II/peptide combination (D/b) than that of the blood donor-derived tTregs can explain prolongation of graft survival in group IV.The specific acquired tolerance in recipients that were transfused with one haplotype-matched DST can be explained by MHC-II recruitment by the endothelial/epithelial cells of the transplanted organ. From here, two pathways, which are not mutually exclusive, can support Tregs in performing their memory function and maintaining allotolerance: (1) semi-direct recognition of MHC-II/peptide complexes transferred directly by epithelial cells to recipient DCs (rDCs) or by epithelial exosomes and membrane vesicles –62; (2) The suggestion that MHC-II recruitment is essential for Tregs to achieve their full regulatory potential, both in transplantation tolerance and peripheral immunological self-tolerance, is consistent with the following discoveries: (1) several studies have demonstrated that endothelial and epithelial cells in human transplants and in mice upregulate their production of MHC-II molecules –66. (2) The attachment of Tregs to endothelial/epithelial MHC-II/peptide complexes may even be a short cut to reactivation of Tregs (memory Tregs) as suggested by Niimi et al. 17 years ago. Although activation of T lymphocytes is closely linked to professional APCs, recent studies have shown that hepatocytes from a transgenic mouse model, expressing stable MHC-II and featuring CD80, are able to activate CD4+ T cells. In this study, the epithelial activation of CD4+ T cells was not associated with autoimmune reactions . AdditioAlthough our hypothesis is about transplantation tolerance induced by donor tTregs, recipients of one haplotype-matched allografts may, like the donor, harbor self-specific tTregs that recognize and bind self-MHC-II/peptide complexes on the donor organ. These hypothetical recipient tTregs may have been a contributing factor to prolongation of graft survival in recipients that were not tolerated for the mismatched antigen in the donor organ, as in group II. However, since tolerance was not achieved with matched pretransplanted homozygous donor blood (A in group VI), recipient and donor tTregs appear to be incapable of inducing tolerance without the presence of an alloantigen, for example, allopeptides from shared minor antigens between the blood donor (haplotype B) and the organ donor (haplotype C) as indicated by the outcome in group II Figure .In this article, we have introduced a novel hypothesis on DST-induced transplantation tolerance based on a re-interpretation of a 17-year-old immunogenetic study by Niimi et al. . The aniBased on the immune status of the mice in the experiment of Niimi et al., two main suggestions have been presented in the hypothesis: (1) blood donor-derived self-specific tTregs can be (re)activated and multiplied by indirectly recognizing MHC/peptide complexes on host APC that correspond to the antigenic background they have been selected on in thymus; (2) the transplanted organ upregulates endothelial/epithelial expression of MHC-II if or when the organ is attacked by the host immune system. It is further suggested that the multiplied donor tTregs suppress bystander allospecific T cells and may induce infectious tolerance by binding directly to the donor organ or semi-directly to professional host APCs.This hypothesis is consistent with recent animal studies which have shown that for long-term transplantation tolerance to occur, there is a need for Tregs that recognize allopeptides presented by self-MHC-II on host and donor APCs.Several studies have indicated that tTregs constitute a more stable and mature cell population than Tregs induced from the naïve T-cell pool. Therefore, approaches that focuses on self-specific donor tTregs with specificity for self-peptides presented by shared MHC-II could be an attractive shortcut to prolongation of allograft survival because many apparently difficult steps in the generation of new Tregs from the naïve T cell pool may already have been completed. Yet, introduction of donor-specific antigens, such as donor blood and donor Tregs, prior to transplantation may increase the risk of immunizing the host.According to our hypothesis, recipients of one haplotype-matched allografts may, just like the donor, harbor self-specific tTregs that can recognize and bind to both pathways of alloantigen presentation. Expansion of such recipient tTregs could minimize the risk of immunizing the host. This mechanism may have contributed the moderate prolongation of graft survival in group II. However, it is crucial to find out why tolerance was not induced with haplotype-matched homozygous donor blood (group VI).To further support the hypothesis proposed in this manuscript, it will be important to test experimentally the longevity of Tregs alive in the recipient and whether these Tregs are essential for tolerance induction. It is also important to test whether the transplanted organ upregulates the production of MHC-II and whether these donor MHC II/peptide complexes are transferred to recipient APCs/DCs. Finally, support of the hypothesis will also require testing of whether linked suppression and infectious tolerance occurs in the donor:recipient combination.We hereby confirm that all the authors’ contribution to this manuscript meets with the criteria of authorship demanded by Frontiers.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.


One third of the data was reserved for independent validation. Models were first fitted to the post-surgery PSA measurements using cross-validation. Major trends were then captured using linear mixed-effect models and a predictive generalized linear model effectively identified early trends connected to BCR. The model generalized for BCR prediction to the validation set with ROC-AUC of 83.6% and 95.1% for the 1 and 3 year follow-up censoring, respectively. A web-based tool was developed to facilitate its use. Longitudinal trends of u-PSA did not display major discrepancies from those of t-PSA. The results support that u-PSA provides useful information for predicting BCR after RP. This can be beneficial to avoid unnecessary adjuvant treatments or to start them earlier for selected patients.Ultrasensitive prostate-specific antigen (u-PSA) remains controversial for follow-up after radical prostatectomy (RP). The aim of this study was to model PSA doubling times (PSADT) for predicting biochemical recurrence (BCR) and to capture possible discrepancies between u-PSA and traditional PSA (t-PSA) by utilizing advanced statistical modeling. 555 RP patients without neoadjuvant/adjuvant androgen deprivation from the Turku University Hospital were included in the study. BCR was defined as two consecutive PSA values >0.2 ng/mL and the PSA measurements were  Prostate-specific antigen (PSA) is the most widely used tool to detect and monitor prostate cancer (PCa)Early detection of BCR is important because salvage radiation therapy (RT) is most efficient when given shortly after BCRPSA doubling time (PSADT) has been used to estimate the risk of disease progression after radical surgery; PSADT of nine months or less is an independent risk factor for prostate cancer specific mortality12The aim of this study was to develop novel tools that reduce the unreliability related to u-PSA. Furthermore, we assessed the potential prognostic significance of u-DT for predicting BCR after RP and applied comprehensive mathematical modeling of u-PSA and t-PSA, in order to establish an accurate predictive link between early measurements of PSA and the risk of BCR.et al.et al. composed of almost identical study population, and assessed optimal u-PSA threshold for upcoming BCR. In the conducted multivariate analysis there were no significant differences between patients with or without ART. According to the Finnish national rules and regulations for medical registry studies with retrospective nature no patient consents are required. Study protocol was approved by the IRB of the hospital district of South West Finland and the study was carried out taking into account all the study guidelines and national laws in Finland.Patients undergoing open RP and limited pelvic lymphadenectomy at Turku University Hospital during 2004–2008 were included (n = 604). The follow-up period was a minimum of 6 years. Open RP was performed as initially described by Walsh The patients were followed every 3 months for the first year after the surgery and semiannually thereafter. The follow-up included a physical examination and u-PSA measurements. Data was collected retrospectively from Turku University Hospital’s medical records and PSA data was obtained from Turku University Hospital laboratory data sources. All the PSA-analyses were done with electrochemiluminescence-immunoassay , which has a lowest limit of detection (LLD) 0.003 ng/mL. The collected data included essential clinicopathological variables, neoadjuvant and adjuvant therapies, and follow-up information, which were later used also in multivariate analysis of a potentially more accurate LASSO penalized prediction model by expanding beyond just PSA-derived information.PSA measurements with non-detected quantities were imputed using the smallest non-zero measurement. Of all the eligible post-surgery measurements, 4502 79.6%) were u-PSA (≤0.1 ng/mL) and 1151 (20.4%) t-PSA (>0.1 ng/mL). Post-surgery PSA nadir was defined as the lowest PSA measurement within a 3 month window after surgery. The 3 month period was chosen because 8 weeks is ample time to allow PSA levels to clear after RP and detectable u-PSA values in 1–3 months after RP are suggested as a marker for BCR progression.6% were To evaluate the generalization ability of the modeling, the data was randomized into 3 subgroups of subjects prior to model development, where factors such as age, BCR status, and Gleason score (GS) were balanced. 