A number of studies has addressed the possible association between patent foramen ovale (PFO) and stroke. However, the role of PFO in the pathogenesis of cerebral ischemia has remained controversial and most studies did not analyze patient subgroups stratified for gender, age and origin of stroke.To address the role of PFO for the occurrence of cerebral ischemia, we investigated the prevalence of right-to-left shunt in a large group of patients with acute stroke or TIA. 763 consecutive patients admitted to our hospital with cerebral ischemia were analyzed. All patients were screened for the presence of PFO by contrast-enhanced transcranial Doppler sonography at rest and during Valsalva maneuver. Subgroup analyses were performed in patients stratified for gender, age and origin of stroke.P < 0.001). PFO was associated with stroke of unknown origin in male (P = 0.001) but not female patients (P > 0.05). After adjusting for age no significant association between PFO and stroke of unknown origin was found in either group.A right-to-left shunt was detected in 140 (28%) male and in 114 (42%) female patients during Valsalva maneuver, and in 66 (13%) and 44 (16%) at rest respectively. Patients with right-to-left shunt were younger than those without (Our findings argue against paradoxical embolization as a major cause of cerebral ischemia in patients with right-to-left shunt. Our data demonstrate substantial gender-and age-related differences that should be taken into account in future studies. Despite numerous studies published in the past two decades, the role of patent foramen ovale (PFO) as a risk factor of stroke remains a matter of debate. A significant correlation between PFO and cryptogenic stroke has repeatedly been shown. However, most studies included only small numbers of patients and thus did not allow to adjust the analysis for gender and age.The aim of our study was to re-evaluate the association between right-to-left shunts (RLS) and stroke subtypes in a patient community large enough to allow multivariate analysis with special consideration of gender-related differences.The records of 973 consecutive patients examined between January 1997 and December 2005 at the Neurovascular Laboratory of the Department of Neurology at the Klinikum Rechts der Isar, Technische Universitaet Muenchen, were retrospectively reviewed. 210 patients without definite diagnosis of cerebral ischemia and those with artificial heart valves were excluded.Complete clinical neurological examination, electrocardiogram, and sonographic examination of the extra- and intracranial arteries were carried out in all patients, as well as a cerebral CT or MRI examination, or both. Echocardiography was performed in 683 patients (89.5%). A 4-lead 24-hour ECG was performed routinely.All baseline ischemic events were classified according to the TOAST criteria using all diagnostic data available,  with oneThe TOAST subtyping was performed by one physician (H.P.) who was blinded for the results of the TCD testing.®) were chosen.For microembolic monitoring, a 2-MHz pulsed-wave transcranial Doppler device  was used for simultaneous insonation of both middle cerebral arteries (MCA) using simultaneous 64-point FFT and bigate technique. An intensity threshold of = 11 dB and a time window of 20 seconds after the start of the injection of galactose (Echovist®) via the right antecubital vein followed by a flush injection of 5 mL of normal saline. Five seconds after start of the injection, patients had to perform a Valsalva maneuver. This was monitored by means of a pressure gauge, which was connected to a flexible tube with a snorkel mouthpiece. The patients were asked to maintain a pressure of 4000 Pa (40 mbar) for 5 seconds. Simultaneous monitoring of the Doppler spectrum allowed us to demonstrate an increased intrathoracic pressure as shown by a reduction in the mean velocity in the MCA of at least 25%. In case of a positive finding, the examination was repeated at rest to discriminate functional large versus small shunts.The patient was placed supine. The transducer was fixed in position with the use of a standard headset. The embolic signals were recorded after bolus injection of galactose (EchovistC-TCD was performed in every case according to our previously published protocol. Except fContinuous data are shown as mean and standard deviation (SD); categorical variables are expressed as absolute and relative frequencies. Differences were tested by chi-square and Mann-Whitney U-test as adequate. Associations between PFO, confounding factors, and different subtypes of stroke were calculated by logistic regression analysis and described using odds ratios (OR) with 95% confidence intervals (CI). The analyses were carried out on the dataset stratified by gender.All calculations were performed using SPSS 13.0 software .Basic characteristics are given in Table P < 0.001). 66 (13%) male and 44 (16%) female patients showed RLS at rest. Both male and female ES-positive patients were younger (P < 0.001) and had fewer traditional vascular risk factors than participants of the same gender without RLS (P < 0.01).RLS was detected in 140 (28%) male and in 114 (42%) female patients during Valsalva maneuver . In male ES positive patients no significant association with atrial fibrillation was found.Female ES positive patients showed a lower prevalence of atrial fibrillation . Multiple logistic regression analysis with adjustment for age leads to a substantial decrease of the effect of the PFO , which is no longer significant. The corresponding values for female patients showed a nonsignificant risk for cryptogenic stroke for ES-positive patients , an effect which completely disappeared after adjustment for age .P < 0.001).Stroke of unknown origin in men and women was associated with younger age . Conflicting with our results, neither the autoptic study of Hagen et al. nor the abovementioned population-based study showed the incidence of PFO to differ significantly between men and women.,12,14 OfOur results furthermore question the common theory of paradoxical embolism: Presuming arterial embolism via PFO secondary to a venous embolic source, the amount of shunt volume would be expected to correlate with the risk of stroke. This thesis has support from prior TEE-based studies.,17 AnzolAnother possible explanation for stroke secondary to PFO but independent of paradoxical embolism is secondary cardiac arrhythmias. We did nOther possible explanations include abnormalities of the endocardial surface of the septum or within the PFO that are a focus for thrombus formation. A substaDespite these limitations, the large number of patients included and the use of c-TCD contribute new arguments, particularly by weakening the thesis of a significant correlation between PFO and cryptogenic stroke and assessing potential gender-related differences that should be taken into account in future studies.The authors declare that they have no competing interests.


