Cardiac safety assessments are commonly employed in the clinical development of investigational oncology medications. In anti-cancer drug development there has been increasing consideration for the potential of a compound to cause adverse electrocardiographic changes, especially QT interval prolongation, which can be associated with risk of torsades de pointes and sudden death. Irrespective of overt clinical toxicities, QTc assessment can potentially influence decision making at many levels during the conduct of clinical studies, including eligibility for protocol therapy, dose delivery or discontinuation, and analyses of optimal dose for subsequent development. Given the potential for serious and irreversible morbidity from cardiac adverse events, it is understandable that cardiac safety results can have broad impact on study conduct and patient management. The methodologies for risk management of QTc prolongation for non cardiac drugs have been developed out of experiences primarily from drugs used to treat non life-threatening illnesses in a chronic setting such as antibiotics or antihistamines. Extrapolating these approaches to drugs for treating cancer over an acute period may not be appropriate. Few specific guidelines are available for risk management of cardiac safety in the development and use of oncology drugs. In this manuscript, clinical and methodological issues related to QTc prolongation assessment will be reviewed. Discussions about limitations in phase-I design and oncology drug development will be highlighted. Efforts are needed to refine strategies for risk management, avoiding unintended consequences that negatively affect patient access and clinical development of promising new cancer treatments. A thoughtful risk management plan generated by an organized collaboration between oncologists, cardiologists, and regulatory agencies to support a development programme essential for oncology agents with cardiac safety concerns. Prolongation of the QT interval associated with the potential fatal arrhythmia known as ‘torsades de pointes’ (TdP) has been a common cause of withdrawal from the market for several drugs . ClinicaTdP was first described in 1966 by the French cardiologist Dessertenne . Until 1Multiple factors have been implicated in causing QT prolongation and TdP. Among these, improper use of QT-interval-prolonging medications deserves special attention ,8. To reThe QT interval on the surface ECG is measured from the beginning of the QRS complex to the end of the T wave . It is tThe ventricular action potential proceeds through five phases. The initial upstroke (phase 0—depolarization) occurs through the opening and closing of Na+ channels. The repolarization process begins with the rapid transient outflow of K+ ions (phase 1). This is followed by the flow of outward current through two delayed rectifier K+ channels  and of inward current through Ca2++ channels, constituting phase 2 or the plateau phase of repolarization. Increasing conductance of the rapid delayed rectifier (IKr) and inward rectifier (IK1) currents completes repolarization (phase 3). Phase 4 represents a return of the action potential to baseline. The LQT1 gene  encodes voltage-gated potassium channel alpha subunits. A tetramer of 4 KCNQ1 alpha subunits co-assembles with the minK gene product (beta regulatory subunit) to form the IKs slowly deactivating delayed rectifier potassium channel . The patBecause the QT interval is prolonged at slower heart rates and shortened at faster heart rates, many formulas have been proposed to adjust for these variations . Yet difAlthough it is convenient to think of QT prolongation as occurring because of either congenital or acquired abnormalities, the phenomenon probably most often involves a gene–environment interaction. Pure congenital prolongation characterized by lifelong, ambient QT prolongation is rare but does carry a high risk of sudden death –27. WhenQT prolongation is a surrogate marker for the risk of developing TdP or other life-threatening arrhythmias. While there is a general qualitative correlation between QTc and risk for TdP, it is not possible to make a quantitative prediction for the risk of TdP for a given QTc prolongation. Thus while it makes sense not to accept a very small risk of TdP for a drug like terfenadine or cisapride, it is more difficult to know what degree of QTc prolongation would lead to such a large risk of TdP so as to make an anti-cancer drug unacceptable. One can measure the efficacy parameters in clinical trials, but regarding the risk for QTc, no study has made a quantitative assessment looking at any medication in the oncology setting. This is particularly difficult to assess because many cancer patients have many other factors that may increase their risk of arrhythmias including medication, cardiac disease, drug-induced heart injury and electrolyte abnormalities. The association of QT prolongation with arrhythmias can be influenced by a variety of factors mentioned in the previous section but can also be drug specific. For example, amiodarone can significantly prolong QTc, but it appears to cause only rare cases of TdP.The decision by a physician to use a drug is predicated on the assumption that the benefits of therapy, however defined, outweigh the risks, however measured. For a cancer patient, the possibility of a cure for the cancer may outweigh the potential risks of QTc prolongation, even when the prolongation is high.2) that had been obtained at baseline and during and following treatment with arsenic trioxide were collected and sent to a central core laboratory for review in a blind test by a single board-certified cardiac electrophysiologist. Baseline ECG abnormalities were observed in 36 patients, including marked sinus tachycardia, bundle branch block, ST-T wave changes, atrial fibrillation, and evidence of previous myocardial infarction. Despite these abnormalities, in 949 (80%) of the ECGs, the RR and QT intervals could be determined, and QTc intervals could be calculated. Compared with baseline, the heart rate-corrected (QTc) interval was prolonged by 30–60 msec in 36.6% of treatment courses, and by more than 60 msec in 35.4% of patients. The degree of prolongation was higher in men than in women during the first course of therapy, and in patients with hypokalemia. In patients receiving multiple courses, QTc intervals returned to pretreatment levels before the second course, signifying that arsenic trioxide does not permanently prolong the QTc interval [Arsenic trioxide (ATO) presents an interesting example of successful risk management, supporting the decision for a patient to accept, or a physician to administer an anti-cancer drug with established liabilities of QTc prolongation and TdP. Although this drug is known to provoke TdP  it is alinterval . In the interval , 45–47. interval . Enhanceinterval ,52 and a2. Standard 12-lead ECG and signal-averaged ECG recordings were performed at baseline and after cumulative doxorubicin doses of 200, 400 and 500 mg/m2. Five patients (18%) developed QT prolongation exceeding 50 msec. The changes in QTc occurred independently of the impairment of left ventricular function. In children who received anthracyclines and thoracic irradiation, analysis of the 12-lead and 24-hour electrocardiograms demonstrated increased frequency of QTc prolongation, supraventricular premature complexes, supraventricular tachycardia, ventricular premature complexes, couplets and ventricular tachycardia when compared with an age-matched healthy population [A prospective study  was desipulation .Preclinical and clinical data demonstrated that tamoxifen can cause prolongation of the electrocardiographic QT interval in humans . The eleThe anti-emetic efficacy of serotonin-type-3 (5-HT(3)) receptor antagonists has been found to be superior to older anti-emetic drugs in many clinical settings. Following the administration of these agents, changes in ECG parameters and increased or decreased heart rates have been demonstrated –59, but New drugs present a special problem, because at the time of approval by the FDA or European Medicines Agency (EMEA), clinical experience with each drug is limited. Many new agents may weakly antagonize IKr or produce a small degree of QT-interval prolongation but there may be limited rigorous data at the high doses that are likely to be given to some patients post-approval. The decision to develop and approve an anti-cancer agent with QTc liability ultimately rests on an estimate of the perceived risk relative to the expected benefits for patients and society. Estimates of benefit are specific to particular indications, but they may include an assessment of whether the specific disease entity itself is associated with morbidity or mortality, the expected favourable impact of a new treatment, and consideration of efficacy and toxicity of other available therapies.FDA and EMEA regulatory guidelines advocate that most new agents, administered systemically, should undergo an electrocardiographic evaluation, beginning early in clinical development, typically including a single trial dedicated to evaluating their effect on cardiac repolarization (‘thorough QT/QTc study’ or TQTS) . Common When one is prepared to take a drug into clinical development for a specific oncology indication, a clinical development plan must consider the degree of risk for QT prolongation. If the risk is small, based on known class effects and preclinical information, then the detailed evaluation of QTc risk can be deferred to the phase-II programme (or phase III), and the phase-I programme can just have routine surveillance ECGs. If the drug has a significant risk of QT prolongation prior to clinical trials, then it is important to evaluate that risk in the phase-I trials. Whatever the plan, it should have feedback and approval in advance from the regulatory agencies at the appropriate times. The degree of QT prolongation that is acceptable when making a decision if a drug should go into development or continue development is highly dependent on commercial, medical and regulatory variables that are not easy to integrate. While more than a 5 msec increase is not acceptable for some drugs, the arsenic trioxide example demonstrates that a drug with a much longer increase can have approval. The clinical development plan must prospectively take into consideration what degree of prolongation would be acceptable, and undertake trials that can document the degree of QT prolongation that the drug actually causes. In other words, the trials must provide the investigators and sponsor the information to continue or stop development, to provide the regulatory agencies the information they need to approve or disapprove the drug, and ultimately provide the physicians the information they need to proceed. All of these decisions need specific considerations in oncology.Several levels of concern can be highlighted in designing phase-I trials in oncology, that may be different from other indications . First of all are patient concerns: (1) the safety of the patient is of paramount importance. Based on drug pharmacokinetics and known preclinical or clinical signals for risk of QTc prolongation, an appropriate safety plan for monitoring QTc and responding to potential changes must be put in place. This can range from periodic ECG data collection and reading, that can have a turnaround measured in days for oral medications with low risk, to rapid QTc assessments based on bedside machine readouts for immediate decisions as to possible IV-drug discontinuation for medication with high risk. The medical