2 of the subgroups were randomly chosen as the exploratory data and fully utilized in model development. Within this exploratory data, generalization ability was maintained through cross-validation. The remaining third of the data was utilized as a validation set, to retain an objective view to the robustness of the final model .log2-transformed PSA would display complex non-linear patterns (low λ) or linear patterns (high λ).Cubic penalized splines were used in the exploratory set with a wide range of values for the spline smoothing parameter λ. The optimal smoothing parameter was identified by minimizing the cross-validation Median Squared Error (MSE) of the spline fits. Penalized splines provided a flexible approach to explore whether the log2-transformed PSA, a linear mixed-effects model was built. The parameter estimates of the model for the log2-PSA nadir and PSADT were used for detecting differences between the BCR and non-BCR patients. A clinical risk assessment tool was further derived using generalized linear mixed-effects models as a binary classifier for BCR using parameter derivatives from the patient-wise nadir and PSADT. Furthermore, we then subjected the binary classification task of nadir and PSADT along with clinical parameters from Based on the observed highly linear patterns of the psplineslme4glmnetThe mathematical modeling was conducted using the R statistical software (version 3.2)log2-transformation in log2-transformation a unit change corresponded to PSA doubling in the original PSA scale. For detailed modeling results, see the Detailed patient characteristics are reported in The major PSA trends were effectively captured readily by linear components in the model based on the optimality of high values of the smoothing parameter λ  as well log2-PSA patterns, we fitted linear regression models to perform parametric inference for the population effects. The focus was on the log2-PSA nadir and PSADT. Patient-wise estimates for these coefficients are shown in Since splines suggested that linear model families were suitable for modeling the Finally, generalized linear models were used as binary classifiers to connect the patient-wise characteristics from log2-PSA nadir model parameter, multiple associations were identified, excluding GS, indicating that multiple clinical parameters may be associated with the sensitive detections possible only in the u-PSA range  and the optimal model was fitted using penalization parameter within a single standard error of minimal CV error , the mulOne representative third of the data was left for objective validation of the modeling procedure in a wider context  with thehttp://compbiomed.shinyapps.io/u-pa/). The tool allows automated analysis of novel measurements with the existing methodology, and is provided with the exploratory dataset for illustrative purposes. Its design allows clinicians to conveniently run the pipeline and generate PDF-based risk reports for new patients. A typical workflow of the GUI is presented in In order to provide the analysis pipeline widely accessible to clinicians, a graphical user interface (GUI) was implemented using the R Shiny (RStudio Inc) platform with the underlying mathematical methodology outlined in the log2-transformation, after which the linear trends may be captured using conventional tools widely available in any statistical or spreadsheet software. According to previous studies the specificity of u-PSA is poorIn the current study we applied mathematical modeling to investigate the role of u-PSA as means of follow-up after RP. Based on our results, u-PSA provides useful information for predicting BCR after RP and we developed an easily applicable prediction platform , which tlog2-PSA after the first follow-up year. Motivated by the spline analysis supporting linear trends , we tested parametric linear inference using both a 1 year and a 3 year post-nadir window. A 3 year time window was also utilized by Malik et al., in a study where they assessed non-detectable vs. detectable u-PSA with the threshold of 0.05 ng/mLet al., but in their study the presence of a detectable u-PSA level earlier than 2 years from surgery did not reliably predict the subsequent clinical course of BCR12et al. and 3 year utilized by Malik et al. may already be well established by the end of the first year post-nadir10Based on our analysis of the second order spline derivatives, major trends in u-PSA curvature were established by the end of the first year and only slight individual variation occurred thereafter . Stabilihttp://compbiomed.shinyapps.io/u-pa/ In our exploratory dataset, the median time between two subsequent post-surgery PSA measurements was 152 days. Therefore, the first year of follow-up mostly consisted of 3 measurements. This amount is the minimal number of observations required to fit a linear regression model. The 3 year follow-up period was less sensitive to the nadir point and more likely holds a more realistic amount of observations for reliable PSADT estimation. However, it remains to be validated to what extent the doubling trends are established by the end of the first year, and for this purpose more intensive coverage of PSA trends would be already required for the early follow-up.et al. demonstrated a poor correlation between PSADTs, possibly due to inconsistency of u-PSA measurements134Accurate methods to determine the clinical risk represented by a rising PSA value are critical to develop rational treatment strategies. So far no studies have demonstrated that u-PSA triggered therapy will improve outcome. On the other hand, u-PSA kinetics may provide predictive information. Only few studies have compared DTs in traditional and ultrasensitive ranges1011151011427Major strength of this study is the extensive mathematical modeling of both the u-PSA and t-PSA measurements, all of which is offered as an easy to use web-based graphical user interface (GUI) platform. All the PSA measurements were done with the same PSA assay, reducing error caused by varying assays. A limitation of the study is that all the patients were from the same hospital district, and thus a larger sample size and longer follow-up is needed for more accurate validation of these findings in order to guarantee generalizability.2log-transformation of the data, and makes our conclusions and estimates comparable to any study utilizing the well-established PSADT as an end-point32Our results indicate that u-PSA provides useful information for predicting BCR after RP. The utilized approach of considering PSADT was easily achieved using How to cite this article: Laajala, T. D. et al. Longitudinal modeling of ultrasensitive and traditional prostate-specific antigen and prediction of biochemical recurrence after radical prostatectomy. Sci. Rep.6, 36161; doi: 10.1038/srep36161 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Functional imaging studies using BOLD contrasts have consistently reported activation of the supplementary motor area (SMA) both during motor and internal timing tasks. Opposing findings, however, have been shown for the modulation of beta oscillations in the SMA. While movement suppresses beta oscillations in the SMA, motor and non-motor tasks that rely on internal timing increase the amplitude of beta oscillations in the SMA. These independent observations suggest that the relationship between beta oscillations and BOLD activation is more complex than previously thought. Here we set out to investigate this rapport by examining beta oscillations in the SMA during movement with varying degrees of internal timing demands. In a simultaneous EEG-fMRI experiment, 20 healthy right-handed subjects performed an auditory-paced finger-tapping task. Internal timing was operationalized by including conditions with taps on every fourth auditory beat, which necessitates generation of a slow internal rhythm, while tapping to every auditory beat reflected simple auditory-motor synchronization. In the SMA, BOLD activity increased and power in both the low and the high beta band decreased expectedly during each condition compared to baseline. Internal timing was associated with a reduced desynchronization of low beta oscillations compared to conditions without internal timing demands. In parallel with this relative beta power increase, internal timing activated the SMA more strongly in terms of BOLD. This documents a task-dependent non-linear relationship between BOLD and beta-oscillations in the SMA. We discuss different roles of beta synchronization and desynchronization in active processing within the same cortical region. Regular time intervals reflect fundamental characteristics of rhythmic events that the brain uses to optimize perception and motor behavior. Anticipation of future events after having internalized temporal regularities in the sensory input is called internal timing  or on only every fourth identical auditory stimulus . While the tapping rate changed for slow and fast conditions, the auditory stimulus did not change across conditions. We hypothesized contrasting the internal generation of a slow rhythm during slow tapping against simple auditory-motor synchronization during fast tapping revealed effects associated with internal timing, because the slow tapping rate was generated internally in the presence of a constant stream of auditory stimuli.To test for effector effects on SMA activity, tapping was either performed with the left and the right hand. While effects of hand have already been shown for the primary motor areas in electrophysiological and fMRI studies  were included in the EEG-fMRI study. Participants had normal or corrected-to-normal visual acuity, no neurological deficits and were right-handed according to self-reports and their scores on the Edinburgh inventory of manual preference . For each hand, a downward arrow indicated slow tapping, an upward arrow fast tapping, and a dot a stationary hand . Four auditory beats of higher pitch primed the fast tapping rate and tapping onset. An inter-block-interval, during which a fixation-cross was presented, was jittered between 5.5 s and 8 s. All eight conditions were presented six times in randomized order, resulting in a total of 48 blocks lasting 22 min.RRID: SCR_002521). Participants were asked to restrict their gaze to the center of the screen during the task. Two pneumatic Biopac pressure sensors  were used to record participants’ tapping pressure. These were attached to the pads of participants’ index fingers. Participants were lying in a supine position