Plasmodium development in the mosquito is crucial for malaria transmission and depends on the parasite's interaction with a variety of cell types and specific mosquito factors that have both positive and negative effects on infection. Whereas the defensive response of the mosquito contributes to a decrease in parasite numbers during these stages, some components of the blood meal are known to favor infection, potentiating the risk of increased transmission. The presence of the antimalarial drug chloroquine in the mosquito's blood meal has been associated with an increase in Plasmodium infectivity for the mosquito, which is possibly caused by chloroquine interfering with the capacity of the mosquito to defend against the infection.Anopheles gambiae genes that are differentially regulated by the presence of chloroquine in the blood meal, using an A. gambiae cDNA microarray. The effect of chloroquine on transcript abundance was evaluated separately for non-infected and Plasmodium berghei-infected mosquitoes. Chloroquine was found to affect the abundance of transcripts that encode proteins involved in a variety of processes, including immunity, apoptosis, cytoskeleton and the response to oxidative stress. This pattern of differential gene expression may explain the weakened mosquito defense response which accounts for the increased infectivity observed in chloroquine-treated mosquitoes.In this study, we report a detailed survey of the Plasmodium at the level of gene expression and highlight the need for a better understanding of the impacts of antimalarial agents on parasite transmission.The results of the present study suggest that chloroquine can interfere with several putative mosquito mechanisms of defense against  Plasmodium development in the mosquito vector involves several critical steps and the sporogonic cycle needs to be completed successfully within the mosquito for the parasite to be transmitted to the vertebrate host. Mosquito physical and chemical barriers that are represented by the epithelia and the mosquito immune response are important factors contributing to the decrease of parasite numbers during this cycle Plasmodium infectivity for the mosquito Plasmodium species. In an attempt to elucidate further the components and mechanisms underlying this effect, we have used Real-Time PCR to demonstrate for the first time that chloroquine down-regulates some immune-related serine proteases and antimicrobial peptides in Anopheles gambiae independently of the presence or absence of Plasmodium berghei infection, midgut bacterial flora or the bactericidal effect of chloroquine Plasmodium yoelli genes that are upregulated during transmission in the presence of chloroquine Plasmodium invasion of midgut cells Anopheles serine proteases cascade, thereby interfering with immune signal transduction pathways that control the transcription of effector genes. However, the mechanism underlying the down-regulation or the extent of chloroquine interference with the multifaceted defense system of mosquito remains unknown.Chloroquine ingestion at the time of the blood meal has been associated with an increase in Plasmodium falciparum resistance, chloroquine remains a commonly used agent against Plasmodium vivax and other human malarias in most endemic regions P. falciparum isolates from some regions of the People's Republic of China and Malawi have decreased or disappeared several years after the discontinuation of chemotherapy Plasmodium defence is emerging as an issue of major importance for the future of malaria treatment strategies. With the present study we aimed to expand our current understanding of the impact of chloroquine on gene expression in the mosquito host by conducting a genome-wide transcript analysis. An A. gambiae cDNA microarray platform comprising 20,000 EST clone inserts from various developmental and tissue specific EST libraries Although the use of chloroquine in malaria chemotherapy has been abrogated in many regions due to Anopheles gambiae s.s. (SUAKOKO strain) were reared at 25°C and 75% humidity with a 12-hour light/dark cycle and were maintained on a 10% glucose solution. At 5–6 days post-emergence, mosquitoes were blood fed on either: i) BALB/c mice (Mus musculus) that 17 hours earlier had been treated intraperitonally with a curative dose of chloroquine (50 mg/kg); and ii) BALB/c mice infected with Plasmodium berghei ANKA clone 2.34 previously treated with 50 mg/kg chloroquine, as described in detail by Abrantes et al. i) non-infected BALB/c mice; and ii) BALB/c mice infected with P. berghei ANKA, respectively. For each group analyzed, mosquitoes were collected 24 hours after blood feeding  comprising 20,000 EST clone inserts from various developmental and tissue-specific EST libraries The http://www.ansorge-group.embl.de/chipskipper; accessed October 2004). The intensities of the hybridized targets and associated background were estimated. The pre-processing analysis was performed using the Chipskipper software. Spots of poor quality were removed and the background was subtracted. The data were transformed in log2-ratio  and normalized within-array using the locally weighted linear regression (LOWESS) method M-value , and a suitable cut-off  was determined for genes with expression greater than a 1.5 fold change. The microarray data were submitted to ArrayExpress .Microarray slides were scanned on a TECAN LS 300 Scanner  and the images were analyzed with the Chipskipper software version 1.1. . The final expression data for ESTs that belong to the same EST cluster were averaged.Identification of ESTs and assignment of putative gene functions were performed according to AnoEST database version 5, oxidative stress-related genes , and a protein synthesis-related gene . Gene-specific primers were designed with the Primer Express software (Applied Biosystems), and are represented in the supplementary material  and reverse-transcribed with M-MLV-RT (Invitrogen- Life Technologies) in the presence of the Oligo(dt)15 primer . Five genes were used for validation of the expression data and included immune genes  of cDNA was used as template. The cycle conditions were: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds and annealing at specified temperature for 1 minute. The optimized concentrations of MgCl2 and annealing temperatures are summarized in S7 gene utilizing a standard curve method for quantification.Quantitative analysis of the expression of these genes was done by Real-Time PCR with the qPCR core kit for SYBR Green  using the iCycler iQ™ (Bio-Rad). The reactions contained 1x reaction buffer, 200 μM dNTP's, 0.3 μM of each primer, 0.025 U/μl of Hot GoldStar enzyme and 1∶66000 dilution of SYBR Green in a final volume of 20 μl. One microliter (A. gambiae midgut transcript abundance was assessed by comparing the gene expression levels of i) mosquitoes fed on a blood meal that contained chloroquine and those fed on a normal blood meal (Chl 50), and ii) mosquitoes fed a P. berghei-infected blood meal that contained chloroquine and those fed a normal P. berghei-infected blood meal (Chl 50Pb).The effect of chloroquine on P. berghei parasites committed to sporogonic development was assumed to be the same as in the previous work The presence of A. gambiae ESTs were up or down-regulated by at least 1.5-fold. The M-value and the average log-intensity (A) for the final datasets are shown in MA-plot Chl 50), with 94 genes being down-regulated and 32 genes up-regulated. P. berghei- infected mosquitoes (Chl 50Pb) showed modulation of expression of 206 genes upon chloroquine exposure: 114 genes were down-regulated and 92 were up-regulated. Complete lists of affected genes are available as supplementary material  and elongation factor 1 alpha (ENSANGG00000015883) were confirmed as being down-regulated in Chl 50 mosquitoes, while the genes for serpin sp121F and C-type lectin CTL4 were confirmed as being down-regulated in Chl 50Pb mosquitoes. Regulation of the elongation factor 1 alpha gene in Chl 50Pb mosquitoes was the only exception observed, since the decrease of expression observed by QRT-PCR results contrasted with the weak up-regulation observed in the microarray analysis. This discrepancy may be due to alternative spliced transcripts or cross-hybridization with similar genes. Although not shown in gambicin. As reported previously by our group gambicin is down-regulated in infected and non-infected mosquitoes fed on a blood meal containing chloroquine.Five genes were subjected to QRT-PCR analysis to confirm the robustness of the microarray data . These gWe will focus on the description of genes involved in the mosquito defense response. Data concerning other biological processes are detailed in the Supporting information file  and in STRX1; GenBank accession number: AL934763) and zinc-superoxide dismutase , and two products associated with detoxification or hormone metabolism . In contrast, the CYP450 gene (AL696888) from the CYP6 gene family was up-regulated, suggesting different cellular functions for the different CYP families. Differential induction of superoxide dismutases  has been associated with broad physiologic differences between refractory and susceptible strains of A. gambiaeOxidative stress-responsive and detoxification genes represented the major functional classes that responded to chloroquine, with 11 genes being down-regulated and 2 being up-regulated . Down-reAL694579), and a leucine-rich repeat (LRR) domain protein (AL934484) were found to be less abundant than in the non-chloroquine-exposed blood-fed controls. On the other hand, transcripts coding for a C-type lectin (AL694890), an angiotensin converting enzyme (ACE) precursor (AL692902), and the phagocytosis-related apolipoprotein D precursor (AL932044) were expressed at higher levels in chloroquine-treated mosquitoes than in the reference controls. Besides being implicated in immunity, C-type  lectins and proteins that contain LRRs are involved in a variety of other important biological processes, such as cell adhesion, cell signaling and apoptosis. The transcript levels of an IAP repeat-containing inhibitor of apoptosis (the direct inhibitors of caspases-apoptosis executors), and of a sphingomyelin phosphodiesterase precursor were decreased 24 hours after a blood meal that contained chloroquine. Sphingomyelin phosphodiesterases are implicated in the metabolism of ceramide, a well-defined intracellular mediator of apoptosis in vertebrate defense against intracellular pathogens P. berghei invasion of mosquito midgut cells Activation of mosquito innate immune response genes has been associated with the ingestion of a blood meal Chl 50Pb were oxidative stress, immunity, protein synthetic machinery, transport (see Supplementary Information Chl 50Pb) but not by chloroquine in the absence of P. berghei; this may be related to the elevated infection levels and associated cytoskeletal reorganizations The major functional classes of regulated genes in Of the 12 differentially regulated oxidative stress/detoxification genes, 7 were down-regulated and 5 were up-regulated by chloroquine treatment . Among tAnopheles midgut cells that are undergoing apoptosis in response to Plasmodium invasion Insect peroxidases are involved in detoxification, stabilization of extracellular matrices and insect immunity, mediating the nitration of BX468701) and the PGRPLB (AAAB01008987). The antimicrobial peptide gambicin gene (AJ237664), the Spi21F, which encodes a fragment of the serpin SRPN10 (BX468769), prophenoloxidase PPO3 (AF004916), and a gene product of unknown function but with similarities to a transferrin-like protein were found to be repressed in chloroquine-treated infected mosquitoes (Chl 50Pb).The presence of 11 gene transcripts with putative immunity functions reinforces our previous data that chloroquine has a significant impact on mosquito immunity-related genes. Among the 11 chloroquine-regulated genes, 2 genes encode pattern recognition receptors, the C-type lectin CTL4 (BX469333), a putative serpin, the suppressor of cytokine signaling SOCS (AL695179), and a leucine-rich repeat domain protein were induced in chloroquine-exposed infected mosquitoes.In contrast, transcripts that encode a lysozyme, the CLIP domain serine protease CLIPA2 , the A. gambiae caspase 3 precursor gene, CASPS3 (Al934916), and a sphingomyelin phosphodiesterase precursor gene. Another repressed gene was a cathepsin B cysteine proteinase precursor, which is known to play an important role in extracellular matrix (ECM) remodeling .Apoptosis-related gene transcripts that were decreased by chloroquine treatment included an IAP  and Plasmodium-infected conditions (Chl50Pb). While chloroquine affected mosquito genes that are implicated in very diverse functions, the present study focused on those genes that have been linked to innate immunity and processes that are known to be affected by Plasmodium infection. Chloroquine induced differential regulation of a plethora of genes implicated in immunity, apoptosis, the cytoskeleton, adhesion, and oxidative stress, confirming our previous findings Plasmodium defense processes.In the present study, we report the first detailed survey of the Chl 50Pb mosquitoes is consistent with this process, as transferrin is a eukaryotic iron-binding glycoprotein that controls the levels of free iron in biological fluids Several theories have been proposed to explain the mechanism of chloroquine action on biological processes. Most of the described effects of chloroquine on metazoan cells appear to be attributable to alterations of the intravesicular pH, which interferes with membrane and recycling processes Plasmodium invasion of the mosquito midgut CTL4 gene which acts as a protective factor of the parasite against melanization CTL4 is a parasite agonist, one might expect a decreased level of P. berghei infection, in contrast to what was observed. However, Frolet et al.A. gambiae anti-parasitic response has two distinct periods, whereby a preinvasion period characterized by basal immunity regulated by REL1 and REL2 members of the NF-κB transcription factors precedes a post-invasion period characterized by the transcriptional up-regulation of antiparasitic genes. Plasmodium berghei development is impaired when the mosquito basal immunity is boosted, and CTL4 seems to be regulated in a NF-kB-independent manner CTL4.In the present study, most of the immune-related genes that were differentially regulated by chloroquine have previously been shown to respond strongly during A. gambiae cells during parasite invasion of the midgut epithelia 24h after the ingestion of an infected blood meal. The down-regulation of important apoptosis-related genes, such as the sphingomyelin phosphodiesterase precursor, which is a mediator of apoptosis that is also known to mediate chloroquine inhibition of NF- κB in epithelial cells Plasmodium midgut invasion Concordant with the results of previous studies of the rats intestinal mucosa Chl 50Pb but not in Chl 50 mosquitoes is in agreement with the reported designation of cytoskeleton dynamics and remodeling as a local epithelial reaction to midgut invasion by PlasmodiumP. berghei-infected mosquitoes. Knockdown of some of those actin-cytoskeleton-architecture-related genes was shown to increase oocyst numbers Regulation of cytoskeleton- and adhesion-related genes in PlasmodiumAnother highly prevalent group of genes showing regulation by chloroquine was that associated with oxidative stress. This was not surprising, as one of the proposed mechanisms of chloroquine action is interference with reactive oxygen species detoxification systems Plasmodium, down-regulating important immune-, apoptosis-, cytoskeleton-, adhesion- and oxidative stress-related genes. This may contribute to the suppression of the well-established defense response system of Anopheles vectors in turn leading to the increased infectivity and modulation of parasite gene expression Taken together, our data show that chloroquine has a significant impact on the transcript abundance of mosquito genes implicated in defense against Plasmodium infection by the potential simultaneous activation of different pathways and modulation of both intravesicular pH-dependent and independent steps. We propose that chloroquine acts by modulating basal immunity, down-regulating apoptosis, and remodeling epithelial cells, thereby permitting more efficient parasite invasion and consequent infectivity changes.Even though we are still not able to dissect the chloroquine mechanism of action underlying this effect, the extent of the impact of chloroquine on mosquito responses is evident from the present study, and it suggests that chloroquine impairs mosquito defenses against P. berghei and P. falciparum parasites P. falciparum and anti-P. berghei activities have been noted for many immune genes P. vivax, P. malariae, P. ovale, and P. knowlesi infections, and the reintroduction of chloroquine as a potential component of P. falciparum combination therapy has been proposed. The present work highlights the need for a better understanding of the impacts of antimalarials on vector and parasite transmission.The impact of chloroquine modulation on murine malaria transmission is evident from our studies. It is however unclear whether these results can be extrapolated to human malaria. Differences in the transmission of rodent and human malaria cannot be disregarded, although similar ookinete invasion routes and midgut epithelial responses have been observed for Table S1Primers and amplification conditions used in QRT-PCR.(0.02 MB DOC)Click here for additional data file.Table S2Detailed annotations of differentially regulated Chl 50 genes.(0.07 MB XLS)Click here for additional data file.Table S3Detailed annotations of differentially regulated Chl 50Pb genes.(0.09 MB XLS)Click here for additional data file.Table S4List of genes demonstrating transcript level changes in response to presence of chloroquine on the blood meal (cont.).(0.24 MB DOC)Click here for additional data file.Figure S1Validation of DNA Microarray results using Quantitative Real-Time PCR analysis. Effects of chloroquine on the transcript abundances of five genes, as assessed by microarray analysis (in black) and QRT-PCR (grey): cytochrome c oxidase (ENSANGG00000019581), thioredoxin: (TRX1), and elongation factor 1-alpha (ENSANGG00000015883) in non-infected treated mosquitoes (Chl 50); and serpin (spi21F), C-lectin (CTL4), and elongation factor 1-alpha in P. berghei-infected treated mosquitoes (Chl 50Pb). Data were normalized using the A. gambiae ribosomal protein S7 gene expression levels. The Y-axis values represent the mean fold-changes obtained for three independent experiments comparing the chloroquine-treated expression levels to the untreated expression levels.(0.06 MB TIF)Click here for additional data file.Text S1(0.04 MB DOC)Click here for additional data file.