team should have sufficient experience and training for an optimal safety assessment and subsequent decision-making; (2) patient capabilities and compliance in tolerating trial demands must be considered—the timing of the ECG is usually concomitant to pharmacokinetic blood sampling and requires outstanding compliance from the patient enrolled into the study; (3) for drugs with a significant risk of QTc prolongation, rapid assessment and decision making during administration of the drug can induce psychological distress that needs to be mitigated through education and preparation including the informed consent process.Another level of concern is related to protocol issues. Trial protocols described in E14 have many features feasible in healthy volunteers or in patients from many therapeutic areas, but are not consistent with conventional care of patients with advanced malignancy enrolled in oncology trials. Oncology trial design issues can be divided into trial design issues and technical ECG issues. The trial design issues come from defining the purpose of the study and how the potential QT evaluation will affect the trial. For example, if there is no known QT prolongation risk for the drug, then routine surveillance ECGs can be done at baseline, at peak drug concentrations, and at a delayed time in case the QT prolongation is related to a metabolite of tissue accumulation. Another example would be to establish QT prolongation only for what might be life-threatening levels (>500 msec) without trying to document small changes. The definition of dose-limiting toxicities (DLTs) is important in phase I trials. The CTCAEv3 definition of Grade 3 QT prolongation is defined as QTc >500 msec without life threatening arrhythmias. Definition of clinical trial inclusion criteria is another important topic. In two studies, the appropriate reference values for adult patients with solid tumours, enrolled across various phase-I programmes were evaluated ,62. DataMany technical protocol decisions related to the ECGs need to be made. These issues can be approached essentially in the same way in oncology trials as in trials in any other area, except that the special needs of oncology patients must be recognized. E14 addresses these technical issues in general ways, and more details are provided in other publications . MeasureClinicians are increasingly faced with both old and new anti-cancer agents that have the potential to prolong the QT interval; a laboratory finding that is associated with the risk of significant arrhythmia. As more sensitive methods for electrocardiographic testing are applied, an increasing number of anti-cancer agents or treatments for supportive care will likely be described with some risk for QTc prolongation. For many agents, the frequency of QTc prolongation may be common while the risk of clinical arrhythmia (TdP) may be very rare. To enable the development and application of future treatments, detailed analyses and publication about TdP events, observed in cancer treatment settings, should continue to support rational strategies for risk management with broad relevance. Oncologists have demonstrated their ability to identify and manage complicated cardiac risks in clinical investigations and general practice. Across multiple geographic regions, successful risk management is exemplified by the published experiences with arsenic trioxide, highlighting the value of systematic ECG testing and attention to concomitant medications, electrolyte abnormalities, and co-morbidities, all likely contributing to the low frequency of clinical cardiac toxicities reported post-approval. Given the growing introduction of promising agents, designed to address unmet medical needs, efforts are needed to promote strategies for risk management, avoiding unintended consequences that can impede development, regulatory approval, and patient access. More research is needed to assess and manage cardiovascular safety of patients treated with anticancer agents, building from organized collaborations between oncologists and cardiologists, and these efforts should have broad relevance to therapeutics designed for treatment or supportive care.Risk–benefit assessments might be relatively straightforward in non-oncological studies and are provided in the ICH E14 document. In principle, risk–benefit must be interpreted in the context of the nature and severity of the disease and conservative approaches will delay or prevent patient access to innovative treatment. Currently, different criteria are used in defining which prolongation of the QT-QTc time or change over baseline is feasible. The use of uniform thresholds to describe changes of concern for all protocol applications can simplify study conduct and subsequent data collection. Grading according to the Common Toxicities Adverse Events version 3 (CTCAE v3) might be considered a uniform guideline in which grade 3 QT-QTc prolongation is defined as a value exceeding 500 msec. In oncology trials, treatment related events of grade 3 or higher are often considered for decision making and dosing decisions. Asymptomatic prolongation of the QT-QTc time exceeding 60 msec over baseline may be too sensitive to guide dosing in oncological studies, since these changes can already be observed as a diurnal variation, even in healthy volunteers, and might even be larger in oncological patients. Of utmost importance is that the data generated in oncological studies are pooled to provide additional information on the QT/QTc interval in cancer patients and to guide us in broadening the eligibility criteria of studies in the oncologic setting, to implement the concomitant use of oncology relevant medication, to standardize dose-modification and discontinuation criteria, to search for alternatives in study design and result in timely approval of drugs by the regulatory authorities providing access for patients to promising new anti-cancer agents.Material included in this review was identified from computerized searches of PubMed (1966 to September 2008), EMBASE (1974 to September 2008) and Current Contents (1998 to September 2008) using the medical subject headings (MeSH) terms ‘QTc prolongation or QT interval or arrhythmia, anticancer drugs. Free text terms ‘QTc prolongation* AND chemotherapy AND phase I’ were also used and combined with ‘clinical trials’, ‘epidemiologic studies’, ‘animal studies’, and ‘in-vitro studies’. Only peer-reviewed English-language papers were eligible for inclusion. Additional information and studies were obtained by checking the reference lists from retrieved papers. EMEA and FDA guidelines for QTc prolongation assessment were also considered in this review.


Transcranial electrical stimulation (tES) is a neuromodulatory method with promising potential for basic research and as a therapeutic tool. The most explored type of tES is transcranial direct current stimulation (tDCS), but also transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS) have been shown to affect cortical excitability, behavioral performance and brain activity. Although providing indirect measure of brain activity, functional magnetic resonance imaging (fMRI) can tell us more about the global effects of stimulation in the whole brain and what is more, on how it modulates functional interactions between brain regions, complementing what is known from electrophysiological methods such as measurement of motor evoked potentials. With this review, we aim to present the studies that have combined these techniques, the current approaches and discuss the results obtained so far. Non-invasive brain stimulation (NiBS) techniques use externally applied stimulation for inducing neuroplastic changes in the human brain. Sub-threshold transcranial electrical stimulation (tES) is a specific subgroup of NiBS using low-intensity electrical current  via two conductive electrodes placed on the scalp   at 1mA the extent of activation of the left sensorimotor cortex was decreased but no other significant changes were found  showing coherent activity at low-frequencies (0.01—0.1 Hz), which are functionally relevant, and comply with the underlying anatomy . These behavioral findings were accompanied by a significant reduction of the BOLD response in the left IFC, including the Broca's area. In a more recent study,  improved semantic word generation performance, by increasing the number of the correct answers during active vs. sham tDCS. Interestingly, similar to the findings of Holland and colleagues, this behavioral enhancement was also associated with a selective reduction of the BOLD-response in the left ventral inferior frontal gyrus (vIFG) and with an increased language network connectivity evidenced by a graph-based eigenvector centrality mapping data analysis approach, this latter result suggesting an enhanced efficacy in information processing at the neural network level.Only two studies have directly investigated the effect of stimulation on cognitive functions with the combination of neuroimaging, and both studies used the concurrent application of tDCS and fMRI. Holland et al.  targetedAt the present, it is difficult to draw a conclusion from these two studies. The experimental evidence so far suggests that in both cases the tDCS-induced changes in higher cognitive functions were associated with reduced BOLD activity in highly-specific task-related brain regions. These findings are converging despite the strikingly different stimulation protocols concerning the stimulation duration and the intensity. A remaining issue for future studies would be providing further evidences for the relationship between altered cognition and the associated neural changes in the functionally relevant brain regions.The aim of this review was to summarize results so far obtained by combining tES and fMRI. Taken together, the studies presented here provide valuable insight to the potential of tES, as a tool to modulate brain activity. Modeling studies estimate a spatially wide distribution of the electric field induced during tDCS (e.g., Bikson et al., Other tES modalities, namely tACS and tRNS are not expected to act by the same mechanisms as tDCS, not only regarding local physiological changes, but also at a functional and network level. To our knowledge, no study combined fMRI with tACS and only two studies combined it with tRNS (Chaieb et al., What also remains to be further elucidated at a whole-brain level, is the temporal evolution of the tES-induced changes, not only in terms of duration of after-effects, but also regarding the influence of protocols and repeated stimulation sessions. As a clinical tool, tDCS has found application in several neurological conditions (Nitsche et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The mysteries of early development of cortical processing in humans have started to unravel with the help of new non-invasive brain research tools like multichannel magnetoencephalography (MEG). In this review, we evaluate, within a wider neuroscientific and clinical context, the value of MEG in studying normal and disturbed functional development of the human somatosensory system. The combination of excellent temporal resolution and good localization accuracy provided by MEG has, in the case of somatosensory studies, enabled the differentiation of activation patterns from the newborn’s primary (SI) and secondary somatosensory (SII) areas. Furthermore, MEG has shown that the functioning of both SI and SII in newborns has particular immature features in comparison with adults. In extremely preterm infants, the neonatal MEG response from SII also seems to potentially