in the MR bore and were asked to tap with their index fingers on their ipsilateral thigh. The pressure sensitivity of the sensors was 0.01 cm H2O with a sampling rate of 1 kHz. Pressure data were inspected for tapping errors. They were below 2% in each participant; thus, no participant was excluded from further analyses.A projector was used to display the visual stimuli on a screen that participants viewed via an MR compatible mirror and auditory beats were presented using MR-compatible headphones . The auditory beats and the visual instructions were presented with Presentation software .During scanning, EEG was recorded using a BrainAmp MR EEG amplifier  and a BrainCap electrode cap  with 30 EEG and 2 EOG channels. Ag/AgCl EEG ring electrodes were positioned according to an extended 10/20 system with a reference electrode placed between Fz and Cz as used in Viola et al. . The imp−1 and a maximum of 512 steps was applied for matrix decomposition. Finally, all channels were referenced to the average of all EEG channels. For each participant, the length of all visually marked artifacts was summed up and set in relation to the total block duration. The entire data set was discarded when more than 10% of the data were affected by artifacts  according to standard preprocessing procedures . These electrodes were defined using an independent EEG/MEG measurement in 17 participants from which eight participants also participated in the current EEG/fMRI study. MEG was recorded using a whole-head system  with 275 channels at a sampling rate of 1200 Hz. Simultaneous EEG was recorded with a custom-made cap equipped with 64 MEG-compatible AG/AgCL electrodes . Participants performed the identical finger tapping paradigm during parallel EEG/MEG recording which allows for proper source analyses. MEG and EEG data were filtered off-line with fourth-order Butterworth 300 Hz low-pass and 2 Hz high-pass filters. Line noise at 50 Hz was bandpass-filtered. Recorded data were down-sampled to 1000 Hz. Blocks containing muscle and SQUID artifacts were removed using an automatic artifact rejection algorithm . More detailed results of the MEG data will be presented elsewhere.For source identification, the mean activity of the left and the right extensor digitorum communis muscle during the bimanual fast tapping condition was used as an external reference signal to detect coherent MEG sources in the brain . Due to residuals of scanner artifacts in high-frequency ranges  separately for mean power differences in low (14–24 Hz) respective high (25–35 Hz) beta. Second, condition differences were investigated using a 2 × 2 ANOVA with timing  and hand  as factors. Post hoc t-tests were additionally performed for significant main effects . All analyses were performed in SPSS for low and high beta separately .First, all tapping conditions were compared with fixation baseline using paired 3 isotropic size) were obtained. To reduce head motion, a vacuum cushion was used .Image acquisition was performed on a Siemens Trio 3 Tesla magnetic resonance system  equipped with a circular polarized Send/ Receive head coil with an integrated preamplifier. Functional images were obtained with a gradient-echo T2*-weighted transverse echo-planar imaging sequence  = 2.08 s; echo time (TE) = 29 ms; flip angle = 90°; 32 axial slices in descending order; 3 mm × 3 mm × 3 mm isotropic voxel size). Additionally, high-resolution T1-weighted anatomical scans . After eliminating the first four volumes in each participant due to field inhomogeneity of the scanner in the beginning of each run, standard preprocessing was performed  standard brain template, and smoothing with an 8 mm full-width at half-maximum Gaussian kernel). The preprocessed images were analyzed within the framework of general linear models (GLM) for time-series data  in addition to four regressors of no interest  were modeled by convoluting the onsets and durations of conditions (modeled by boxcar functions) with the canonical hemodynamic response function to obtain predicted BOLD responses. Additional nine regressors of no interest were capturing the variance associated with the instructions for each conditions and an additional tap participants usually made after the last metronome click in fast conditions. Six non-convolved regressors were modeling head-motion-related effects.RRID: SCR_009605). Analyses were performed for the left and right SMA separately in SPSS .For group-level analyses, the four regressors of interest, modeling condition-specific tapping effects, were included in a 2 × 2 ANOVA, similar to the aforementioned EEG analysis. The SMA coordinate  reflected the MNI coordinate of the MEG source (see above). Because the coordinate falls in the inter-hemispheric fissure, two cubic ROIs were used to extract fMRI beta values from the left and right SMA, separately . For each of the four conditions of interest, average beta values for the two ROIs were extracted from all 20 subjects with their respective standard error of the mean . Post hoc t-tests in the low beta band revealed higher low beta power in slow than in fast tapping conditions . In the high beta band, post hoc t-tests revealed stronger power decreases for the left than for the right hand . Yet, the hand effect resulted primarily from the F0 condition, which showed the strongest high beta power decrease .To investigate BOLD signal condition differences in the SMA, ROI analyses were performed. As expected, all conditions showed more activation compared to fixation baseline Figures . A main Our study revealed four key findings in the SMA. First, the comparisons against baseline revealed the well-known condition-independent task-related desynchronization of low and high beta oscillations together with a concomitant BOLD activation. Second, within the overall suppressed beta state of the SMA during tapping, generation of an internal rhythm relatively increased beta amplitude in parallel to a concordant increase in BOLD activation. Third, and as a consequence of finding one and two, no general linear relationship between the EEG signal in the beta band and the BOLD signal was found. Fourth, tapping with the left compared to right hand decreased beta power in the SMA, particularly for fast tapping, while this effect was not observed in BOLD.When investigating SMA activity-sensitive EEG electrodes for condition effects in relation to baseline, beta desynchronization was found for all conditions in the low (14–24 Hz) and high beta band (25–35 Hz). Since these effects were condition-independent they likely relate to a general task-related activation of the SMA. Indeed, BOLD analyses documented such activation that has previously been related to movement effects in fMRI finger tapping studies  were interpreted as active processes. A recent neurodynamic model focusing on cortico-hippocampal interactions during memory encoding proposes an innovative perspective on the active role of beta desynchronization even in non-motor cortices (Hanslmayr et al., Earlier studies associated the simultaneous occurrence of cortical synchronization and desynchronization in alpha and low beta bands with processing in thalamo-cortical networks (Pfurtscheller and Lopes da Silva, The diminished beta band desynchronization for slow compared to fast finger tapping could potentially also be interpreted as a consequence of different tapping rates. Yet, if reduced motor activity in slow compared to fast tapping would relate to the observed beta power increase, consequences of rate effects would also apply to the fMRI results. Higher tapping rates are linearly related with BOLD increases, however, in primary motor cortices (Hayashi et al., While effects of internal timing were observed in the low beta band, effects of hand were limited to the high beta band. This is not only in accordance to previous findings, which associate finger movement to oscillations in the high beta band in the SMA (McFarland et al., Our results indicate that beta suppression cannot be simply equated with increases in BOLD signal. While the relationship between beta power and BOLD activation may differ from brain region to brain region (Kujala et al., Internal timing-related effects have mostly been associated with oscillations in the beta band (Gerloff et al., Our study design included eight conditions and was thus not ideally designed for classical EEG-fMRI analyses of correlations between the BOLD signal and EEG power fluctuations over time, especially with respect to analyses of correlations between the beta power envelope beyond task-related desynchronizations with BOLD activity. Such an analysis requires larger time windows for correlation compared to the block length used in this study. With the knowledge of internal timing-related effects for unimanual conditions, a reduced task design with only unimanual conditions and longer tapping blocks could be more appropriate to capture such effects using standard analyses.The EEG electrode selection was based on significant correlation with MEG source signal in the SMA. We are thus confident that the signal reflects activity in the SMA. Yet, we cannot exclude that these electrodes also picked up signal from neighboring cortices like slightly more lateral aspects of the dorsal premotor cortex. We believe their contribution is marginal since time courses in the left and right dorsal premotor cortices did not correlate with the set of EEG electrodes reflecting activity in the SMA.In conclusion, our findings suggest internal timing-related low beta synchronization occurs in the context of movement-related beta desynchronization in the SMA. The non-linear relationship between beta power effects and BOLD activation is suggestive of active contributions of both regional beta desynchronization and subregional beta synchronization to active processing.This article was written by FG, AP, HL and CAK. FG recorded, preprocessed and analyzed the EEG-fMRI data with help of CAK. AP recorded, preprocessed and analyzed the EEG-MEG data and programmed scripts. HL provided the EEG-fMRI equipment and gave methodological support for EEG-fMRI acquisition and subsequent 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.