Constraint-based flux analysis of metabolic network model quantifies the reaction flux distribution to characterize the state of cellular metabolism. However, metabolites are key players in the metabolic network and the current reaction-centric approach may not account for the effect of metabolite perturbation on the cellular physiology due to the inherent limitation in model formulation. Thus, it would be practical to incorporate the metabolite states into the model for the analysis of the network.iAF1260 in silico metabolic model of Escherichia coli, we demonstrated that this novel concept complements the conventional reaction-centric analysis.Presented herein is a metabolite-centric approach of analyzing the metabolic network by including the turnover rate of metabolite, known as flux-sum, as key descriptive variable within the model formulation. By doing so, the effect of varying metabolite flux-sum on physiological change can be simulated by resorting to mixed integer linear programming. From the results, we could classify various metabolite types based on the flux-sum profile. Using the Metabolite flux-sum analysis elucidates the roles of metabolites in the network. In addition, this metabolite perturbation analysis identifies the key metabolites, implicating practical application which is achievable through metabolite flux-sum manipulation in the areas of biotechnology and biomedical research. Cellular metabolism is more often than not represented and analysed based on a stoichiometric modelling framework under the stationary assumption of the metabolic network . Such stMetabolites and biochemical reactions in the metabolic network can be graphically represented by nodes and edges connecting the nodes respectively. Based on this graphical representation, it is obvious that there can be two approaches in the analysis of the network, focusing on the flow of materials through either the nodes (metabolite-centric approach) or edges (reaction-centric approach). Typical FBA can be conducted based on a reaction-centric approach where constraints were introduced to restrict the range of reaction flux values so as to define a feasible solution space . This anE. coli system using i AF1260 in silico metabolic model , H2O [p] and H2O [e] indicate water found in three different compartments. The cellular objective was assumed to be the maximization of biomass production. The reaction reversibility constraints were set as given by [Escherichia coli in glucose minimal medium. All these settings were based on previous observation for glucose and oxygen uptake rates and experimentally determined ATP requirement for maintenance [The genome-scale given by . The nonntenance ,21. The iAF1260 model by solving (P1), resulting in 4.20 and 23.9 for average (μFS) and standard deviation (sd) of the flux-sum values respectively. About 70% of the metabolites have zero flux-sum in the base case while metabolites with high , allowed us to identify different types of metabolites. Firstly, we found blocked metabolites with maximum flux-sum equal to zero. The consumption and production of these metabolites were blocked due to reaction pathway dead-ends  with constraint (C1) for these 394 basal active metabolites, thereby giving rise to the flux-sum attenuation profile  with constraint (C2), we generated the flux-sum intensification profile .Click here for fileEscherichia coli.Demonstration of flux-sum analysis for increasing succinate production in Click here for file


Drosophila we modulated the function of Shaw, a widely expressed neuronal potassium (K+) channel known to regulate RMP in Drosophila central neurons.In addition to the molecular feedback loops, electrical activity has been shown to be important for the function of networks of clock neurons in generating rhythmic behavior. Most studies have used over-expression of foreign channels or pharmacological manipulations that alter membrane excitability. In order to determine the cellular mechanisms that regulate resting membrane potential (RMP) in the native clock of 1, DN2 and DN3), or in subsets of clock neurons  increases locomotor activity at night. In free-running conditions these manipulations result in arrhythmic locomotor activity without disruption of the molecular clock. Reducing Shaw in the DN alone caused a dramatic lengthening of the behavioral period. Changing Shaw levels in all clock neurons also disrupts the rhythmic accumulation and levels of Pigment Dispersing Factor (PDF) in the dorsal projections of LNv neurons. However, changing Shaw levels solely in LNv neurons had little effect on locomotor activity or rhythmic accumulation of PDF.We show that Shaw is endogenously expressed in clock neurons. Differential use of clock gene promoters was employed to express a range of transgenes that either increase or decrease Shaw function in different clusters of clock neurons. Under LD conditions, increasing Shaw levels in all clock neurons (LNv, LNd, DNBased on our results it is likely that Shaw modulates pacemaker and output neuronal electrical activity that controls circadian locomotor behavior by affecting rhythmic release of PDF. The results support an important role of the DN clock neurons in Shaw-mediated control of circadian behavior. In conclusion, we have demonstrated a central role of Shaw for coordinated and rhythmic output from clock neurons. These feedback loops, and the behavioral rhythms controlled by them, can be reset cell-autonomously by light-induced TIM degradation via the blue-light photoreceptor protein Cryptochrome (CRY) and through light inputs to the pacemaker neurons from the eyes. This central molecular clock mechanism operates in a set of pacemaker neurons located in the fly brain A 24 hour (circadian) cycle of rest and activity exists in almost all animals, persisting even in complete darkness. In flies, this behavior is dependent on the rhythmic expression of oscillating genes under control of a molecular clock that consists of interlocked molecular feedback loops of transcription including the Drosophila may involve ion channels under direct transcriptional control of the clock gene products or post-translational modulation of ion channels by clock controlled proteins + channels are key regulators of the intrinsic excitability in all neurons, and therefore they are crucial for output rhythms of clock neurons under free-running conditions in the Bulla eye + channels has not been extensively explored in Drosophila central clock neurons.Control of electrical activity has been postulated to be important for the function of the clock Drosophila artificial expression of truncated dORK channels has also been shown to hyperpolarize RMP and to decrease the firing rate of clock neurons Current models of the mammalian SCN predict RMP to be rhythmically regulated by the clock Drosophila clock neurons or if they are regulated by the circadian clock. A number of Drosophila ion channels have been implicated in clock function, these include Narrow Abdomen (NA), a Na+/Ca++ channel ++-sensitive K+ channel These studies did not, however, shed any light on the actual cellular mechanisms that regulate RMP in the native clock. So far it is not known which endogenously expressed ion channels and membrane proteins influence electrical membrane properties of Drosophila, Shaw is a member of the Shaker family of voltage-gated K+ channels and encodes a slowly activating and non-inactivating K+ current. These channels can be open at normal cell RMP and cause hyperpolarization by allowing K+ efflux. In Drosophila, the Shaw K+ channel is widely expressed in the nervous system and helps regulate RMP in Drosophila central neuron + channel to endogenously regulate RMP in clock neurons. In this study we reveal a circadian function of Shaw K+ channels in Drosophila and demonstrate that they are required for rhythmic output from clock neurons.These studies suggest that electrical membrane properties maybe similarly important in the fly clock and mammalian SCN. Moreover, mammalian Shaw homologues, Kv3 channels, are widely distributed within the SCN and the magnitude of their current varies between the day and the night, even under free-running conditions. Blocking Kv3.1b and Kv3.2 currents prevents the daily rhythm in firing of SCN neurons CantonS, Pdf-GAL4tim-GAL4 insert 27 and 67 cry-GAL4 insert 13 8.0-luc:9DUAS-Shaw-FLAG insert 12B and UAS-ShawTRuncated-FLAG insert 332 (truncated at residue 369) Shaw transgene. The Pdf-GAL80 and cry-GAL80Flies were grown at similar density in bottles on standard medium at 25°C in 12 hr∶12 hr LD cycles. The following strains were used: UAS-Shaw RNAi construct contains a 720 bp fragment of the 3′ end of Shaw starting at nucleotide 881 in Exon 8 through to the end of the gene including approximately 110 bp of 3′ untranslated sequence. This fragment was cloned into the multiple cloning site of the SympUAST transformation vector Drosophila germline by P-element mediated transformation. Stable lines were established and a line containing the insert homozygous on both chromosome 2 and 3 was used.The Locomotor activity of adult males placed in an