predict developmental outcome: those lacking SII responses at term show worse motor performance at age 2 years than those with normal SII responses at term. In older children with unilateral early brain lesions, bilateral alterations in somatosensory cortical activation detected in MEG imply that the impact of a localized insult may have an unexpectedly wide effect on cortical somatosensory networks. The achievements over the last decade show that MEG provides a unique approach for studying the development of the somatosensory system and its disturbances in childhood. MEG well complements other neuroimaging methods in studies of cortical processes in the developing brain. Around the time of full-term birth, the central nervous system (CNS) of a human newborn is developing dramatically Figure . Transiein vivo studies of the developing CNS. Advanced magnetic resonance imaging (MRI) techniques [such as voxel-based morphometry and diffusion tensor imaging (DTI)] allow not only the visualization but also the quantification of gray and white matter structures  and tactile stimulation (SEFT) consist of an initial deflection with an underlying current source pointing anteriorly . The following sections discuss how and when such somatosensory response patterns are attained in infancy and what kind of underlying processes this development might reflect.Somatosensory responses can be evoked by electrical stimulation of a peripheral nerve  or by tactile stimulation of the skin . Stimulation of the median nerve at the wrist activates a mixture of afferent and efferent fibers, including those innervating many types of cutaneous receptors in about two thirds of the palmar side of the hand. In most of the experiments reviewed here, the tactile stimulation was provided with an inflatable plastic diaphragm driven with pulses of compressed air. Such a stimulus feels like a gentle tap on the fingertip and activates mainly slowly adapting mechanoreceptors in a relatively localized skin area. Compared with median nerve stimulation, the early somatosensory evoked field (SEF) deflections to tactile stimulation have usually lower response amplitudes, and slightly longer latencies Figure , partly In the following sections, we first review different aspects of somatosensory development and then discuss the relevant findings of developmental somatosensory MEG studies.in vivo DTI data show a basic pattern of white matter maturation both before and after term-age. As a function of gestational age, mean diffusivity decreases and fractional anisotropy  increases in a posterior-to-anterior and a central-to-peripheral order  recorded on the scalp, or with SEFs recorded extracranially with MEG. In preterm infants, median nerve SEPs are recordable on the scalp already by the 25th GW , though with considerable inter-individual variability  can be explored MN30, the initial SEF to median nerve stimulation in full-term newborns peaks at around 30 ms (sefMN30). The MEG data together with earlier EEG findings suggest that this earliest cortical response in newborns reflects activation of a similar cortical ensemble as the earliest SEFMN response in adults (SEFMN20 peaking at 20 ms) .In term-age infants, primary somatosensory responses have also been studied with MEG. In accordance with the neonatal sepMN30 and sefT30 latency decreases and slightly increases thereafter , and fractional anisotropy decline   of the post-synaptic neuron due to high intracellular Cl− concentration  on the post-synaptic neuron. In the adult brain, GABA is an inhibitory neurotransmitter. At early stages of development, however, GABAin vivo is not straight forward, but some inferences can be made from careful examination of neurophysiological data. The deflections following the adult SEFMN20/SEPMN20 or neonatal sefMN30/sepMN30 are considered to represent a “higher” level of information processing either within local neural circuits of the primary cortical area or in higher-order cortical areas. In a developmental SEP study, Laget and coworkers  is generally agreed to represent thalamo-cortical excitation of pyramidal cells in area 3b of SI . Such intracellular current could be generated by either inhibition in deeper cortical layers  responses in contra- and ipsilateral SI without significant differences between the hemispheres. This was interpreted as immature lateralization of somatosensory processing in newborns , posterior parietal cortex, and mesial paracentral lobule , in young infants it may present as generalized and non-specific symptoms  may present with generalized hypotonia or hypertonia, hypokinesia, or hyperexcitability)  and grade III–IV IVH picked up by cranial ultrasound  median nerve sepA recent MEG study has expanded the somatosensory response evaluation from SI to SII, thus including cortico-cortical processing assessment. SEFs were recorded at term-age from 39 extremely preterm infants (born <28 GW) whose neurodevelopmental outcome was assessed at 2 years corrected age  hemisphere as demonstrated by MEG  was disrupted in both hemispheres by an additional peak, SEFMN25, with posterior current orientation, preceding a delayed SEFMN35. Second, within SI, the cortical representations of contralateral digits II and V were located abnormally close to each other in both hemispheres. Unilateral spatial alterations in the hand representation area have been detected by fMRI, where the area activated by passive hand movement showed larger inter-individual spatial variability in the affected than unaffected hemisphere in patients with CP .  In three CP patients, Gerloff et al.  observedDiscoveries of the large-scale alterations in the wiring of the sensorimotor brain networks in people with CP clearly demonstrate the power of multimodal functional investigation of the developing brain. MEG is an essential tool in the brain development investigation toolbox, particularly when used in combination with other non-invasive methods, such as DTI, fMRI, and TMS. The puzzle of brain network organization after early insults of different scale, timing, and etiology, however, still remains only partly solved. The heterogeneity in this patient population calls for large multi-methodological studies to reveal the principles of vulnerability vs. plastic potential in brain organization after early brain insults.Studying children – infants in particular – with MEG or most of the other new non-invasive brain research methods is not and will never become as straightforward as studying adults. First, up to a certain age, only passive paradigms are applicable. Moreover, as most of the new research methods require staying either relatively  or completely  still, the youngest subjects can only be studied during sleep (or anesthesia), which requires a lot of time and patience and may alter the results. Furthermore, when studying brain function during sleep, the sleep stage needs to be carefully monitored since the background neural activity changes dramatically between different sleep stages, thus the possible effect of sleep stage on the phenomena of interest cannot be ignored . To compare groups, the recordings need to be done at the same vigilance and/or sleep stage, which usually lengthens the recording times. In our experience, most of the failures in infant MEG measurements result from the infant not falling asleep within the reserved time slot.In addition, we have faced challenges in head position measurement more often in measurements of infants than adults or older children. These problems are likely to arise from the disproportionately large size of the adult sensor helmet compared with the newborn’s head, resulting in a longer distance between some of the position indicator coils and the MEG sensors and, consequently, worse signal-to-noise ratio for the head position measurement. For reliable SEF recordings, the head of a newborn/infant needs to be close to the surface of the measuring helmet (Gaetz et al., Possible head movements during the measurement constitute another important issue in developmental MEG studies. Conducting recordings during sleep in the smallest subjects compensates for most of the movements, and continuous head position measurement likely deals with twitches during sleep (Taulu et al., As a clinical tool in our hospital, MEG is only used in a very limited number of patients, mainly those considered for epilepsy surgery to localize epileptic foci (e.g., Wilenius et al., In the last decade, MEG has provided a wealth of new information about normal and abnormal development of somatosensory cortical processing in early infancy, which would have been very hard to infer using other methods. Thus, a solid ground has been established to investigate the developing brain using MEG. With the somatosensory system, new information related to SII responses provides opportunities for studying higher cortical processing in neonates at risk for adverse outcome. Since neonatal care has significantly improved in the last decades, there is a growing need to detect the subtle disturbances that possibly cause problems in higher cortical functions but are evident clinically only later in life, e.g., during school years. MEG as a technique offers great possibilities for studying the pathophysiologic mechanisms underlying such subtle disturbances from a neuroscientific perspective.Päivi Nevalainen had the main responsibility of writing the manuscript and preparing the figures. Leena Lauronen and Elina Pihko helped to design the outline and content of the manuscript and edited the manuscript and figures. Elina Pihko also prepared Figures The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The accurate quantitation of proteins and peptides in complex biological systems is one of the most challenging areas of proteomics. Mass spectrometry-based approaches have forged significant in-roads allowing accurate and sensitive quantitation and the ability to multiplex vastly complex samples through the application of robust bioinformatic tools. These relative and absolute quantitative measures using label-free, tags, or stable isotope labelling have their own strengths and limitations. The continuous development of these methods is vital for increasing reproducibility in the rapidly expanding application of quantitative proteomics in biomarker discovery and validation. This paper provides a critical overview of the primary mass spectrometry-based quantitative approaches and the current status of quantitative proteomics in biomedical research. Quantification in a proteomics setting relies on the ability to detect small changes in protein and peptide abundance in response to an altered state . DiffereQuantitative proteomics comes in two forms: absolute and relative. Relative quantitation compares the levels of a specific protein in different samples with results being expressed as a relative fold change of protein abundance . AbsolutTraditional proteomic quantitation approaches rely on high-resolution protein separation by 2D gels. The use of dyes, fluorophores, or radioactivity to label proteins allows visualization of spots/bands with differential intensities , 4. ThesThe ultimate aim of biomarker discovery is to develop a simple differential test to be used as a clinical evaluation tool. This requires a lengthy and difficult process which involves candidate discovery, verification, validation, and translation to clinical laboratory use , 13. Cur12-fold concentration range, from millimolar for albumin, down to attomolar ranges, and further for cytokines [A challenge facing biomarker development is the sheer complexity and range of concentrations