Disruptions in chromatin structure are necessary for the regulation of eukaryotic genomes, from remodelling of nucleosomes at the base pair level through to large-scale chromatin domains that are hundreds of kilobases in size. RNA polymerase is a powerful motor which, prevented from turning with the tight helical pitch of the DNA, generates over-wound DNA ahead of itself and under-wound DNA behind. Mounting evidence supports a central role for transcription-dependent DNA supercoiling in disrupting chromatin structure at all scales. This supercoiling changes the properties of the DNA helix in a manner that substantially alters the binding specificity of DNA binding proteins and complexes, including nucleosomes, polymerases, topoisomerases and transcription factors. For example, transient over-wound DNA destabilises nucleosome core particles ahead of a transcribing polymerase, whereas under-wound DNA facilitates pre-initiation complex formation, transcription factor binding and nucleosome core particle association behind the transcribing polymerase. Importantly, DNA supercoiling can also dissipate through DNA, even in a chromatinised context, to influence both local elements and large chromatin domains. We propose a model in which changes in unconstrained DNA supercoiling influences higher levels of chromatin organisation through the additive effects of DNA supercoiling on both DNA-protein and DNA-nucleosome interactions. This model links small-scale changes in DNA and chromatin to the higher-order fibre and large-scale chromatin structures, providing a mechanism relating gene regulation to chromatin architecture in vivo. Supercoiling is a transition from the relaxed state of the DNA double helix to one that is more under- or over-wound Fig. a. In DNANucleosome core particles connected by linker DNA are the fundamental unit of chromatin structure Fig. . Arrays In eukaryotes most DNA supercoiling is generated by the transcription machinery  DNA Champoux . EukaryoThe presence of unconstrained supercoiling in the linker DNA introduces free energy into the double helix Fig.  which prUnderstanding how the free energy of DNA supercoiling influences DNA–protein interactions through changes in DNA structure is key to identifying the effects of DNA supercoiling on chromatin architecture. Protein–DNA interactions that require changes to the structure of the DNA double helix, including melting and bending, are generally facilitated by an under-wound DNA structure  of the human At promoter regions an under-wound DNA structure may facilitate the formation of a disrupted, alternative or melted DNA structure that influences DNA protein binding. The TATA-box DNA sequence element is predicted to be sensitive to under-wound DNA supercoiling, and the structure of TATA-box binding protein bound to DNA identifies that the DNA has an under-wound structure  dependent manner. This influences the accessibility of linker DNA, which provides a reservoir of unconstrained DNA supercoils, and the capacity of supercoiling to promote/inhibit DNA–protein interactions.The next level of chromatin organisation above the nucleosome array is the higher-order fibre Fig. , which iThe higher-order chromatin fibre is influenced by the underlying distribution of nucleosomes on the nucleosome array and by other DNA–protein interactions. A fibre containing regular repeats of the Widom-601 nucleosome positioning sequence has a uniform structure which has been determined by X ray crystallography  gene. Measuring under-wound DNA with a biotin psoralen probe and visualising by immunofluorescence with streptavidin-green fluorescent protein, these same authors demonstrated that DNA becomes under-wound prior to expression of the HSP70 gene. This result indicates that transcription-generated DNA supercoiling primes large-scale chromatin domains prior to productive gene expression and together with other results supports a role for unconstrained DNA supercoiling in the structure of large-scale chromatin domains in vivo.The large-scale influence of DNA supercoiling on chromatin structure and genome regulation can also be observed cytologically, through changes in the compaction of large-scale chromatin structures. Fluorescence in situ hybridisation studies on human tissue culture cells revealed that under-wound DNA supercoil domains are cytologically decompacted compared to gene-poor over-wound domains and that this decompaction is lost in the presence of transcription inhibitor or DNA-nicking reagents  and condensin Hirano , but furUnderstanding the influence of DNA supercoiling on chromatin structure and gene regulation is in its infancy, despite almost 30 years of research following the publication of the twin-supercoil domain model (Liu and Wang In this model there is the potential for local amplification of unrestrained DNA supercoiling and gene expression in regions containing multiple active genes Fig. . At the To further understand how DNA supercoiling influences protein–DNA interaction, chromatin structure and gene regulation it is necessary to determine the properties of promoters and regulatory elements that are sensitive to DNA supercoiling. The FUSE element of c-myc has been the classic example of a supercoil sensitive protein-binding sequence element, and the TATA-box sequence has been implicated as supercoil sensitive at promoters, but it is unclear how prevalent supercoil-dependent regulation is in eukaryotic genomes. A recently described single-stranded DNA sequencing technique for mapping melted DNA structure in human cells provides a starting point for understanding the prevalence of supercoil-sensitive sites in the genome (Kouzine et al. Drosophila polytene chromosomes (Kouzine et al. Above the scale of individual genes, the importance of DNA supercoiling on chromatin structure in vivo has so far been determined by the addition of transcription inhibitors, topoisomerase inhibitors or DNA-nicking agents or through heat shock in Taking these approaches to understand how transcription and DNA sequence function together, through DNA supercoiling, to facilitate protein binding and chromatin structure will offer fresh insight into the role of DNA structure in the chromatin fibre. The model we propose identifies DNA supercoiling as a key factor regulating general principles of chromatin architecture, in addition to individual protein–DNA interactions, by transmitting information about gene regulation from the site of transcription through the chromatin fibre and over large-scale domains.


It is a 25-amino acid residue peptide blocking N-type voltage-gated calcium channels. Its FDA definition reads that ziconotide is intended for the treatment of chronic severe pain in patients that are intolerant or refractory to systemic analgesics or intrathecal morphine [Bothrops jararaca led to the development of Captopril, even prior to Prialt®. Captopril, and its follow-up compounds, are widely used as inhibitors of angiotensin converting enzyme (ACE) [®, which is a synthetic version of a hormone called exendin-4 isolated from the saliva of the glia monster [® has been approved by the FDA since 2005 [Venoms from marine and terrestrial animals  can be seen as an untapped cocktail of biologically active compounds, being increasingly recognized as a new emerging source of peptide-based therapeutics. Venomous animals are considered specialized predators that have evolved the most sophisticated peptide chemistry and neuropharmacology for their own biological purposes by producing venoms that contain myriads of toxins with an amazing structural and functional diversity. These neurotoxins have been shown to be highly selective and potent ligands for a wide range of ion channels and receptors. Therefore, they represent interesting lead compounds for the development of novel medicines, for example, analgesics, anti-cancer drugs, and drugs for neurological disorders. A successful example of venom-derived peptides used is the drug Prialtmorphine . Charactme (ACE) ,4. Anoth monster . Exenatince 2005 . In this special issue, “Toxins in Drug Discovery and Pharmacology”, we have attempted to provide the reader with a comprehensive overview of new toxins and toxin-inspired leads. This issue focuses on the mechanism of action, structure–function, and the evolution of pharmacologically interesting venom components, including, but not limited to, recent developments relating to the emergence of venoms as an underutilized source of highly evolved bioactive peptides with clinical potential. The following is a short synopsis of the six reviews and 15 research papers that constitute this special issue.Phoneutria nigriventer that activates μ-opioid receptors without inducing β-arrestin2 recruitment. PnPP-19 is the first spider toxin derivative that, among opioid receptors, selectively activates μ-opioid receptors. The lack of β-arrestin2 recruitment highlights its potential for the design of new improved opioid agonists. Agwa and colleagues  have expDong et al.  describeThe anti-cancer properties of snake venom have been investigated by Osipov et al. by verifying the anti-tumor effects of nerve growth factor from cobra venom. This work suggests that the antitumor effect of nerve growth factor in vivo depends critically on the normal status of the immune system. The nerve growth factor antitumor mechanism may cause an increase in lymphocytic infiltration in the tumor, a rise in the levels of IL-1β and TNF-α in the serum of tumor-bearing mice, and an increase in aerobic glycolysis . Sales eAnother source for venom-based drug discovery is bee venom. Bee venom given subcutaneously attenuates allodynia in mice models of CPIP without notable adverse effects. The anti-allodynic effects were closely associated with a significant decrease in NK-1 receptor expression in DRG. These findings suggest that repetitive bee venom therapy could be a useful therapeutic modality for the treatment of complex regional pain syndrome type I . Shin anLepiarczyk and colleagues  describeVespula vulgaris with potent antimicrobial activity against Salmonella infection. However, there exist some limits for its practical application due to the loss of its activity in an increased bacterial density and the difficulty of its efficient production. Ha et al. [Escherichia coli secretion system efficiently producing active mastoparan V1.The mastoparan V1 is a mastoparan from the venom of the social wasp a et al.  modulateShort toxin-like proteins from insects are often overlooked in drug discovery. The study performed by Linial et al.  indicateTwo dipeptides with anticoagulant activity have been isolated from scorpion venom . This stPark and Park  providedRoyal and Motoba  review tThe anti-metastatic mechanisms of snake toxins can be divided into three molecular targets. These are the inhibition of extracellular matrix components-dependent adhesion and migration, the inhibition of the epithelial–mesenchymal transition and the inhibition of the migration by alterations in the actin/cytoskeleton network. The molecular mechanisms in which snake toxins target metastasis are reviewed by Urra and Araya-Maturana .Conus has become an important genetic resource for conotoxin identification and drug development. The many challenges of drug discovery from cone snail venom are reviewed by Gao et al. [The genus o et al. .Animal toxins are valuable tools to study ion channels such as TRPV1. A comprehensive summary of the advancements made in TRPV1 research in recent years by employing venom-derived peptide toxins is provided by Geron and colleagues . In this