infrared beam-cross counting apparatus was monitored automatically and analyzed as in Wülbeck et al. Whole-mounted adult brains were processed and stained with a rabbit anti-Shaw C terminal (amino acids 413–498) antibody (pre-absorbed and used at 1∶1000), mouse anti-FLAG , rabbit anti-crab PDH  objectives of a Leica TCS SP2 confocal microscope. Care was taken to keep all intensity readings within the linear range below saturation and all double immunofluorescent images were scanned sequentially. Quantification was performed on 1 µm sections with pixel intensity readings taken in a given region of interest for PDF-Alexa488  with a surrounding background measurement subtracted using the Leica TCS SP2 quantification software. Quantification was performed blind to genotype and experimental condition. Statistical analysis was performed in Excel (Microsoft) and JMP (SAS). Significance levels in figures were determined by one-way ANOVA unless otherwise specified.8.0-luc:9D transgene was measured, analyzed, and plotted as described in Veleri et al. Luciferase expression of individual flies carrying the g and then 14,000× g for 15 min at 4°C to remove debris 2–3 day old flies were entrained to 12 hr∶12 hr LD cycles for 2–3 days and frozen in liquid nitrogen. After decapitating by vortexing, equal numbers of frozen heads from each genotype were homogenized and solubilized in ice-cold RIPA buffer for 30 min. Homogenates were spun at 500× GAL4 drivers timeless . Pdf-GAL4 is expressed only in the small and large ventral lateral neurons (s- and l-LNv). Using GAL80 to suppress GAL4 activity tim-GAL4; Pdf-GAL80 animals, only LNd and Dorsal Neurons should express UAS transgenes Pdf-GAL80 weak reporter gene expression , likely due to the ability of the endogenous and truncated channels to multimerize and the fact that the C-termini of Shaw channel complexes are required for correct localization To visualize endogenous Shaw expression, whole brains of the GAL4/UAS system to manipulate channel function and level in different subsets of clock neurons. Three approaches were used: i) targeted over-expression of Shaw using a full-length transgene; ii) targeted expression of a dominant-negative C-terminal truncated form of Shaw  .To explore the role of Shaw in the clock we employed the e above) ; and iii+ current in clock neurons, full-length Shaw was over-expressed in the tim-GAL4 pattern  cycles. To increase non-inactivating K pattern . This wotim-GAL4/UAS-Shaw; Pdf-GAL80 to tim-GAL4/UAS-Shaw to remove Shaw over-expression in the LNv has no effect on the hyperactive tim-GAL4/UAS-Shaw phenotype  increased night activity persisted  driven Shaw over-expression resulted in increased activity at night, suggesting that the cry expressing Dorsal Neurons also contribute to this phenotype , no activity increase was observed, indicating that the +Pdf  cells also contribute to this phenotype . In summary, our results indicate that the DNs are sensitive to Shaw levels, and to some extent the LNv too. These findings are in good agreement with the endogenous Shaw expression detected in these cells  behavior immediately after the transition to DD  maintain rhythmicity in DD albeit with reduced amplitude compared to controls  for 9–16 days following the LD portion of the experiment . Expresson to DD ; Table 2 compare . Flies tcontrols , Table 1tim-GAL4/UAS-Shaw flies is similar to that seen with over-expression of an open-rectifier K+ channel (dORK) in clock neurons Pdf-GAL4/UAS-Shaw flies remain rhythmic  maintain rhythmic behavior  appears to result in a small increase of rhythmically behaving flies compared to expressing Shaw in all clock neurons (tim-GAL4/UAS-Shaw) suggesting that Shaw in the LNv influences DD behavior to some extent . Although not all the flies were arrhythmic in DD, more than 30% were, and the rhythmic flies exhibited rather weak rhythmicity  lead to a dramatic period lengthening of 3.5 to 4 hr  and introducing a dominant negative form of Shaw, which seems to multimerize with wild-type Shaw subunits in cytoplasmic organelles , in which (Per-Luc) is predominantly expressed in the DN1, DN2, and DN3 neurons with occasional expression in a small subset of the LNd neurons Shaw expression in all tim expressing cells did not have any drastic effect on 8.0-luc reported PER expression in the DNs, neither in LD nor in DD. This indicates that although all clock neurons are electrically inactivated, the molecular feedback loops operate more or less normally at least in the DNs. We did observe a slight increase in period length when Shaw was over-expressed in the 8.0-luc background indicating some influence on the central clock mechanism  and determined the abundance and phosphorylation state of endogenous PER , which are approximately the same at ZT1 and ZT21, and reaching peak and trough levels at ZT9 or ZT13, respectively (p<0.00001). Expression of dominant negative Shaw causes lower PDF levels at day and night compared to wild-type (ZT9 p<0.00001), but levels are somewhat higher at ZT21 and ZT13 compared to ZT9 (p<0.001 and p<0.05 respectively). Reducing Shaw in all clock neurons using Shaw RNAi also results in an overall reduction of PDF levels  and peak levels are also reached during the night (p<0.0001). We cannot rule out that there might still be some PDF rhythm after Shaw over-expression, and possibly still residual, but improperly phased rhythms in flies with reduced Shaw.A mechanism by which Shaw KThe results suggests that increasing hyperpolarizing Shaw throughout the clock circuit causes LNv to display decreased probability of neuropeptide release resulting in the accumulation of PDF in the LNv terminals. In turn, this could explain the increased LD activity levels displayed in this genotype. Genotypes with a reduction of functional Shaw throughout the clock have lower PDF levels in their LNv terminals, perhaps caused by an accelerated neuropeptide release, which in this case is not correlated with a reduction of behavioral activity.Pdf-GAL4/UAS-ShawTR and wild-type are the same, while Pdf-GAL4/UAS-Shaw (p<0.01) and Pdf-GAL4/UAS-ShawRNAi (p<0.001) show weakly increased PDF abundance.Surprisingly, when Shaw is manipulated only in the PDF expressing LNv, rhythmic accumulation of PDF is not disrupted. PDF in the LNv terminals remains higher during the day (ZT1 or ZT9) than at night , as in the controls . FurtherThis suggests that changing Shaw levels in LNv neurons alone has little effect on either rhythmic accumulation or levels of PDF in the LNv terminals. These data are consistent with the lacking effect of changing Shaw levels in LNv neurons on locomotor activity under LD conditions. Instead it seems that changing Shaw levels in the DN cells has the greatest effect on locomotor activity –2 and rhWe show that Shaw-mediated changes in clock neuronal electrical activity severely affect the fly's locomotor activity in LD and DD conditions. Mammalian Shaw homologues have been shown to have a central function in clock neurons, and were recently demonstrated to be the first circadianly-regulated intrinsic voltage-gated currents in mammalian cells Shaw K+ channels  results in an increase in locomotor activity at night. Therefore our data suggest that under LD conditions appropriate Shaw-mediated electrical activity is not required for rhythmic output from the clock, but rather regulates overall behavioral activity levels with the DN neurons being particularly important for this regulation. Similarly in SCN neurons of the mammalian system, decreased electrical activity is correlated with increased behavioral activity In LD conditions, over-expression of In DD, over-expression of Shaw in clock neurons lead to arrhythmic behavior, consistent with previous reports, in which clock neurons were electrically silenced tim-GAL4/UAS-Shaw; cry-GAL80  caused a dramatic period lengthening of 3.5–4 hr, confirming that endogenous Shaw contributes to clock function under constant conditions.Again as for Shaw mediated changes in LD, the Dorsal Neurons seem to have a special role in regulation of DD behavior and are particularly sensitive to changes in the level of Shaw. Restricting over-expression of Shaw to subsets of the Dorsal Neurons with Shaw in a subset of the clock neurons even though we find endogenous Shaw in all clock-neuronal groups, strongly suggests that Shaw may mediate neuronal properties of all clock neurons. However reducing Shaw levels equally throughout the whole clock neuronal network has little effect, perhaps because the communication between the individual cells with respect to each other is not changed and their electrical properties are all changed in the same direction. If only a subset of the network is altered , communication can be influenced or disrupted, resulting in long period rhythms. Alternatively, the clock may be able to compensate for equal increases in electrical activity throughout its neural circuit and preserve its functional output. If however activity is only changed in part of the hierarchy of clock neurons (the DNs), then the clock circuit cannot compensate as a whole and behavior becomes compromised, in