within the human proteome , 16. Humytokines  and otheDisease-specific proteins, including low-mass peptides, can be low in abundance and difficult to detect amongst a diverse “sea” of proteins . CombineThere are a number of novel techniques that allow for the fractionation, depletion, enrichment, and equalisation of complex samples to assist in improving the proteome coverage and number of peptide ions targeted for MS/MS within an instrument's detection range. Fractionation techniques can be applied to cut samples into subgroups of fewer proteins  and are Data analysis is yet another significant challenge associated with MS-based proteomics. With the enormous volumes of proteomic data generated, expert manual analysis would be inconsistent and unfeasible . Thus, bProtein mass spectrometry is not inherently quantitative. There are many reasons as to why the amount of analyte compared to the MS signal intensity does not always show a linear relationship , 44. BecTwo widely used label-free quantitative methods are spectral counting and peptide peak intensity measurement. Spectral counting requires proteins to have sufficient peptides (both in number and abundance) to trigger MS/MS data for quantification and identification. The approach is based on the observation that more abundant proteins will produce more MS/MS spectra than less abundant proteins, and abundant peptides are sampled more often in fragment ion scans than are low abundance peptides. Relative quantitation by spectral count thus involves comparing the number of identified spectra from the same protein between different samples . SpectraRelative quantitation using peptide peak intensity measurements involves comparing the MS peptide ion intensities belonging to a given protein . The ionStable isotope labelling techniques are based on the introduction of a differential mass tag which affects only the mass of a protein or peptide without changing the chemical properties during chromatography or MS . Relativ15N labelling can only compare two samples in one experiment, high-throughput quantitation was developed in the form of stable isotope labelling by amino acids (SILAC) [in vivo and also combines light and heavy samples prior to sample preparation to significantly reduce sample handling and thus quantitative errors, allowing very small changes in protein levels as well as protein modifications to be detected.Metabolic labelling involves the introduction of stable isotopes to whole cells through the growth medium, which enables the labels to be incorporated during normal cell growth and division . Differe (SILAC) . SILAC iIn chemical labelling, the isotope label is introduced to proteins or peptides by a chemical reaction, such as with isotope-coded affinity tags (ICAT)  and isotThe development of isobaric mass tags such as tandem mass tag (TMT)  and isobMultiple reaction monitoring (MRM) is the main current approach for highly confident protein and peptide quantification. MRM targets specific peptides in complex samples by typically using a triple quadrupole mass spectrometer or hybrid triple quadrupole/linear ion trap mass spectrometer. These instruments have two mass filters that can select a predefined peptide ion and a combination of its specific fragment ions to analyse and monitor over time for accurate quantitation , 83. ComAbsolute quantitation can be achieved when MRM is incorporated with isotopically labelled synthetic peptide internal standards, which are designed to be identical to target peptides . For MRM5 [MRM has a greater sensitivity towards low abundance peptides and relatively good quantitative precision compared to other methods discussed . It is c5 , 86. The5 . MRM remMRM has been used to quantify major plasma proteins and target biomarkers for a range of diseases. Further instrument developments have taken advantage of the high resolution and mass accuracy of the TOF and orbitrap analysers and combined them with the selectivity of the quad analysers by replacing the third quad with either an orbitrap or a TOF analyser. These high resolution/accurate mass (HR/AM) instruments are addressing the challenge of eliminating cofiltering interfering ions, while taking advantage of the accuracy afforded by these instruments. In experiments similar to MRM called parallel reaction monitoring (PRM), it is possible to detect all product ions of a peptide in parallel rather than just few transitions per peptide. This allows an increased number of peptides to be quantitated in the one experiment. This combination of analysers firstly uses the quadrupole to select a restricted m/z range , and the MS/MS mode provides further selectivity and accuracy utilizing the orbitrap or TOF analyser to achieve higher resolution and mass accuracy in both MS and MS/MS scanning modes . A reducThe use of multiparametric assays is becoming an increasing necessity in quantitative studies to overcome a variety of challenges associated with properties of the marker and/or the techniques including immobilisation efficiencies, detection, signal-to-noise . ProteomQuantitative proteomic analysis has been a point of discussion for the last four decades, with comparative and once limited MS-based techniques heralding the advances that would forge the necessary connection between the dynamic biology of a system and its quantitative proteomic content. The major advances in quantitative MS proteomics have been exceptionally demonstrated over the last decade with the introduction of compatible and reliable label and label-free techniques. These advances now require further developments in bioinformatics and downstream validation, technologies that are required to make sense of complex data and enable researchers to infer more meaningful data that will transform into clinical benefit for years to come.


Magnetoencephalography (MEG) provides a direct, non-invasive view of neural activity with millisecond temporal precision. Recent developments in MEG analysis allow for improved source localization and mapping of connectivity between brain regions, expanding the possibilities for using MEG as a diagnostic tool. In this paper, we first describe inverse imaging methods  and functional connectivity measures, and how they can provide insights into cortical processing. We then offer a perspective on how these techniques could be used to understand and evaluate auditory pathologies that often manifest during development. Here we focus specifically on how MEG inverse imaging, by providing anatomically based interpretation of neural activity, may allow us to test which aspects of cortical processing play a role in  auditory processing disorder [(C)APD]. Appropriately combining auditory paradigms with MEG analysis could eventually prove useful for a hypothesis-driven understanding and diagnosis of (C)APD or other disorders, as well as the evaluation of the effectiveness of intervention strategies. Magnetoencephalography (MEG) is a non-invasive human physiology technique that records magnetic field patterns from hundreds of sensors simultaneously with millisecond precision. Like electroencephalography (EEG), MEG principally measures neural activity from large populations of  cortical pyramidal neurons firing with close temporal and spatial alignment Okada, , but meaMagnetoencephalography has been used for over 30 years to better understand auditory processing. About a decade after the first reported MEG recording of human brain activity Cohen, , researcHere we will provide a perspective on potential ways in which MEG could, with additional time, effort, and validation, influence clinical practice for developmental pathology. In terms of methodology, we will specifically focus here on inverse imaging methods because they provide an accurate mapping from sensor readings onto underlying neural anatomical structures. Additionally, connectivity measures derived from these inverse imaging methods can also be informative in identifying interactions between nodes of brain networks. In terms of application, we will focus on how MEG could come to influence practice surrounding  auditory processing disorder [(C)APD], which often manifests during childhood as difficulty hearing in noisy environments  despite normal audiometric measurements . We speculate about how MEG, auditory paradigms, and specific analysis methods could be combined to diagnose or help guide treatment of (C)APD, or, by extension, other disorders that manifest during development.A common method of MEG analysis is to examine trial-averaged signals directly at the sensor-level, commonly known as event-related fields or potentials (ERF/Ps), which could prove useful for multiple clinical purposes  or ECD modeling of time series, and discuss the potential advantages these methods could confer for clinical use.a priori to form what is known as a source space, and the time-varying amplitude of each source is estimated to account for the observed sensor data  measurements is a mathematically under-constrained problem, as there are infinite solutions that could give rise to the observed electro-magnetic patterns . However, we suggest here that MNE is the best all-around choice for exploring the cortical dynamics in auditory tasks, where potentially extensive cortical networks could be involved  approach is perhaps the most prominent distributed inverse imaging method  regions of significant activation, or latency differences between areas or conditions. Although such time-series analyses have received the largest focus in past studies, a rapidly growing area of research in neuroimaging is in connectivity analysis, which seeks to determine the relationships between two or more neural populations APD. Note that although (C)APD is most often diagnosed in children, most of the ideas presented here apply equally well to experiments that involved adults or children with (C)APD.Listeners with (C)APD often experience difficulties understanding speech in noisy acoustic environments despite having normal audiological pure-tone thresholds. Around 5% of children referred to audiological clinics for listening problems have normal-hearing thresholds, leading to (C)APD diagnoses APD is based on observed difficulties in the perceptual processing of auditory information, as demonstrated by a patient having poor performance in one or more of the following skills: sound localization and lateralization; auditory discrimination; auditory pattern recognition; temporal aspects of audition; or performance with competing acoustic signals and with degraded acoustic signals  during an auditory task better accounted for individual listening ability APD. To our knowledge, MEG has not been used to examine the cortical processing involved in (C)APD, perhaps in part because focus has been predominantly on sub-cortical processing in (C)APD. MEG may thus serve as a complement to auditory brainstem response measurements, which have previously been suggested as a potential diagnostic tool APD with other disorders (such as attention deficit hyperactivity disorder or autism spectrum disorder) may complicate behavioral diagnosis of (C)APD, MEG may prove useful as a tool to help isolate the extent to which different neural structures are involved in each of these pathologies. Other measurements such as ABR would also serve as a useful complement to MEG by measuring the function of midbrain and earlier auditory structures. This would provide a more complete perspective on how the brain may influence subject behavior APD, and eventually evaluate the neural outcomes of various treatments.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.