Dv) and stomatal density (SD) have been shown to be strongly positively correlated in response to a range of environmental variables in more recently evolved plant species, but the extent of this relationship has not been confirmed in earlier diverging plant lineages. In order to examine the effect of a changing atmosphere on the relationship between Dv and SD, five early-diverging plant species representing two different reproductive plant grades were grown for 7 months in a palaeo-treatment comprising an O2:CO2 ratio that has occurred multiple times throughout plant evolutionary history. Results show a range of species-specific Dv and SD responses to the palaeo-treatment, however, we show that the strong relationship between Dv and SD under modern ambient atmospheric composition is maintained following exposure to the palaeo-treatment. This suggests strong inter-specific co-ordination between vein and stomatal traits for our study species even under relatively extreme environmental change. This co-ordination supports existing plant function proxies that use the distance between vein endings and stomata (Dm) to infer plant palaeo-physiology.In order to be successful in a given environment a plant should invest in a vein network and stomatal distribution that ensures balance between both water supply and demand. Vein density ( Plants are a critical component of the hydrological cycle, influencing the amount of water vapor that is returned to the atmosphere via the process of transpiration  is the length of veins per leaf area (mm mm-2), and in angiosperms it is determined predominantly by the minor veins, as they make up >80% of the total vein length of the leaf (Kx) and outside xylem conductivity (Kox), parameters that determine leaf hydraulic conductance (Kleaf) (Stomata are microscopic pores on a leaf surface that regulate gas exchange. COthe leaf . Stomatathe leaf . Stomata genetic  and envi genetic . A chang genetic . A netwothe leaf . Minor v (Kleaf) . Thus itDv and SD in response to light (T) across a range of ferns, conifers, and angiosperms from both tropical and temperate ecosystems  in a diverse range of Proteaceae species (Dv and gmax (Dm) between vein endings and stomata. In multi-veined species, veins should be optimally placed to minimize Dm ensuring maximum photosynthetic capacity  and demand (SD) traits is critical to plant success and the relationship between the two seems to be conserved across the major plant groups under present day atmospheric conditions (2) and CO2 in the atmosphere change. Co-ordination between these two morphological traits may have been critical throughout the past 400 million years during times of fluctuating atmospheric O2 and CO2. Maintaining this balance between water supply and demand may have allowed certain species to operate more efficiently in their environment. For example, it has been widely proposed that the co-evolution of leaf traits (an increase in Dv and SD) during the Cretaceous decline in atmospheric CO2 allowed angiosperms to outcompete other plant groups as they transitioned from predominantly moist to drier habitats , this study examines for the first time the effect of changing atmospheric conditions on the relationship between Dv, SD, and gmax. Using four angiosperm and one fern species, the plasticity and co-ordination of these morphological plant traits was assessed in a low O2/high CO2 atmosphere. In the context of this study, co-ordination in plant traits refers to either inter-specific or intra-specific co-ordination. Inter-specific co-ordination is taken to mean an observable trend in morphological plant traits across the experimental species. Intra-specific co-ordination on the other hand, is when the direction of change of plant traits is the same within a single species. We acknowledge that the number of species studied here is relatively small and experimental conditions limited to one palaeo-treatment, and as such, results discussed here are merely intended to be a suggestion of the possible behavior of early diverging plant species under changing atmospheric conditions. Further studies using a wider range of species and palaeo-treatments will build on the current study and will allow more robust conclusions to be drawn.Using a range of early diverging plant species  is a plant functional trait calculated using both SD and anatomical measurements of stomatal geometry  of ancient fossil species. The extent of the relationship between gmax and operational stomatal conductance (gop) has been shown in two angiosperm species (gmax and gop were found to be strongly related (r2 = 0.54), with a scaling relationship of gop = 0.25 gmax. Therefore gmax was used in the current study as a means to infer changes in physiological behavior with a change in the concentration of O2 and CO2 and to relate this to changes in Dv.Results were used to determine the robustness of plant function proxies that rely on co-ordination in morphological traits (such as the use of ion rate ), specifgeometry . This try models  to infer species  and more species . Across Cyathea australis) and angiosperms , were grown for approximately 7 months in Conviron  BDW-40 walk-in plant growth chambers at PÉAC , Rosemount Environmental Research Station, University College Dublin. For each of these plant groups, the earliest diverging species obtainable from each plant family was used in order to follow the nearest living relative (NLR) protocol  and a palaeo-treatment of low O2/high CO2 . These conditions represent prehistoric modeled atmospheres  and using Image J software the length of veins in these areas was manually traced (using a Wacom Intuos4 pen tablet) and Dv calculated in Excel. Minor Dv is independent of leaf size and accounts for the majority (>80%) of total vein length per area in most angiosperms  attached and Syncroscopy Automontage  digital imaging software was used to impose grids and scale bars on each image.SD and protocol . Leaves iosperms , therefogmax quantification, anatomical measurements of 90 to 120 stomata per species and per treatment were obtained using the same images used for SD determination . gmax was calculated using the following diffusion equation (For equation :2 s-1), ν = molar volume of air (0.0224 m3 mol-1), SD = Stomatal density (m-2), pamax = max stomatal pore area (m2), pd = stomatal pore depth (m). The maximum stomatal pore area was calculated (treating the pore as an ellipse) by using stomatal pore length as the long axis, pore length/2 as the short axis and taking the stomatal pore depth as being equal to the width of a fully turgid guard cell  and ferns  were grown for 18 months in walk-in Conviron growth chambers in both an ambient and low O2/high CO2 atmosphere . These species have either parallel or dichotomously branching major veins, therefore it was not necessary to clear and stain leaves for Dv observation. Dv was calculated on one leaf per plant and three plants per species per treatment. Leaves were imaged using a Nikon SMZ1000 stereomicroscope with Leica DFC490 camera attached , and using Image J software the length of veins was manually traced and Dv calculated in Excel in an area between 20 mm2 and 60 mm2.In order to assess whether other non-angiosperm species show a change in Dv, and gmax between treatments. Minitab (version 16.1.1) statistical software was also used for correlation tests, boxplot representation of data, and to graphically display percent changes in SD, Dv, and gmax. RStudio (version 0.99.489) was used for Standardised Major Axis (SMA) regression analysis and for scatterplot representation of data.Data were first checked for normal distribution and Generalized Linear Models were run in Minitab (version 16.1.1) statistical software to investigate differences in SD, Dv, and gmax to the palaeo-treatment . A significant decrease in SD is seen in two species (Chimonanthus praecox shows a 24% and Zantedeschia aethiopica a 34% decrease), Magnolia delavayi and Cyathea australis show a non-significant yet noticeable decrease , and another angiosperm (Cornus capitata) shows a small (9%), but non-significant increase . Dv shows a similar mix of responses, three of the species show a significant decrease , Magnolia delavayi shows a very slight (2%) yet non-significant increase, and Cornus capitata shows a significant (14%) increase . Two of the angiosperm species (Chimonanthus praecox and Zantedeschia aethiopica) show a significant decrease in gmax  in response to the palaeo-treatment, with Magnolia delavayi showing a non-significant decrease (11%), Cornus capitata a non-significant increase (15%), and Cyathea australis a non-significant decrease (2%) . See Supplementary Table F and associated p-values).Species show a varied and species-specific response in SD, Chimonanthus praecox, Cornus capitata, and Zantedeschia aethiopica) and the fern species Cyathea australis demonstrate intra-specific co-ordination of Dv, SD, and gmax in response to the palaeo-treatment . This co-ordination is evident even though one angiosperm species (Cornus capitata) shows an increase in all three parameters while the remaining species show a decrease. Magnolia