this case indicated by a lengthening of the free-running period.The fact that we observe this phenotype only when manipulating − clock neurons to cause arrhythmicity. Second, blocking output of TIM but not PDF neurons by either transgene increases activity at night during LD conditions. This correlation suggests that Shaw somehow affects chemical signaling between clock neurons in non-PDF cells. In turn this could explain the alteration of PDF accumulation in LNv terminals observed with tim-GAL4 (but not Pdf-GAL4) driven Shaw over-expression and down-regulation.Our results are similar to those obtained after blocking pacemaker synaptic output with tetanus toxin (ttx) tim expressing cells had no gross effects on PER oscillations in LD in peripheral clock cells  caused DD arrhythmia but TIM cycling in all clock neuronal types  remained normal in tim-GAL4/UAS-ttx flies under LD and DD It is not understood how temporal information from the molecular clock in the nucleus is conveyed to the membrane, and how rhythmic physiological events in the membrane feedback to the molecular clock. So far this question has been addressed by expression of Drosophila used a series of mutations in narrow abdomen (na), another channel encoding gene expressed in clock neurons na mutations would be expected to hyperpolarize RMP and result in DD behavioral arrhythmicity. However unlike the case of dORK, this was not correlated with a loss of molecular clock protein cycling + channel (dORK) and then used semi-quantification of intensity of PER and TIM antibody staining of the LNvs to judge the status of the molecular clock. In contrast, we electrically inactivated all clock neurons using an endogenous non-inactivating K+ channel and directly measured per-luciferase oscillations of the DNs in the intact animal.Another study that addressed the relationship between electrical activity and the molecular clock of Drosophila membrane properties may only influence clock output. In addition, the fact that we observed Shaw-dependent period-lengthening of behavioral rhythms and PER oscillations suggests that altered membrane properties may modulate the molecular clock. Such a Shaw-dependent lengthening in circadian period in our luciferase or locomotor activity data also reiterates a key current concept in the study of circadian systems: the temporal readout to behavior does not dependent solely on a cell autonomous molecular timekeeper, but coordinated clock output is a property that arises from many clock cells acting together in concert with electrical activity and neurochemicals mediating intercellular signaling and synchronization. In this respect it will be interesting to determine the molecular period of the different subsets of clock neurons in the long period flies caused by reduced Shaw levels in the DN only.Based on these studies, it seems uncertain if changes of membrane properties feedback to influence circadian clock gene expression and if they are indeed required for molecular clock function. If true, this would imply that in Drosophila clock cells is circadianly regulated with resting membrane potential and associated electrophysiological parameters varying between subjective day and subjective night Drosophila study that demonstrated that intracellular Ca2+ regulates clock cell oscillations Drosophila brain clock and the mammalian SCN.Our data lends further support to recent studies that show the electrical activity of cry-GAL4 negative DN1-3) appear to be particularly important for this behavior. Alternatively , the s-LNvs could be the “most important” Shaw cells, but the cry-GAL4 and Pdf-GAL4 drivers maybe too weak (compared to tim-GAL4) to observe an effect of Shaw expression on the circadian clock. Either way our data suggest that PDF negative clock neurons participate in regulation of DD behavior. The importance of the DN in circadian rhythms has been reiterated in recent studies demonstrating that these cells regulate rhythmic behavior in LL Under free-running conditions appropriate levels of Shaw-mediated electrical activity are required in the clock network to maintain rhythmic activity, whereby the DN  clock neurons, which control circadian locomotor behavior by affecting rhythmic release of PDF. We have demonstrated a central role of Shaw KFigure S1cry-GAL4/Shaw flies exhibit a Shaw-dependent activity increase during the night (see text and Behavior of flies in light: dark cycles over-expressing Shaw or a dominant-negative form of Shaw in different subsets of clock neurons. Histograms show daily averages of locomotor activity in the LD portion of the experiment (7 days). Open and black bars indicate activity levels during 30 min intervals when the lights were on and off, respectively. All genotypes exhibit bimodal behavior, showing the characteristic anticipation of the lights-on transition in the morning, and the lights-off transition in the evening. Note that only the (6.01 MB TIF)Click here for additional data file.


To elucidate the molecular mechanism, we investigated the effects of oxaliplatin and its chloride complex [Pt(dach)oxCl]- on the voltage-gated Shaker K channel expressed in Xenopus oocytes. The recordings were made with the two-electrode and the cut-open oocyte voltage clamp techniques.Oxaliplatin is a platinum-based chemotherapeutic drug. Neurotoxicity is the dose-limiting side effect. Previous investigations have reported that acute neurotoxicity could be mediated via voltage-gated ion channels. A possible mechanism for some of the effects is a modification of surface charges around the ion channel, either because of chelation of extracellular CaTo our surprise, we did not see any effects on the current amplitudes, on the current time courses, or on the voltage dependence of the Shaker wild-type channel. Oxaliplatin is expected to bind to cysteines. Therefore, we explored if there could be a specific effect on single (E418C) and double-cysteine (R362C/F416C) mutated Shaker channels previously shown to be sensitive to cysteine-specific reagents. Neither of these channels were affected by oxaliplatin. The clear lack of effect on the Shaker K channel suggests that oxaliplatin or its monochloro complex has no general surface-charge effect on the channels, as has been suggested before, but rather a specific effect to the channels previously shown to be affected. Most of the patients recover a few months after treatment discontinuation --free extracellular solution with the high-resolution cut-open oocyte technique where the oocyte is sandwiched between two holes of two Perspex chambers. The cut-open oocyte technique reduces the capacitive current and thereby allows analysis of the activation time course in detail - that exerts effect. Therefore similar experiments with the two-electrode voltage clamp technique were carried out with oxaliplatin added to a Cl- -containing (0.1 M) extracellular solution. Fig. GK(V), curves calculated as described in Methods. Since most experiments were carried out immediately after the addition of oxaliplatin, a mixture prepared as described in Methods was added to the extracellular fluid to be certain that the oocytes would be exposed to [Pt(dach)oxCl]-. Again, the K channels remained unaffected by this treatment. Totally 31 oocytes expressing wild-type channels were investigated with concentrations of oxaliplatin in the range from 60 to 1000 μM. No shift of the GK(V) curve was seen.We first tested 60 μM oxaliplatin in a Cln detail . The rean = 2; data not shown).For Na channels, it has been reported that the fast inactivation is slowed down considerably ,8. To sen = 8).Oxaliplatin is expected to bind to cysteines . Therefo2+, have a much larger effect on two neighbouring cysteines than on a single cysteine 2) methanethiosulfonate bromide) , was applied continuously in the bath solution by a gravity-driven perfusion system. The cysteine reagent was applied to saturation (1 mM for 1 minute) followed by carefully washing off MTSET.GK(V) was calculated as GK(V) = IK(V)/(V - EK), where IK(V) is the steady-state current,V is the membrane potential measured in the bulk solutions, and EK is the equilibrium potential (-80 mV).The steady-state K conductance Xenopus oocytes, we tested the effects on oocytes with the vitelline layer mechanically removed. No difference was seen between the recordings with or without the vitelline layer (n = 3). Because removal of the vitelline layer makes the oocytes fragile we kept it intact throughout the rest of the experiments.To make sure that the platinum-based substances reach the ion channels and do not get stuck in the vitelline layer surrounding the -, Fig. 2  [Oxaliplatin Fig.  was purcout 10%)  This mixThe authors declare that they have no competing interests.AB carried out the electrophysiological experiments and drafted the manuscript. EJ prepared the platinum compounds and drafted the manuscript. JY initiated the study and helped to draft the manuscript. HE participated in the design of the study and helped to draft the manuscript. FE conceived of the study, and participated in its design and coordination and helped to draft the manuscript.