Active video games (AVGs) have gained interest as a way to increase physical activity in children and youth. The effect of AVGs on acute energy expenditure (EE) has previously been reported; however, the influence of AVGs on other health-related lifestyle indicators remains unclear.This systematic review aimed to explain the relationship between AVGs and nine health and behavioural indicators in the pediatric population (aged 0–17 years).Online databases  and personal libraries were searched and content experts were consulted for additional material.Included articles were required to have a measure of AVG and at least one relevant health or behaviour indicator: EE , adherence and appeal , opportunity cost , adiposity, cardiometabolic health, energy intake, adaptation (effects of continued play), learning and rehabilitation, and video game evolution .51 unique studies, represented in 52 articles were included in the review. Data were available from 1992 participants, aged 3–17 years, from 8 countries, and published from 2006–2012. Overall, AVGs are associated with acute increases in EE, but effects on habitual physical activity are not clear. Further, AVGs show promise when used for learning and rehabilitation within special populations. Evidence related to other indicators was limited and inconclusive.Controlled studies show that AVGs acutely increase light- to moderate-intensity physical activity; however, the findings about if or how AVG lead to increases in habitual physical activity or decreases in sedentary behaviour are less clear. Although AVGs may elicit some health benefits in special populations, there is not sufficient evidence to recommend AVGs as a means of increasing daily physical activity. The majority of children and youth around the world do not meet current physical activity guidelines and are considered to be inactive High levels of habitual sedentary time  are associated with a range of negative health and behavioural indicators including poorer measures of body composition, fitness, self-esteem, self-worth, pro-social behaviour, and/or academic achievement AVGs have the potential to increase habitual PA and improve measures of cardiometabolic health among children and youth who would otherwise be spending time in sedentary, screen-based activities. Manipulating the gaming environment as an intervention tool for increasing PA is reinforced by recent findings showing that playing AVGs acutely increases EE compared to sedentary video games a-priori ranking of health indicators and quality assessment of the evidence. Quality of evidence for each health indicator was assessed based on study design, risk of bias, consistency of results, directness of the intervention, precision of results, and possibility of a dose-response gradient. Details on data extraction are presented in the following sections. Details on GRADE methodology can be found elsewhere The GRADE  framework was used to guide our review including a priori by an expert panel (paper authors) and prioritized based on group consensus  or indirect  measurement, and a measure of at least one relevant health or behaviour indicator. Relevant health and behaviour indicators were chosen onsensus .All published, peer-reviewed studies were eligible for inclusion; no date limits were imposed, but due to feasibility, studies in languages other than English or French were excluded. Studies were excluded if the mean age of participants was greater than 17.99 years; if the study examined only passive video games; if there was more than one aspect to the intervention that may have confounded the results ; or if the outcome of interest was not included in our list of relevant health and behavioural indicators.The following electronic bibliographic databases were searched: MEDLINE, EMBASE, psycINFO, SPORTDiscus and Cochrane Central Database. The search strategy was created and run by AGL see . DatabasTitles and abstracts of potentially relevant articles were screened by two independent reviewers , and full text copies were obtained for articles meeting initial screening criteria. Full text articles were screened in duplicate for inclusion in the review ; any discrepancies were discussed, and resolved by the reviewers. In addition to our search, seven key content experts were contacted and asked to identify what they deemed important papers in the field.Data extraction was completed by one reviewer and checked by another for accuracy . One reviewer (AGL) independently assessed the quality of evidence for all studies Studies were grouped depending on if they examined habitual activity , or acute EE  .2) after a 12-week DDR intervention.Eleven randomized controlled trials (RCTs), and five observational studies examined the relationship between AVG play and habitual PA. The majority of the `RCTs reported that an AVG intervention had no effect on time spent engaging in total PA 2max) after a 30-week DDR intervention; however, Owens et al. The observational studies provided inconsistent results. Bethea et al. 2), heart rate, and rating of perceived exertion while playing Wii boxing than when compared to rest or light treadmill walking (1.5 mph) Two RCTs examined the effect of AVG on acute EE. The first reported no significant difference in average time spent pedaling (min/session), EE , time spent in MVPA (60–79% peak heart rate/session), or average distance pedaled (km/session) between a GameBike intervention group and music only exercise group Seven intervention studies and 12 cross-sectional studies examined the EE of AVGs compared to rest or to sedentary video games and all reported significant increases in EE Studies were grouped depending on if they examined adherence to playing AVGs , or appeal of AVG  .Of the eight RCTs that assessed adherence to AVG play, four reported high levels of adherence at the midpoint of the study, but significantly lower levels by the end of the interventions (interventions ranged from 10–12 weeks) Two intervention studies and one cross-sectional study reported on adherence to AVG play. Bethea et al. One RCT presented qualitative data reporting that in general, children like AVGs, and things they did not like were game-specific This review identified one RCT and two observational studies reporting on adverse events associated with AVGs. The RCT reported that none of the adverse events that occurred during the study period were related to the AVG intervention (EyeToy) Six RCTs (from seven papers), and three intervention studies were included. Two RCTs and one prospective cohort study reported on the relationship between AVGs and cardiometabolic health. After a 12-week DDR intervention with overweight children, Murphy et al. Two studies examined energy intake during AVG play This review included two RCTs and seven observational studies examining the relationship between AVG and learning and rehabilitation. The first RCT used Nintendo Wii along with standard physiotherapy to treat those with cerebral palsy , and saw significant improvements in upper limb function This systematic review is the first to provide a comprehensive understanding of the influence of AVGs on multiple health and behavioural indicators in children and youth. Existing evidence suggests that AVGs are able to increase EE above rest and when compared to playing passive video games. The studies included in the systematic review also showed that AVGs do not make a significant contribution to enable children and youth to meet guidelines of 60 minutes of moderate- to vigorous-intensity physical activity on a daily basis a priori which helps to limit potential bias throughout the review. Furthermore, all steps of the review  were done in duplicate to minimize error. Further, the systematic review has been completed as per PRISMA guidelines  but showed a trend towards significance. Future work should aim to use larger sample sizes to avoid being underpowered, and focus on using both direct  and indirect  measures to assess total AVG use. Both measures are needed to reflect the nuances associated with capturing AVG play such as body position or intensity of play. Moreover, multiple follow-up measurements over longer time periods are required so the longitudinal effects of AVG use can be better understood. It is also important that future work aims to harmonize methods for data collection and analysis so that meta-analyses can be performed. Moreover, the review included studies that were largely based on what could be deemed “first generation” AVGs, as such there will be a need to re-evaluate the evidence in the future as AVGs evolve .Other reviews in this area have shown similar results to ours in that some AVGs are able to acutely increase light- to moderate-intensity PA in some children and youth but unable to elicit PA of high enough intensity, or volume to enable children to meet physical activity guidelines While controlled laboratory studies clearly demonstrate that a motivated player can obtain some light- to moderate-intensity PA from most AVGs, the findings are inconsistent about whether, or the circumstances under which, having an AVG results in sustained PA behaviour change, or for how long the behaviour change persists. Some of these games offer nuances on game play that could be related to increased PA or decreased sedentary behaviour. AVG technology is innovating at a rate that outpaces the related research. Higher quality research is needed that tests conceptual models of how different AVGs may relate to the initiation and maintenance of increased PA or decreased sedentary behaviour and understand their effects on health outcomes to resolve these inconsistencies.Appendix S1Search strategy.(DOC)Click here for additional data file.Appendix S2PRISMA presubmission checklist.(DOC)Click here for additional data file.


Frontiers in Physiology-Systems Biology. The authors have provided an interesting investigation about the successful use of an environmentally friendly product derived from plants as a larvicidal agent to control mosquito populations as well as a substance that could alter the vector competence of mosquitoes for arthropod-borne viruses (arboviruses). Specifically Eastep and collaborators used coffee extracts (with and without caffeine) to try to answer two hypothesis: first, coffee extracts could have good results as a mosquitocidal compounds applied in larval biotopes and second, virus replication can be modulated in adult mosquitoes by exposing the larvae to sublethal concentrations of coffee extracts. Detailed information about results and methodology can be found in that article, but we can anticipate that both hypotheses were affirmative confirmed. Although some aspects remained unknown and must be further analyzed, e.g., the specific identification of the active agent or agents responsible for the observed effects, these preliminary results provide a new and hopeful way of fight against mosquito-borne viruses, since two flanks of the complex eco-epidemiological cycle of arbovirus can be attacked: density decrease of disease vectors and reduction/inhibition of the virus replication mosquitoes. However, the influence of coffee extracts exposure on vectorial capacity (VC) of mosquitoes must be carefully analyzed, since VC depends of a great variety of parameters such as vector density, host feeding preference, number of gonotrophic cycles, adult survival, or extrinsic incubation period, among others. Despite Eastep and colleagues detected high mortality in mosquito larvae exposed to coffee extracts, it must be noted that the same authors also evidenced that females reared in coffee tended to lay more eggs than control females, which could theoretically lead to a higher risk of virus transmission by increasing the vector density at medium or long term. Therefore, possible adverse implications of larval biotopes alterations by applying coffee extracts should be deeply and meticulously studied.This commentary highlights key points, basic ideas, and future outlooks presented by Eastep et al.  in FrontAedes albopictus , which is currently one of the most important threats for public health all over the world due to its invasive behavior and potential to transmit a great broad of diseases including some devastating viruses for human population such as Dengue, Yellow Fever, or Chikungunya (Bueno Marí and Jiménez Peydró, It is interesting to note that mosquito species selected to carry out the assays was Bacillus under special conditions (Paris et al., The paper of Eastep and collaborators affects to an important issue nowadays for public institutions and private companies responsible for mosquito control campaigns: the development of natural insecticides with scanty resistance effects and low toxicity to humans and non-target organisms of aquatic environments. Mosquito resistance is currently an increasing problem due to the almost exclusively use of chemical insecticides to manage mosquito pests in many territories during decades. Resistance may develop due to changes in enzyme systems of mosquitoes, resulting in more rapid detoxification or sequestration of the insecticide, or even due to mutations in the target site preventing the insecticide-target interaction (Hemingway et al., Global change and human vulnerability to vector-borne diseases” published by Frontiers in Physiology-Systems Biology which includes many researches related with the use of other natural larvicides against mosquitoes (Kumar et al., Finally I would like to acknowledge Eastep and colleagues for his valuable contribution to classical strategies of fight against mosquito-borne diseases and also highlight that manuscript of Eastep et al.  is one ohttp://www.frontiersin.org/Systems_Biology/researchtopics/GLOBAL_CHANGE_AND_HUMAN_VULNER/323.