delavayi shows co-ordination in two plant traits in response to the palaeo-treatment (SD and gmax), however, co-ordination is lacking between both of these parameters and Dv.Three out of four angiosperm species  is strong under ambient conditions  and it persists in the palaeo-treatment . Similarly, Dv and gmax  show a strong relationship under both the ambient  and palaeo-treatment . The slopes of the regression lines between Dv and SD and Dv and gmax are not significantly different in the ambient and palaeo-treatments  in response to growth in 35% O2  have vein densities higher than 6 mm mm-2 , the ‘critical vein density’  is interesting , as it is thought that non-angiosperm species are incapable of altering their vein architecture in the same way that angiosperm species can . Examination of archived leaf material of three gymnosperms and one fern species from a 2009 palaeo-experiment , but has no effect on Dv in the remaining species (two gymnosperms and one fern). These non-angiosperms have vein densities below 2 mm mm-2 and all have either parallel or dichotomously branching major vein networks, implying that this vein configuration may lack developmental plasticity. The major veins of non-angiosperms are generally thicker in diameter and their xylem anatomy is distinct from that of the angiosperms, lacking an important feature that is believed to be paramount in the proliferation of minor veins, vessels with simple perforation plates. Only angiosperms evolved these less resistive perforation plates, and this in conjunction with the development of thinner minor veins may have allowed this plant group to outperform non-angiosperms  demonstrates inter-specific co-ordination in two morphological plant traits that determine hydraulic supply and demand across different plant lineages and under a changing atmosphere. Furthermore, the robust relationship observed between Dv and gmax  demonstrates that morphology has the potential to influence the physiological behavior of these species, via the strong relationship already found between gmax and gop . Three out of four angiosperm species and the fern species show intra-specific co-ordination in Dv, SD, and gmax in response to the palaeo-treatment. Co-ordination between traits that determine the water relations (supply and demand) of a plant is critical for its survival. For example, an increase in SD and/or gmax would increase the evaporative demands of the plant and without a corresponding increase in Dv (to match the increase in water demand with an increase in hydraulic supply) the plant would be mal-adapted to its environment and would most likely not survive. The opposite scenario would not be as detrimental to plant survival, however, a decrease in SD and/or gmax without a corresponding decrease in Dv would result in a waste of resources, the construction of veins being costly to the plant  by evolving smaller stomata . It is interesting, however, that examined non-angiosperm species from the 2009 palaeo-experiment show limited plasticity in Dv as well as in SD (see SD results for these non-angiosperm species reported in The current study supports these theories by demonstrating a higher degree of plasticity in 2 and declining CO2 (2 and high CO2 conditions. The robust positive relationship observed here between the density of veins and stomata strongly supports the theory suggested by Dm) is minimized. Although Dm was not directly measured in this study, the morphological co-ordination observed suggests that any change in Dv will elicit a corresponding change in SD or vice versa, allowing the leaf to minimize the distance between veins and stomata, and to maximize photosynthetic performance and operational efficiency of the leaf.Furthermore, these results suggest that angiosperms are not only capable of showing morphological plasticity in response to rising Oning CO2 , but alsDv and SD persists . Furthermore, the positive relationship observed between Dv and gmax  demonstrates the link between hydraulic and gas exchange/diffusional processes in these species, as shown in previous studies (Dm is most likely maintained under changing atmospheric conditions (due to the intra-specific co-ordination between Dv and SD) has important implications when attempting to understand plant–atmosphere interactions throughout the last 400 million years of plant evolution. A lack of co-ordination in Dv and SD on developmental time-scales would result in a plant that is morphologically and physiologically out of sync, negatively impacting operational efficiency and overall fitness under changing atmospheric conditions. Furthermore, plant species that exhibited plasticity in these morphological traits under a changing atmosphere would likely have had an ecological advantage over plant species that were morphologically inflexible, being able to maximize their photosynthetic capacity as the surrounding environment changed (Dm)   remains  (Dm) (2  since th (Dm) (2 . The curstimates , howeverDv, and gmax to growth in an experimental low O2/high CO2 palaeo-atmosphere. Regardless of this variation in responses, a strong relationship is observed between Dv and SD and Dv and gmax under both the ambient and palaeo-atmosphere. Gymnosperms studied here appear to lack the same degree of developmental plasticity in Dv compared to the angiosperms, at least on short experimental time-scales. The ability to increase their range of Dv values may have contributed to the success of angiosperms during the Cretaceous decline in CO2; a high degree of plasticity in this trait possibly provided early diverging angiosperms with a competitive advantage over other seed plant groups in more changeable environments. The tight relationship observed between Dv and SD in the palaeo-treatment suggests that Dm is likely maintained under environmental change and lends confidence to existing palaeo-CO2 proxies that use this parameter in their models. Further studies examining the robustness of these plant trait relationships under a range of O2:CO2 ratios are needed in order to elucidate the full spectrum of plant–atmosphere interactions throughout the last 400 million years.Species show a varied response in SD, gmax calculation, statistical analysis, and drafted the manuscript. AP contributed SD and gmax data for Cyathea australis. CE-K was involved in the 2009 palaeo-experiment and therefore provided dried leaf material for vein density analysis. JM is the principal investigator. All authors read, revised, and approved the final manuscript.CE-F carried out vein density and SD analysis, anatomical stomatal measurements for 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.


Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome. cis-regulatory elements to control gene expression. Here, mouse-specific TEs are shown as binding sites for multiple collaborating transcription factors in embryonic stem cells, and act as cis-regulatory modules in synergistic fashion.The gene-battery model posits transposable elements (TEs) may be  Transposable elements (TEs) are widely known for their capability to affect gene expression, beginning with Barbara McClintock's seminal maize experiments1346789111213cis-regulatory modules of TF-binding sites and coordinating the transcription of multiple genes at the same developmental stage1516cis-regulatory modules encoded in TEs.A few studies have demonstrated that TEs indeed can rewire existing gene regulatory networks, supporting Britten and Davidson's hypothesis15cis-regulatory sites, but also cis-regulatory modules. TE-derived modules of TF-binding sites make an interesting model for studying the regulatory role and innovation contributed by TEs to a genome. A TE-derived cis-regulatory module represents a collection of TF-binding sites that together regulate the expression of nearby target genes. Although there are no well-characterized cases of TE-derived cis-regulatory modules that we know of, this will provide important support to the regulatory innovation contributed by TEs to a genome. The benefit of TE-derived cis-regulatory modules is that TEs form an efficient evolutionary mechanism for the rapid spread of cis-regulatory modules, as opposed to alternatively acquiring the cis-regulatory module via point mutations in the sequence. Here we study and characterize a module of binding sites for pluripotency TFs in mouse embryonic stem cells (ESCs) in conjunction with the associated ability of these TEs to regulate gene expression. We observe that indeed the TE-derived TF-binding modules enhance gene expression synergistically. Interestingly, our results indicate that the TEs likely acquired the potential to regulate gene expression from their ancestral sequences.An interesting part of the Britten and Davidson hypothesis is the theoretical description of TEs containing not only in vivo binding sites for these five TFs occur in TEs (10We used chromatin immunoprecipitation-sequencing (ChIP-seq) to analyse the binding of five pluripotency TFs in mouse ESCs192021222325r in TEs . The majin TEs 10. Overallin TEs 10. Interesin TEs 10.P value<0.001; in vivo TF binding  of various individual TF's binding. The six TE subfamilies enriched for multiple TF-binding motifs  of binding sites for various numbers of TFs in each TE subfamily (see Methods). Certain TE subfamilies showed strong enrichment  sequence, the sequence of the two LTRs flanking the ERV is identical, but post insertion, the LTRs evolve independently and their sequence diverges. By estimating the sequence variation between two LTR sequences of a nearly intact ERV sequence, the time of insertion of the element can be calculatedNext, we set out to define an epigenetic signature of the TEs bound by multiple TFs (see Methods). As expected, candidate TEs that encoded multiple TF-binding sites were marked by increased H3K4me1 and H3K27ac, bound by P300, and had reduced DNA methylation, which are known signatures of transcriptional enhancers . InteresWe then compared the epigenetic signatures of TEs bound by two or more TFs in mouse ESCs, with other mouse cells to determine the cell-type specificity of this enhancer epigenetic signature (see Methods). The enhancer signature of TEs bound by multiple TFs was specific to mouse ESCs and absent in other cell types . CorrespAkap12 , and no significant change in nearby genes Next, we determined the effect of