However, the relation of hemodynamic responses in the resting phase after a preceding cognitive challenge remains relatively unexplored. We test the hypothesis that the degree of activation of the DMN following cognitive challenge is influenced by the cognitive load of a preceding working-memory task.The default-mode network (DMN) is a functional network with increasing relevance for psychiatric research, characterized by increased activation at rest and decreased activation during task performance. The degree of DMN deactivation Twenty-five healthy subjects were investigated with functional MRI at 3 Tesla while performing a working-memory task with embedded short resting phases. Data were decomposed into statistically independent spatio-temporal components using Tensor Independent Component Analysis (TICA). The DMN was selected using a template-matching procedure. The spatial map contained rest-related activations in the medial frontal cortex, ventral anterior and posterior cingulate cortex. The time course of the DMN revealed increased activation at rest after 1-back and 2-back blocks compared to the activation after a 0-back block.We present evidence that a cognitively challenging working-memory task is followed by greater activation of the DMN than a simple letter-matching task. This might be interpreted as a functional correlate of self-evaluation and reflection of the preceding task or as relocation of cerebral resources representing recovery from high cognitive demands. This finding is highly relevant for neuroimaging studies which include resting phases in cognitive tasks as stable baseline conditions. Further studies investigating the DMN should take possible interactions of tasks and subsequent resting phases into account. Resting-state networks (RSN) have recently attracted increased attention in neuroscience research. In contrast to task-related increases of brain activation in functional networks, usually the focus of neuroimaging research, RSNs have been characterized as functionally distinct networks that represent the baseline activity of the human brain in the absence of stimulated neuronal activity e.g. . RSNs alOne prominent RSN is the so-called default-mode network (DMN) that comprises medial frontal regions, the ventral anterior cingulate cortex (vACC), precuneus, the posterior cingulate cortex (PCC) and the lateral parietal cortex during task performance be confirmed? ii. Does the load of a working-memory task exert an influence on the activation of the DMN in the subsequent resting phase?While many studies investigated the activation of the DMN during a challenging task, much less is known about the potential impact of a cognitive task on the subsequent resting phase. A functional connectivity analysis of specific DMN regions in resting phases before and after an orthographic lexical retrieval task revealed relatively small increases and decreases in functional connectivity between both resting sessions Twenty-five healthy right-handed volunteers participated in this study . An initial telephone screening was conducted to ensure inclusion criteria, to exclude medical and neurological diseases or MRI contraindications. The SCID-I-interview was performed to exclude any current or previous psychiatric disorders The working-memory task was part of a larger fMRI and neuropsychological study of memory processes Each n-back block  lasted 36 s, each subsequent rest block 21 s plus 3 s instruction for the next task . N-back blocks and rest blocks were presented in a fixed order . During the resting phase, participants looked at a white fixation cross on a black screen. Subjects were instructed to relax and wait until the next n-back-block starts. Letters were presented in white in the centre of a black screen for 500 ms and an interstimulus interval of 2500 ms .Functional images were acquired on a 3 Tesla whole-body scanner . We used a circularly polarized transmit/receive birdcage head coil with an HF reflecting screen at the cranial end for spin excitation and resonance signal acquisition. One hundred and twenty whole-brain T2* weighted single shot echo planar (EPI) images were acquired . Thirty-six transversal slices orientated to the AC-PC line were acquired.Responses and response latencies (in ms) for the n-back performance were recorded during fMRI scanning. Performance is reported as accuracy rate (percentage of correct answers) for each n-back condition. We performed repeated-measures analyses of variance (ANOVAs) with one within-subject factor (working-memory load: three levels) for accuracy rate and response latency.http://www.fmrib.ox.ac.uk/fsl) Data were pre-processed using tools from the FMRIB Software Library  To select a component corresponding to the DMN, components with motion related artifacts or with a mean power above 0.1 Hz were excluded from further analysis. Furthermore, since TICA is able to extract the subject-/session-domain specific effect magnitude for each spatio-temporal functional activation pattern, we also excluded all components driven by a single outlier subject. For the detection of a rest-related component we used an approach described by A repeated measures ANOVA with two within-subject factors LOAD  and PHASE  was calculated for the dependant variable DMN activation with six repetitions , enabling the investigation of interaction effects of LOAD and PHASE in the resting state component. Post-hoc tests were conducted to test specifically the hypothesis that resting activity after a cognitive demanding working-memory task  has a greater magnitude compared to resting activity after 0-back. Since other studies reported that inter-subject variability of DMN activation can be fairly large Analyses of variance of accuracy and on response latency showed a significant main effect of working-memory load  = 8.75, p<0.001) and  = 17.36, p<0.001), respectively. With increasing working-memory load, accuracy decreased and response latency increased . Post-hoTICA was applied to 25 healthy subjects. The temporal domain of the data was reduced to 31 independent components explaining 80% of the total variance. Fifteen components did not meet the criteria for RSN maps described in the The goodness-of-fit score for the best-fit component was 4.13 and for the second best-fit component 1.60. A two-sample t-test showed that both components differ significantly (p<0.001). Note, that a goodness-of-fit score represents the average z-score of voxels falling within the template minus the average z-score of voxels falling outside the template. Hence, the largeness of the value depends on the range of the z-scores.The selected resting-related component showed activations in the vACC and in posterior parietal and medial occipital areas . The corThis study applied TICA to 25 healthy subjects to investigate the influence of cognitive load in a working-memory task on subsequent resting phases. We determined the DMN using an unbiased template matching procedure From a methodological perspective, a model-free approach such as TICA is an efficient method for identifying time courses of activation of such functional networks. Unlike the analysis based on the general linear model (GLM), the definition of a stable baseline is not necessary. TICA enables the investigation of resting phases between cognitive tasks, which in the GLM are usually considered to correspond to a stable baseline. Since the time-course of the DMN, extracted by ICA, does not represent any relative differences to a certain condition, the increased resting-state activation does apparently not just reflect the negative difference between a baseline and a demanding task. Therefore this approach may reveal task-related shifts in embedded rest phases. The use of TICA is therefore warranted even in designs when the GLM might also be applicable.As a first result of our study, we confirm that deactivation of the DMN during task performance increases with task difficulty, which is in line with other studies investigating the DMN in working-memory paradigms e.g. . McKiernAs the main result of