Hordeum vulgare ssp. spontaneum) in geographic and environmental space in the context of current, past and potential future climates is important for conservation and for breeding the domesticated crop (Hordeum vulgare ssp. vulgare). Spatial genetic diversity in wild barley was revealed by both nuclear-  and chloroplast-derived (5 cpSSR) markers in 256 widely-sampled geo-referenced accessions. Results were compared with MaxEnt-modelled geographic distributions under current, past  and mid-term future  climates. Comparisons suggest large-scale post-LGM range expansion in Central Asia and relatively small, but statistically significant, reductions in range-wide genetic diversity under future climate. Our analyses support the utility of ecological niche modelling for locating genetic diversity hotspots and determine priority geographic areas for wild barley conservation under anthropogenic climate change. Similar research on other cereal crop progenitors could play an important role in tailoring conservation and crop improvement strategies to support future human food security.Describing genetic diversity in wild barley ( Ecological niche modelling of the distributions of crop wild relatives in present, past and future climates can provide important insights into the past evolution and future trajectories of crop progenitors and domesticates Hordeum vulgare ssp. spontaneum), the progenitor of the agriculturally important domesticated H. vulgare ssp. vulgareWild barley  is the most comprehensive geo-referenced collection of de novo at 24 of the barley nSSR loci described by Ramsay et al. de novo at five of the cpSSR loci designed for Hordeum by Provan et al. Three molecular marker data sets were analysed in the current study. First, SNP data derived from two Illumina barley oligonucleotide pool assay platforms were used . After trial runs, K was set at five because log Pr(X/K) values in STRUCTURE had started to plateau at this point K.) The ‘no admixture’ model in STRUCTURE was used to assign single states to individuals. Other analysis options were kept at default settings. STRUCTURE was run five times for each data set and the most common group assignments used as the basis for the interpretation of results (most accessions placed in the same groups in separate runs).STRUCTURE analysis was not designed for predominantly inbreeding species such as barley, but it has been widely applied to cultivated and wild barley populations to reveal interesting genetic features (see discussion in www.diva-gis.org) and ArcGIS 10 www.esri.com/software/arcgis/). Two approaches were used, the first based on allelic (or haplotype) richness and the second based on K groupings. In the first, allelic (BOPA SNP and nSSR) and haplotype (cpSSR) richness estimates were calculated for groups of accessions. Groups were circumscribed using a circular neighbourhood diameter of four degrees and a grid size of 30 minutes (method described in K groupings (K = 5) revealed by STRUCTURE for BOPA SNPs and nSSRs were used instead of allelic/haplotype richness estimates, in order to reveal genetic differentiation at a local geographic scale. Apart from this, the method of analysis was the same as applied in the first approach.To overlay genetic diversity onto geographic maps we employed DIVA-GIS 7.3 Circular neighbourhood analysis with rarefaction, as conducted in both the above approaches, has the advantage of allowing unbiased comparisons of genetic diversity across geographic space. However, it necessarily excludes accessions from analysis where sampling intensity is low. In the current study, a total of 38 accessions were thereby excluded .www.worldclim.org/) , modelling was based on CCSM and MIROC models . The decrease in similarity observed with geographic distance is, however, not a simple trend for any of our three data sets. Differences in profiles are also observed between marker types. For both BOPA SNPs and nSSRs, an increase in similarity at a distance class of around 1,000 km is observed, after which similarity declines again. For cpSSR haplotypes, an obvious increase in similarity is also observed at a distance class of around 500 km. Spatial autocorrelation analysis therefore indicates that a simple isolation-by-distance model does not fully explain genetic structure across the geographic range of wild barley tested  in the Eastern Mediterranean than in Central Asia  comparing wild barley from the Eastern Mediterranean region and environs with Central Asia. For example, Volis et al. Hordeum. The results of our ecological niche modelling are presented in Our intention in this study is to compare patterns of genetic variation in wild barley with the modelled distributions of the taxon under all three conditions of current, past and future climates, something which to our knowledge has not been undertaken before for any member of the genus Considering first the modelled present-day geographic distribution of wild barley, visual assessment confirms that the accessions included in our study for genetic analysis provide good coverage of most of the taxon's range in the Fertile Crescent and Central Asia compare . The mosThe apparent relatively recent range expansion of wild barley in Central Asia as revealed by ecological niche modelling is consistent with our findings of lower levels of genetic diversity in the region compared to the Eastern Mediterranean, in which latter region it can be postulated that a continuous presence of the taxon has led to the accumulation of genetic diversity  for each chromosome, and then averaged across chromosomes, in EXCEL. Results were then expressed in graphical form comparing LD estimates across sub-samples   .Our comparison of LD estimates indicates that at shorter chromosome distance intervals values of LD are relatively higher in Central Asia than in the Eastern Mediterranean, but that with increased distance along chromosomes values become more equal. Linkage disequilibrium values are difficult to interpret because of the many influencing factors, including population structure, selection pressures, mating patterns and changes in population size and range in situ conservation measures A comparison of present-day plant distributions with predictions for the 2080s is useful for devising responses to anthropogenic climate change N = 155, i.e., excluding accessions in ‘lost’ habitat, as indicated in N = 243). We chose nSSRs as the estimator for this analysis because of their high allelic variability and hence sensitivity in describing diversity differences. Our analysis indicated a relatively small reduction in allelic richness under climate change . Further assessment, based on BOPA SNPs and cpSSRs, revealed a relatively modest 108 SNP alleles (4.6% of SNPs in the comparison) and four chloroplast haplotypes unique to 2080s ‘lost’ habitat. Our analysis therefore suggests that, overall, mid-term future losses in genetic diversity due to climate change are expected to be relatively low. A comparison of modelled current, past and future distributions  populations included in the same study and sampled at the same dates. This suggests different responses to climate change by different cereals in the Eastern Mediterranean region. Third and finally, our current assessment was based on genetic markers that are presumably (mostly) neutral with regard to phenotype, so we are not able to determine whether or not there will be important losses in functional genetic diversity under anthropogenic climate change.It is important to consider a number of provisos when interpreting our findings. First, we have not considered in our analysis that existing wild barley populations in habitat that will be lost under climate change could migrate to (newly) environmentally-matched sites. Such migration will presumably be easier in areas with greater micro-geographic environmental heterogeneity, as the distances to be moved are then smaller. This would suggest migration is more feasible in the Eastern Mediterranean region see . WhetherOur analyses are consistent with the view that climate change has played a role in determining the levels of present-day genetic variation observed in wild barley in different portions of its natural range. Our data support the utility of ecological niche modelling of current and past plant distributions for predicting geographic areas of high genetic diversity, and suggest limited future losses of genetic diversity in wild barley under mid-term future climate change. We have explored the use of chromosome-position-mapped SNPs for discriminating between different hypotheses to explain diversity patterns in wild barley, but more research is required on this topic, ideally using SNPs that have been physically positioned in the genome. With the promise soon of a complete genome sequence for barley Click here for additional data file.


We compared results of decomposing thirteen 71-channel human scalp EEG datasets by 22 ICA and BSS algorithms, assessing the pairwise mutual information (PMI) in scalp channel pairs, the remaining PMI in component pairs, the overall mutual information reduction (MIR) effected by each decomposition, and decomposition ‘dipolarity’ defined as the number of component scalp maps matching the projection of a single equivalent dipole with less than a given residual variance. The least well-performing algorithm was principal component analysis (PCA); best performing were AMICA and other likelihood/mutual information based ICA methods. Though these and other commonly-used decomposition methods returned many similar components, across 18 ICA/BSS algorithms mean dipolarity varied linearly with both MIR and with PMI remaining between the resulting component time courses, a result compatible with an interpretation of many maximally independent EEG components as being volume-conducted projections of partially-synchronous local cortical field activity within single compact cortical domains. To encourage further method comparisons, the data and software used to prepare the results have been made available ( Brain-generated EEG data are generally considered to index synchronous aspects of local field potentials surrounding radially-arrayed cortical pyramidal cells in vivo (‘phase cones’) in vitro  in silicoEmergence of near-synchronous field activity within small cortical domains has been observed and modeled and spatial distributions of cortical (and other) source areas of recorded scalp signals using an electrical forward head model , ICA directly models What distinct signals are contained in the volume-conducted scalp data, and returns the relative projection strength of each maximally independent source to the scalp sensors, thereby also greatly simplifying the problem of determining Where in the brain each EEG source signal is generated BSS and, in particular, ICA methods are now widely used for separating artifacts from scalp-recorded electroencephalographic (EEG) and related data Scalp-recorded EEG data also include non-brain or ‘artifact’ signals that are linearly mixed with brain EEG source activities at the scalp electrodes. ICA has been found to efficiently separate out several classes of spatially stereotyped non-brain signals: scalp and neck muscle electromyographic (EMG) activities, electro-oculographic (EOG) activities associated with eye blinks Though ICA algorithms all have the same root goal To date, however, suitable measures have not been demonstrated for comparing the components returned by different ICA/BSS algorithms applied to actual (as opposed to simulated) EEG data for which ‘ground truth’ source signals and scalp projections are not available. In particular, components produced by ICA decompositions that minimize mutual information between simultaneously recorded signal values have not been much compared to components produced by BSS algorithms that simultaneously minimize component signal redundancy at multiple time delays Here, we use three measures – the amount of mutual information reduction (MIR) between the recovered component time courses relative to the recorded data channels (in kbits/sec), the mean remaining pairwise mutual information (PMI) between pairs of component time courses (in kbits/sec) The motivation for the first two measures (MIR and PMI) is clear; they test how well the results of the decomposition approach the instantaneous independence objective. MIR, introduced here, is a direct  measure of the statistical distinctness of the activities of the resulting components, the absence of dependency entailing, in particular, suppressing the strong linear mixing of EEG signal source signals by common volume conduction to the electrodes. PMI is a partial measure of MIR that can also be directly (though less efficiently) computed from the data. MIR takes into account multi-component dependencies whereas PMI only considers pairwise dependencies. Both can be said to index the relative success of ICA/BSS in finding component processes with fixed scalp projection patterns and near-independent time courses whose projections sum to the data.The motivation for the third measure (dipolarity) is the assumption that brain and non-brain EEG sources have spatially fixed source locations and orientations, as well as temporally distinct, independently varying time courses. This assumption is reasonable at least for scalp muscle, ocular, and electrode artifact signals and, as described above, for many cortical source processes as well, whose volume-conducted potentials recorded at the scalp represent far-field projections of signals each generated within a compact patch of coherent cortical field activity , ranking its output midway between the most efficient ICA method (AMICA) and the least efficient . The mean percentage of near-dipolar (ND) components for SOBI (24%) was also midway between the highest (AMICA) and lowest (PCA) results. While completely uncorrelated by definition, the sets of PCA components were not as highly independent, retaining higher-order dependencies.3) of equivalent dipoles for components with near-dipolar (r.v.