TF-binding sites on the TE's ability to regulate gene expression. We identified that three out of the six TE subfamilies that could regulate gene expression had binding sites for Esrrb, Klf4 and Sox2 (EKS) in the upstream part of their sequence . We sougt-test P value<0.05). Although mutating any one motif reduced the regulatory potential of the wild-type sequence, the extent to which the mutation reduced the regulatory potential was different for different motifs and TE sequences. Importantly, the reduction in regulatory potential caused by mutating one motif is comparable to mutating all three motifs. Our results show that the effect of the motifs in the EKS cluster is greater than the sum of each motif. Since the regulatory potential of the TEs is dependent on the presence of all three motifs, it suggests that this EKS motif module in TEs work cooperatively to enhance gene expression. We also observed that mutating all motifs in the EKS motif module reduced the expression of the wild-type sequence and made it comparable to TEs from the same subfamily  that lacked the EKS motifs . This suggests that the EKS motif module drives the regulatory potential observed from these TEs, and more importantly the EKS motifs work as a module that synergistically enhance gene expression.We identified that mutations to the EKS motifs reduce the expression of the wild-type TE sequence . MutatinThe ability of TEs to act as transcriptional enhancers is well documented6101142434418142931To investigate the evolution of EKS module in these TE subfamilies, we scanned the RepBase-consensus sequence for TF-binding motifs  between the genomic TE and RepBase-consensus sequences  that were enriched for multiple TFs-binding sites , making them candidates to study TEs' role in evolving pression . Using tession 30, but alsession 30, just liAkap12 is an example of a gene regulated by RLTR9E specifically in mouse ESCs. Mouse and human ESCs are known to have differences in their signalling pathwaysAkap12 is one gene involved in signalling pathways and specifically upregulated in mouse ESCs. Taken together, our results highlight a role for TEs in depositing modules of pluripotency TF-binding sites and contribute towards mouse ESC-specific gene expression patterns.Genomic TEs most likely inherited the regulatory potential and the module from the ancestral TEs. However, we also observe that the genomic TEs have lower regulatory potential in comparison with the ancestral state. This is consistent with the hypothesis that when TEs are co-opted to serve a host function their potential can be tinkered . The total number of peaks used in the analyses for this paper is tabulated in We used ChIP-seq peaks from Chen patserP value<0.001) that matched the motif's consensus.To identify TF-binding motifs, we used position-weight matrices for the five TFs from JASPARTo determine clusters of TF-binding sites , we identified regions that had multiple TF-binding sites that were within 100 bp of each other. For ChIP-seq peaks, we used the centres of the peaks as the reference point for each peak, while for TF-binding motifs we used the starting position of the motif.intersectBed tooli' TF-binding sites  in TE subfamily ‘j', using a log odds ratio:We overlapped these clusters of TF-binding sites with RepeatMasker-annotated TEsWe plotted TE subfamilies that had at least 10 elements that overlapped genomic regions with ‘i' TF-binding sites.blast toolFor the evolutionary analyses of these TE subfamilies, we used three methods. First, we used blastp) of each element compared to the RepBase-consensus sequence, from the RepeatMasker output (rmsk file) on the UCSC genome browser. The substitution rate was calculated using the formula:Second, we used sequence divergence to estimate the substitution rate of each element in a particular TE subfamily using the Juke-Cantor evolutionary modelblast2  cells to determine the epigenetic signature of TEs (from the six candidate TE subfamilies) bound by various number of TFs . We downloaded ChIP-seq data for histone modification marks  from the mouseENCODE projectP value<0.1) compared to mouse ESCs. Among these, 1,868 genes were upregulated in specifically in mouse ESCs . We quantified the expression level of Akap12 transcript in wild-type mouse ESCs (RW4 cell line) and cells with the CRISPR-mediated deletion, using the SYBR Green Master Mix (Bio-Rad). The quantification of the expression levels of Akap12 gene, and other neighbouring genes as control (Lrp11 and Nup43), was performed in the wild-type and CRISPR-deleted cells . We used Gapdh as the normalizing gene for these experiments (primers listed in ΔΔCt) for each gene were calculated by comparing the cycle-time (Ct) values of each gene to Gapdh Ct levels (ΔCt) in the same replicate, and then comparing ΔCt values between wild-type and CRISPR−/− cells (ΔΔCt). Results are representative of three independent experiments.To determine the effect of the RLTR9E-derived TF-binding module on the expression of −1 leukemia-inhibiting factor (Sigma) and 0.1 mM β-mercaptoethanol (Gibco).For experimental validation of the enhancer function of TEs in mouse ESCs, we used the RW4 cell line, and cultured it as previously describedTo estimate the regulatory potential of TE and non-TE sequences in mouse ESCs, we used the Dual-Glo luciferase assay (Promega). For the luciferase assay, we cloned TEs into the pGl4.23 vector . We co-transfected the TE-plasmid with pRL-TK (containing renilla luciferase) using lipofectamine (X-GENE-HD at 1 μl for 1 μg of plasmid DNA) into RW4 cells plated in 0.1% gelatin-coated 96-well plate. After 24 h, we assayed the luciferase levels according to the Dual-Glo reporter assay system (Promega).We performed each luciferase experiment in triplicate, and repeated each experiment three times. The luciferase fold change is estimated as the ratio of firefly and renilla luciferase for each TE, and then normalized by the empty vector (pGl4.23 with no insert).dsRed and a Pou5f1 minimal promoter.). Each test TE CRE sequence ; the low correlation score can be attributed to the technical differences in the two assays, including differences in the lengths of the sequences tested (82 bp in CRE-seq compared with ∼300 bp in the luciferase assay). We obtained high correlation between the three biological replicates (R2=0.9). The expression level of a CRE sequence is measured as the average of the ratio of sequencing read counts of cDNA to DNA, per CRE barcode, across biological and technical replicates. We normalized the expression level of each CRE to basal CREs  to make comparisons between CREs. To determine the impact of mutations to the EKS module on the wild-type CRE's expression, we normalized the expression level of the mutant sequence to its corresponding wild-type sequence's regulatory potential.We sequenced each CRE barcode to a depth of 3,000 × coverage, and retrieved on average 96% of the CRE barcodes. The CRE-seq expression levels were correlated with the luciferase assay (We used the QuikChange site-directed mutagenesis protocol (Agilent Technologies) to mutate individual TF-binding motifs in the RepBase-consensus sequence. We designed primers to target each motif on plasmid constructs and incorporate upto four mutations per motif .Mutations were introduced at positions in the motif that had the highest information content and were replaced by the least informative nucleotide at that position. We verified the primers to ensure that the mutations did not create a new TF-binding motif, using the TOMTOM tool from the MEME suite of motif analysisWe used gBlocks Gene Fragments from IDT to synthesize the RepBase-consensus sequences listed in The data sets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.How to cite this article: Sundaram, V. et al. Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus. Nat. Commun.8, 14550 doi: 10.1038/ncomms14550 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-16 and Supplementary Tables 1-12


Concurrent EEG and fMRI is increasingly used to characterize the spatial-temporal dynamics of brain activity. However, most studies to date have been limited to conventional echo-planar imaging (EPI). There is considerable interest in integrating recently developed high-speed fMRI methods with high-density EEG to increase temporal resolution and sensitivity for task-based and resting state fMRI, and for detecting interictal spikes in epilepsy. In the present study using concurrent high-density EEG and recently developed high-speed fMRI methods, we investigate safety of radiofrequency (RF) related heating, the effect of EEG on cortical signal-to-noise ratio (SNR) in fMRI, and assess EEG data quality.The study compared EPI, multi-echo EPI, multi-band EPI and multi-slab echo-volumar imaging pulse sequences, using clinical 3 Tesla MR scanners from two different vendors that were equipped with 64- and 256-channel MR-compatible EEG systems, respectively, and receive only array head coils. Data were collected in 11 healthy controls  and 13 patients with epilepsy . Three of the healthy controls were scanned with the 256-channel EEG system, the other subjects were scanned with the 64-channel EEG system. Scalp surface temperature, SNR in occipital cortex and head movement were measured with and without the EEG cap. The degree of artifacts and the ability to identify background activity was assessed by visual analysis by a trained expert in the 64 channel EEG data .RF induced heating at the surface of the EEG electrodes during a 30-minute scan period with stable temperature prior to scanning did not exceed 1.0° C with either EEG system and any of the pulse sequences used in this study. There was no significant decrease in cortical SNR due to the presence of the EEG cap (p > 0.05). No significant differences in the visually analyzed EEG data quality were found between EEG recorded during high-speed fMRI and during conventional EPI (p = 0.78). Residual ballistocardiographic artifacts resulted in 58% of EEG data being rated as poor quality.This study demonstrates that high-density EEG can be safely implemented in conjunction with high-speed fMRI and that high-speed fMRI does not adversely affect EEG data quality. However, the deterioration of the EEG quality due to residual ballistocardiographic artifacts remains a significant constraint for routine clinical applications of concurrent