<5%) scalp maps in two brain slices. The figure reflects the fact that applied to these data sets AMICA returned a larger number of near-dipolar components than extended infomax ICA, FastICA, or SOBI. For this reason, we used AMICA as a standard for exploration of the similarities and differences between components returned by the other decompositions. Source densities for PCA and sphering decompositions are not shown: Very few of the PCA component scalp maps were near-dipolar – for example on average only 3 of 71 at the 10% residual variance (r.v.) cutoff, and those mainly dominated by large eye artifacts. A much larger number of ‘sphering’ components (87%) had near-dipolar scalp maps, as is guaranteed by the nature of the sphering decomposition  activity, frontal midline theta band (near 5-Hz) activity, eye blink artifacts, left and right mu rhythm activities , lateral saccade artifacts, and electromyographic (EMG) activity from a right forehead muscle. We then found the components from four other decompositions of the same data set  whose scalp maps were most correlated to the seven representative AMICA component maps.Results in Next, we found clusters of AMICA components from the other data sets most similar in location and dynamics to each of the original seven components; the four component clusters accounting for non-artifact brain sources are shown in For all of the twelve algorithms that reduced average pairwise mutual information more than sphering  between the mean increase in the independence of the component time courses (in MIR kbits/second) and the number of returned components with near-dipolar scalp maps.Next, we asked whether the algorithms that better reduced the mutual information present in the raw scalp channel time courses also returned more components with near-dipolar scalp maps. 2 and probability (by t-test) of the regression line for residual variance dipolarity thresholds between 2% to 99%. The inset shows that the positive slope of the regression line in the main figure is significant by t-test (at p<10−4) at residual variance (r.v.) thresholds from 2% to over 20%, with the linear fit accounting for more than 96% of algorithm variance at its peak (r.v.< = 6%). The 6% r.v. threshold might not be far above the minimum r.v. error compatible with our use, here, of a simple best-fitting spherical head model and standardized electrode locations to compute the scalp map projections of the equivalent dipoles; using a best-fitting spherical head model instead of an individual head model built from a subject MR head image adds on average a 3% r.v. error . Components with scalp map r.v.< = 6% may thus be quite consistent with the projection of a single equivalent dipole . In brief, more independent linear EEG decompositions include more near-dipolar components, and that this monotonic relationship is on average nearly linear across a large number of ICA and BSS algorithms from PCA to Infomax ICA and AMICA.The inset to pairs of component time courses, compared to pairwise mutual information between EEG scalp channels  for PMI is not as strong as for total MIR, the residual variance cutoff at which this linear trend is maximum is near equivalent  scalp maps, against the mean percentage of mutual information remaining between 2 = 0.74). Thus, on average algorithms that returned more near-dipolar components .To confirm the consistency of these findings, Use of ICA to remove distinct sources of artifact data from EEG and other neuroimaging data is increasingly widely accepted no far-field cortical signals can be recorded at the scalp. Local cortical field activity that is partly or wholly coherent across a compact cortical patch, on the other hand, is volume-conducted to the scalp electrodes as far-field potential.Physiologically, EEG signals originating within the brain are assumed mainly to be associated with near-synchronous field activities within a connected patch (or two densely connected patches) of cortical pyramidal cells sharing a common alignment near-perpendicular to the cortical surface post hoc into the ‘average reference’ potential difference between each scalp electrode signal and the average potential across all the electrodes). Scalp EEG channels record differently weighted sums of the far-field signals that reach them from all brain and non-brain sources, plus any near-field electrode noise generated at the electrode/skin interface.While spatiotemporal dynamics of local cortical field synchronies have not yet been fully observed, investigations using small electrode grids have modeled such field patterns as ‘phase cones’ predominantly or nearly independent from any other such signal arising elsewhere in cortex (with exceptions discussed below). Therefore, decomposing scalp EEG data into component processes with maximally independent time courses should recover component processes whose scalp projection patterns should strongly resemble a single equivalent dipole even though none of the ICA/BSS algorithms we tested here either incorporate or take advantage of any biophysical or topographical information about electrode locations or source conduction patterns.The high anatomic bias in cortical connectivity toward local (<100 µm) connections, as well as the primarily radial connectivity between cortex and thalamus, support the concept that the far-field signal emerging from one domain  of local spatiotemporal field synchrony should be typically , In the idealized case in which the source signals whose far field projections are mixed at the electrodes are truly independent – e.g., in decompositions of synthetic model data of unlimited length – many ICA algorithms may be expected to give equivalent results linearity of this relationship across the 18 decompositions (from PCA to Infomax and AMICA) in We suggest that the linear relationship of mutual information reduction and dipolarity may only be explained by a direct physiological connection between the nature of source processes themselves and single equivalent dipole scalp projections. For the many component processes that originate in cortex, this may indeed reflect synchronous field activity across a cortical patch, while many non-brain (‘artifact’) EEG component projections may also resemble the projection of a single equivalent dipole within or at the surface of the head . Distinguishing between near-dipolar brain and non-brain sources is typically straightforward based on their time courses, spectra, and the position of their equivalent dipole.Interestingly, the five most efficient algorithms in our comparison all use natural gradient descent on approximations of the instantaneous data likelihood. Thus, it appears that this approach may indeed be optimal for efficient separation of component source processes from high-density EEG data per se, and thus the components these algorithms return may have other features of interest relevant to their individual objectives. Nevertheless, the components returned by the most tested of these algorithms, SOBI, tended to resemble components returned by instantaneous ICA  is to lump together as much variance as possible into each successive principal component, whose scalp maps must then be orthogonal to all the others and therefore are not free to model a scalp source projection resembling a single dipole. The necessary orthogonality of principal component scalp maps guarantees that higher-order component scalp maps resemble checkerboards of various densities, while lowest-order principal components may be dominated by single large artifacts  reduce the number of quasi-dipolar component maps they return  both the anonymized EEG datasets and the custom MATLAB  scripts we used to obtain the results in There might be multiple reasons for the inefficient results of the three ICA/BSS algorithms we did not include in ICA decomposition has proven to be a highly useful approach for EEG data analysis, and our results here, as well as relevant reports on invasively acquired data Our results show that other portions of the remaining mutual information between ICA components might reflect the still imperfect decomposition performance of even the best current ICA methods. Step-like changes in spatial source structure and/or transient or sustained periods of spatiotemporal dependence within one or more component subspaces are other possible sources of residual dependence. Spatiotemporal non-stationarities may include source processes that appear to travel long distances across cortex, such as sleep slow waves The ICA approach to EEG source identification contrasts with the long (and still) predominant approach of attempting to identify (only) those EEG sources active during peaks in averaged evoked potential epochs following abrupt onsets of experimentally presented sensory signals, on the assumption that these scalp maps sum the projection(s) of one or at most very few cortical source areas. Unfortunately, effects of sensory signals on the statistics of cortical field activities spread quite rapidly, concurrent with neural cross-talk and feedback between early sensory areas beginning about 30 ms Reduction of the EEG data by response averaging has the additional liability of discarding the large majority of the data, thus not allowing identification of the sources of the great majority of ongoing EEG activity that is not captured in ERP averages. The ICA approach by contrast, when favorably applied to suitable and sufficient data, allows identification of up to dozens of individual cortical source projections, both their maps and activity time courses, and their individual contributions to the ongoing  data, making available a wider range of source and network level analyses while avoiding severe confounds produced by the unavoidable summation of their signals being broadly volume conducted from each brain and non-brain source to most of the scalp electrodes.information contributing to the data. Component processes with near-dipolar scalp projections may also represent physiologically distinct brain sources when and if they can be associated with field activity partially or fully synchronized across a cortical patch . The tight connection between temporal independence and dipolarity demonstrated by our results is compatible with the hypothesis that cortical contributions to scalp EEG in large part sum far-field potentials from emergent islands or patches of near-synchronous cortical field activity. The activities of other (and sometimes many) maximally independent component processes are clearly generated, at least in large part, by non-brain sources typically identifiable as arising predominantly from eye blinks or saccades, scalp or neck muscle electromyographic activity, electrocardiographic contamination, line noise, single electrode noise, etc.Our results confirm that ICA and other BSS algorithms are capable of separating high-density EEG data into as many as dozens of processes with maximally independent time courses and near-dipolar scalp projections. Each unmixed maximally independent component