EEG-fMRI. This might enable improved localization of the sources of the electrical events based on the associated blood oxygenation level dependent (BOLD) response. Concurrent acquisition of fMRI and EEG has also emerged as a new tool for localizing the epileptogenic zone. In medically intractable epilepsy patients, removal of the epileptogenic tissue is a well-documented treatment in cases where the epileptogenic focus can be localized [Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) have in recent years emerged as a new tool in brain research. EEG-fMRI combines the high temporal resolution of EEG in milliseconds range with the high spatial resolution of fMRI in the mmocalized  2] 3], [3],3 ra, [3],3 rocalized  5]. The. The3 raThe acquisition of high quality EEG data inside the MRI scanner environment is challenging and much effort has been spent on deconvolving gradient, motion and ballistocardiographic (BCG) pulse artifacts  7]. EEG. EEG7]. The impact on fMRI data quality with concurrent use of EEG equipment has been investigated in a number of studies, which reported increased inhomogeneity of the static magnetic field in the vicinity of the electrodes causing decreases in cortical signal-to-noise ratio (SNR), but no significant effect on the sensitivity of detecting task-based fMRI signal changes and resting state networks  9] 10] [10]9] [ [10]9] .Recent advances in increasing the temporal resolution of fMRI using high-speed acquisition methods that enable un-aliased sampling of physiological signal fluctuation  have considerably increased sensitivity for mapping task-based activation and functional connectivity, as well as for detecting dynamic changes in connectivity over time  13] [14 [1413] . HoweverA key concern is RF related heating of the EEG leads and electrodes, which depends on many MR acquisition related factors. RF heating has been shown to be mainly related to the specific absorption rate (SAR) of RF energy  17]. Lo. Lo17]. Our present study investigated RF heating in concurrent EEG and high-speed fMRI by comparing three different fMRI pulse sequences  in healthy controls and patients with epilepsy using two different 3 Tesla MRI scanners from 2 different vendors equipped with a 64- and 256-channel MR-compatible EEG system, respectively. RF heating was examined by direct multi-point temperature measurements. The effect of the EEG cap on the signal-to-noise ratio was evaluated in occipital cortex. EEG data quality was assessed using the 64-channel EEG system. Here we investigated to which extent residual gradient artifacts due to the choice of the fMRI pulse sequence (EPI and MEPI compared to MB-EPI and MS-EVI), BCG artifacts and patient movement impact the data quality of EEG and ongoing activity recorded inside an MR scanner.The project was approved by the Ethics Committee of the Capital Region of Copenhagen Denmark (H-KA-20060151 and H-2-2013-038). All subjects (controls and patients) gave written informed consent before participation. The controls were recruited from an online database and by word of mouth. Recordings consisted of MR scans, EEG recordings and temperature measurements between the skin and the EEG electrodes.A total of 11 healthy controls were studied: 8 subjects were scanned using the Philips Achieva , and 3 subjects using the Siemens Prisma scanner . In the subjects studied on the Phillips scanner the EEG quality was assessed in all except 1 and systematic temperature measurements in 3. In all subjects studied on the Siemens scanner the temperature was systematically investigated.A total of 13 patients were scanned  using the Philips Achieva scanner and EEG quality was assessed. Judged by EEG, MR and semiology 7 had temporal lobe epilepsy, 1 had probable temporal lobe epilepsy, 2 had frontal lobe epilepsy, 1 had epilepsy arising from the occipital lobes, 1 had idiopathic generalized epilepsy, and 1 had epilepsy arising from the central region. All patients were on anti-epileptic medication based on best clinical practice. The patient inclusion criteria were: adults (> 18 years), with epilepsy and a history of frequent interictal spikes or sharp waves (aiming for > 5 per 1/2 hour) with high amplitude (aiming for > 70μV), who could cooperate with the examination.Data were collected using two different clinical 3 Tesla MRI scanners from two different vendors: A Philips Achieva scanner  equipped with a 32 channel RF head array coil and a Siemens Prisma Fit  scanner equipped with a 64 channel RF head array coil. The scanners were located respectively at Glostrup and Blegdamsvej campus of Rigshospitalet, Copenhagen, Denmark; The Philips Achieva scanner was equipped with a 32- and a 64-channel MR Cap , which consisted of 63 EEG electrodes, 1 external ECG electrode, 1 ground (AFz), and, 1 reference electrode (FCz). The Siemens Prisma scanner was equipped with a Geodesic EEG System  with 256 EEG channels, 2 external ECG electrodes and 1 reference electrode (Cz).Phantom measurements were performed using a spherical daily quality assurance phantom provided by the manufacturers.BrainCapMR: This device was used on the Phillips Achieva scanner. The system had 64 ring-type sintered nonmagnetic Ag/AgCl electrodes. The EEG cap was selected according to the head size .Geodesic EEG System MR: This device was used on the Siemens Prisma Fit scanner. The system had 256 channels consisting of sponge based electrodes. The EEG cap was selected according to the head circumference .Temperature measurements in the Philips Achieva and the Siemens Prisma scanner were performed using a four channel Luxtron 3100 fiber optic thermometer (LumaSense Technologies Inc.). In the Phillips scanner, an extra 2-channel Veris MR-compatible monitoring system (MedRad Inc.) was used to monitor additionally the scanner bore and room temperature .The acquisition parameters for the different acquisition methods are listed in Philips Achieva scanner, additional data were acquired using partial brain EPI at shorter TR (402 ms), and using whole brain and partial brain multi-echo EPI (MEPI) with 3.2-fold SENSE acceleration, a minimum TE of 9.6 ms, an echo time spacing of 17 ms and TRs of 3034 and 376 ms, respectively. Multi-band (simultaneous multi-slice) EPI data were acquired with 4-fold acceleration (MB4-EPI) using a pulse sequence developed by GyroTools LLC . Data reconstruction was performed offline. The initial version of the MB-EPI pulse sequence suffered significant temporal instability and artefactual inter-slice correlations as a result of RF pulse instability that was resolved with an update of the pulse sequence software in the middle of the study. This instability prevented removal of gradient artifacts in the initial data. Sequential multi-slab echo-volumar imaging (MS-EVI) data were acquired using a custom designed implementation by GyroTools LLC  based on the method described in Posse et al., 2012 . Al. Al24]. The EEG data quality using the 64 channel EEG system during MR scanning was considerably degraded compared to EEG data measured outside of the scanner. However, EEG data quality was not significantly different between the conventional and the fast sequences. The quality of the EEG data was visually evaluated by a trained expert. This could be relevant, since epileptiform discharges are also identified this way . However, this visual approach is subjective, and thus represents a potential bias, although the EEG expert was blinded to the type of MR sequence when evaluating EEG quality. We consider this a limitation of the study.Due to initial timing errors in the EPI and MB-EPI pulse sequences and in the EGI software, which resulted in failure of gradient artefact removal, we have not been able to assess the data quality of the high-density 256 channel EEG array implemented in the Siemens Prisma scanner. This comparison will be performed in a future study. There is now compelling evidence that detection and identification of spikes is significantly improved using electric source imaging with high-density electrode arrays (HD-EEG), with extended coverage including the face and neck areas  28]. Mo. Mo28]. Another possible source of artifacts is RF noise that may be transmitted through the EEG leads. Further, EEG leads and electrodes may distort the transmit RF field (B1+) and the receive RF field (B1-) profiles, which causes regional flip angle variations and spatial variations in sensitivity, resulting in regional variation of fMRI signal intensity and degradation of the SNR . HoweverConcurrent EEG-fMRI is a tool that is of considerable interest for many aspect of neuroscience, e.g., evoked potentials, sleep and epilepsy. Although the electrodes put some pressure on the skin, and especially for the electrodes the subjects were lying on, the use of the EEG cap in our study was well tolerated and did not limit the duration of the studies. Among the potential clinical use of concurrent EEG-fMRI should be mentioned selected patients with pharmacoresistant epilepsy where it is well documented that seizure freedom can be obtained after surgical removal of the epileptogenic tissue . HoweverThis study demonstrates that high-density EEG can be safely implemented in conjunction with high-speed fMRI. Despite these encouraging results one should not forget that placing electrodes and wires into high field scanners that are equipped with high-power RF amplifiers requires constant attention for the safety and comfort of the subjects. We recommend performing temperature measurements with any new pulse sequence. High-speed fMRI does not adversely affect EEG data quality as visually evaluated by a trained expert. However, the deterioration of the EEG quality due to residual ballistocardiographic artifacts remains a significant constraint for routine clinical applications of concurrent EEG-fMRI.S1 FigElectrode display in the 256-channel (left) and the 64-channel (right). The temperature probes were placed underneath the electrodes marked with the red circle, or underneath one of the neighboring electrodes. The ear temperature probe was placed on the left or the right side.(TIF)Click here for additional data file.S1 Data(CSV)Click here for additional data file.S2 Data(CSV)Click here for additional data file.S3 Data(CSV)Click here for additional data file.S4 Data(CSV)Click here for additional data file.S5 Data(CSV)Click here for additional data file.