process can be said to be a concurrently active source of ICA decomposition transforms the problem of EEG analysis from analysis of the locally highly-correlated source signal mixtures recorded at the two-dimensional scalp surface to analysis of the time courses and spatial 3-D source distributions of maximally temporally independent data sources whose separate patterns of projection via volume conduction to the scalp sensors are given by the decomposition. More importantly for neuroscience, the enhanced signal-to-noise ratio of the unmixed independent source time courses of both brain and non-brain component processes allow study of relationships between multiple EEG source processes, behavior, and subject experience through a collection of single trials and/or in the continuous data record. Further, the separated component scalp maps greatly simplify the process of identifying the cortical patch (or non-brain source) involved in generating each identified source activity using a suitable biophysical inverse method.Here we have shown, first, that although some ICA and BSS algorithms return larger numbers of EEG components with more nearly dipolar scalp maps than others, they appear to identify similar, biologically plausible brain and non-brain component processes. Further, we have shown that for many ICA and BSS algorithms applied to our data, their degree of efficiency in reducing mutual information among the resulting component time courses is positively correlated with the number of biologically plausible near-dipolar components they return. Moreover, for 18 such algorithms , this relationship appears to be, on average, linear, a result that invites deeper study since the methods used by these algorithms appear on the surface diverse and dipolarity and MIR would seem to have no inherently simple numerical relationship.what EEG source activity time courses compose the data record from the biophysical inverse problem of finding where these source activities take place, and (b) separating the spatial source projection pattern for each identified source signal, thus simplifying the biophysical inverse problem for sources in the whole data, rather than only in limited response averages drawn from it. We hope that extracting source-level information from high-density EEG data by ICA decomposition brings closer the goal of developing high-density EEG imaging into a true functional 3-D cortical imaging modality, with high temporal resolution and spatial resolution adequate for studying distributed macroscopic cortical brain processes supporting both normal and abnormal behavior and experience.To invite comparisons of the methods tested with other linear decomposition techniques, we are making the anonymized EEG data and custom MATLAB analysis and plotting scripts available for this purpose see . We hopeHuman subject data presented in this article have been acquired under an experimental protocol approved by an Institutional Review Board of University of California San Diego. Written consent was obtained from each subject.Fourteen volunteer participants performed a visual working memory task EEG data were collected from 71 channels  at a sampling rate of 250 Hz with an analog pass band of 0.01 to 100 Hz . Input impedances were brought under 5 kΩ by careful scalp preparation. We initially selected data from 14 out of 23 participants based on the perceived quality of the original ICA decompositions under visual inspection . Of these, we informally judged seven to give ‘better’ extended infomax ICA decompositions , and seven to give ‘poorer’ ICA decompositions (with fewer such component maps). For one of the participants with an unusually ‘poor’ ICA decomposition, for unknown reasons all ICA/BSS algorithms failed to substantially reduce mutual information from the level of the raw scalp channels. Results for this data set were also unreliable across algorithms, so data from this participant were excluded, leaving data sets from 13 participants to be used in the comparisons.Data were analyzed by custom MATLAB scripts built on the open source EEGLAB toolbox number of channels by number of time points), number of ICA components by number of channels), and number of ICA components by number of time points).We tested a total of 22 linear decomposition algorithms, 20 ICA or BSS algorithms plus principal component analysis (PCA) and PCA-related data whitening or sphering. MATLAB code for the ICA algorithms we used can be downloaded from the Internet see . The selICA and BSS algorithms learn the unmixing weight matrix that makes the resulting component time courses or activations as temporally independent from each other as possible. However, the approach of each algorithm to estimating and/or approaching this independence is different. AMICA SOBI Other algorithms including  FastICA maximize the negentropy of their component distributions or their fourth-order cumulants  information in the data instead of, like PCA, characterizing orthogonal directions of maximal variance in the data. Thereby ICA can bypass PCA's spatial orthogonality constraint that invariably gives a complex checkerboard appearance to scalp topographies of high-order PCA components ICA and BSS algorithms differ from principal component analysis (PCA) in that they identify sources of distinct Sphering decorrelates the component pair time courses while leaving each component scalp map centered on an original scalp channel. It is equivalent to rotating the data into their principal component basis, equating data variance along all principal axes (thus ‘whitening’ the data), and then rotating the data back to their original channel basis differential entropy, and is defined byIndependence of a set of random variables can be measured by the mutual information. Mutual information is defined in terms of the entropy (a measure of the degree of randomness or unpredictability) of the data, which for a continuous random vector mutual information between two random variables The combinations of component values are more or less likely than the individual component values would in themselves dictate).The mutual information is always non-negative since the joint entropy of a random vector is always less than or equal to the sum of the marginal entropies, with equality only if the components of the random vector are independent. Larger mutual information indicates that the entropy or “uncertainty” in the joint distribution of For ICA analysis, the observed EEG data are modeled as an independent and identically distributed (i.i.d.) realization of a vector time series In cumulant ICA methods, the source densities are expanded in terms of cumulants  that are estimated empirically from the partially unmixed data as the un-mixing matrix is optimized For the linear transformation The pairwise mutual information matrix We estimate the entropy using the usual binning method, where histograms and a simple Riemann approximation to the integrals are used to compute the entropies. This approach is generally suitable for large sample sizes like those encountered in EEG. Approximate asymptotic variance of the estimate is also available in terms of number of bins and number of samples, allowing us to assess the statistical significance of the results.kth bin. The estimate of the continuous one-dimensional density kth bin, where kth bin, and We choose a fixed number of bins, kth bin and lth bin. The estimate of the joint entropy is then,ix and jx, we have,The two dimensional joint entropy is estimated similarly, with the joint density of The approximate entropy estimator bias is given bymutual information reduction (MIR), i.e. the amount of mutual information removed from set of channels, is given by,The reduction in mutual information achieved by a potential ICA matrix W can be calculated using only one-dimensional density models. Specifically, the After computing all 22 decompositions for each of the 13 EEG datasets, we localized a best-fitting single equivalent dipole corresponding to each returned component using a single equivalent dipole in a best-fitting spherical four-shell model head  using the DIPFIT plug-in (version 1.02) in the EEGLAB toolbox (version 4.515) http://sccn.ucsd.edu/wiki/BSSComparison) together with a ‘README’ file and MATLAB scripts using EEGLAB functions (http://sccn.ucsd.edu/eeglab) that perform the algorithm comparisons including computation of mutual information reduction (MIR) and dipolarity (dipole fitting). Plotting scripts are also included for reconstructing http://sccn.ucsd.edu/eeglab), interested readers may compare MIR and dipolarity of any other linear decompositions of these data, and may compare results of an algorithm of their choice with our results as applied to the same data sets. The authors invite readers to send us results of these comparisons for inclusion on the web page above, following instructions contained in the README file.The 13 anonymized data sets used in the analysis are available for download (


In this study, we use a modeling approach to show how such a silent MPSO can exist in mitral cells and favor the synchronization of cell discharge to the MOB slow rhythmic input.In mammals, the main olfactory bulb (MOB) is driven by air flow respiratory input into the nose. This slow 2-10 Hz) rhythmic input can be observed both at the local field potential (LFP) level, single cell discharge pattern level and intracellular slow oscillations of mitral cells . Using intracellular recordings in freely breathing anesthetized rats, we examined membrane potential slow oscillations (MPSO) in mitral cells and their relationship with their discharge patterns [0 Hz rhytFirst, using a simple integrate-and-fire (IF) model, we showed that the presence of strong and balanced excitatory and inhibitory inputs with similar oscillatory dynamics led the model to display a silent MPSO. This silent MPSO is characterized by a flat membrane potential in spite of strong synaptic conductance oscillations. Second, assuming that the slow oscillatory input could be decomposed as a mean input and stochastic fluctuation with oscillating amplitudes, we showed that the model could display a slow rhythmic discharge similar to our experimental observations.However, MOB mitral cells have a complex anatomy with (i) an apical dendrite and a tuft receiving both excitatory nose input and local inhibitory regulation, and (ii) long lateral dendrites which receive a large recurrent inhibitory input from the MOB network. We thus wondered how our previous modeling results could generalize in this case. To this aim, we used a previously developed detailed mitral cell model and showed with numerical simulations that silent MPSO could be generated in this model. However, the precise dynamics of the oscillations depended on the main inhibitory input of the mitral cell. If most of the inhibition comes from the tuft, then the silent MPSO can quite easily be generated in the cell, and partial imprecision in the excitation-inhibition balance can be hindered by the apical dendrite filtering. However if a fraction of the inhibition is coming from the lateral dendrites, the silent MPSOs are more difficult to stabilize and the resulting MPSOs have complex shapes resembling some of our intracellular recordings.Overall, we found that mitral cell silent MPSOs with respiratory-patterned discharge, that we observed experimentally, can indeed be a consequence of the precise balance of their slow oscillatory excitatory and inhibitory inputs.


One possible explanation for its occurrence is coregulation of ionic current levels, and specifically the changes that appear to take place in such relationships in response to deafferentation  [etc., which all constitute separate objectives in the MOEA, and must be within limits determined in physiological experiments for a model to be deemed acceptable. We consider such models to represent "recovered" neurons, as they function despite neuromodulation deprivation [i.e., maximum membrane conductances) for the AB and PD neurons in isolation, as well as the entire pacemaker.Neurons in the crustacean stomatogastric ganglion (STG) receive neuromodulatory inputs from higher centers through the stomatogastric nerve (stn). After the stn is cut or blocked, the stereotypical bursting activity of the system ceases, as STG neurons initially lose their activity pattern. Interestingly, however, the neurons typically recover their function within 24 to 96 hours and again exhibit activity similar to that in intact STGs . This phivation) . We defiivation) ). Specif