this study, we showed that the DMN was more active in the rest phase after 2-back and 1-back than during rest after 0-back. We hypothesize that DMN activation is influenced by cognitive load not only during but also after cognitive challenging tasks. Previous studies investigating rest phases after cognitive load specifically focused on demonstrating the stability of the DMN and regional connectivity over time. Waites et al. Conclusions regarding the psychological role of the DMN can only be derived indirectly from our data, as the conducted experiment did not include reports of the subjects' mental processes after the n-back blocks. The increased activation of the DMN after 1- and 2-back could be interpreted as increased redistribution of cognitive resources in the subsequent rest phase, reflecting a neuronal correlate of mental fatigue. Previous studies investigated the functional correlates of mental fatigue with respect to chronic diseases, such as fatigue syndrome, but focused only on task-related brain areas or on differences between patients and controls While the n-back task increases working-memory effort parametrically, the decrease and increase of DMN activation seems to be attenuated from 1-back to 2-back. The observed non-parametric decrease during the task condition corresponds to previous findings The functional role of the DMN has been described in the literature with concepts, such as task-unrelated thoughts, stream of consciousness, self-referential processes, verbal and visual imagery, planning and problem solving, internal monitoring and episodic memory encoding Some limitations need to be considered. Although the sample size is high for a functional imaging study and inclusion criteria were rigorous, we cannot completely exclude that our results are influenced by the selection of the cohort. Moreover, the complex behavior of the DMN and the inter-subject variability that has been noticed by us and other groups weaken the results. Another limitation might be the short resting phase between the n-back blocks which does not allow the investigation of the temporal dynamics of the DMN deactivation during a longer period of time. Future studies should integrate a longer resting period in their working-memory task to examine this issue. However, the short resting phase of 24 s in our study underlines the intensity of the described interaction effect demonstrating the modulation of baseline activity by preceding cognitive tasks. Furthermore, we suggest that future investigations should assess psychological variables about self-referential thoughts during the rest phases.In summary, we demonstrated that cognitively challenging working-memory tasks have a significant influence on the activation of the DMN in subsequent resting phases. Thus, we provide evidence that, even at rest, functional processes are affected by the preceding cognitive task. In the framework of a GLM, the reported baseline shifts might lead to overestimated brain activation or deactivation during task performance if they are not included as regressors in the classical multivariate analysis. How this effect is related to self-referential processes in response to a preceding task needs to be investigated in future studies.


Antiplatelet antibodies are known to be present in a wide spectrum of patients, which include chronic Idiopathic Thrombocytopenic Purpura (ITP), infections, etc., including Glanzmann's thrombasthenia (GT) patients who receive multiple platelet transfusions. The presence of natural antibodies to platelet receptors is not studied in cases of GT. We studied the antiplatelet antibodies in 23 patients with GT, 15 of which had received multiple transfusions and eight that had not received transfusions, along with 50 cases of chronic ITP. The prevalence and specificity of platelet-bound antibodies were detected by inhibition assays using O-group platelets on flow cytometry. The mean antiplatelet antibodies in 15 patients of GT who had not received transfusions and eight patients with multiple transfusions was 8427 + 2131.88 and 9038 + 2856 antibodies/platelet, respectively, while in case of the 50 ITP patients studied, it was 22166 + 5616 antibodies/platelet . We conclude that GT patients who have not received transfusions may develop antiplatelet antibodies to the missing/abnormal receptor. Whether this is due to a molecular mimicry or due to some other mechanism needs to be explored. The immune memory recognizes these self-antigens and does not elicit an immune response to them. A classical example of this phenomenon is the blood group agglutinins. However, antibodies may be produced against some such self-antigen epitopes because of a molecular mimicry with some of the natural infectants (virus or bacteria) or intestinal microorganisms. In the early 1980s’, Caen et al. demonstrated the presence of antibodies formed against the glycoprotein receptor IIb/IIIa on the human platelet surface membrane,[Immune recognition to self-antigens is obtained in early fetal life membrane,3 It is nmembrane, GT is a 2 polyclonal anti-IgG-FITC for 20 min and subjected to flow cytometry. The MFI values of samples and controls were extrapolated on the calibration curve and their corresponding molecule numbers were read directly.[Fifteen patients of GT who had not received any transfusions and eight patients of GT with a history of multiple platelet transfusions were studied. Fifty cases of chronic ITP were also included in this study. FITC-labeled rabbit anti-human immunoglobulin (Ig) G  was used for the determination of antiplatelet antibodies. Rabbit anti-mouse IgG-FITC was used as the negative control and mouse anti-human GPIIb/IIIa and GPIb/IX were used for the receptor studies. The receptor studies in the GT patients were carried out using the patient platelets and FITC-tagged receptor-specific antibodies . For the determination of platelet-bound antibodies, patient serum 1:5) was incubated with normal O-group platelets at room temperature for 30 min. After washing thrice in buffered citrated saline, these platelets were incubated with FITC-anti-human globulins for 20 min at room temperature and the fluorescence was measured as mean fluorescence intensity (MFI) by flow cytometry. For the quantitation of antibody, a suspension of calibrated beads precoated with increasing and accurate quantities of Igs was used . These beads were incubated with F(ab’) was incudirectly..HPA-1 genotyping was carried out using allele-specific polymerase chain reaction in GT caA pool of platelets from five O Rh-positive normal healthy subjects was taken. The platelets were washed and the GPIIb/IIIa receptor occupancy was studied using FITC-tagged mouse anti-human GPIIb/IIIa and GPIb/IX antibodies (BD Biosciences and Dako Cytomation) by flow cytometry. The washed platelets were incubated with GT patient serum (1:5) for 30 min at room temperature. Percent inhibition of fluorescence-tagged GPIIb/IIIa or GPIb/IX antibody binding was noted by flow cytometry. O-group-normal platelets were incubated for 30 min at room temperature with GT patient sera (1:5) and then washed thrice to remove unbound excess antibodies.  and 1b sAll the 15 GT patients not having received platelet transfusions showed the presence of antiplatelet antibodies . The absorption of antibodies against normal pooled platelets reduced the GPIIb/IIIa expression by 82.31 ± 4.33% in all the GT patients, showing that the major determinant against which the antibodies were directed in the GT sera was the GPIIb/IIIa complex, whereas in chronic ITP patients, this inhibition was variable. and 1b t6 platelets),(8) whereas by flow cytometry, the measure obtained is antibody/platelet. Comparison studies for the determination of platelet antibodies by ELISA-based assays and immunofluorescence showed that the fluorescence method had a high sensitivity in the detection of platelet antibodies than PAIgG by ELISA-based assays.[The receptor studies in the GT patients were carried out using the patient platelets and FITC-tagged receptor-specific antibodies (BD Biosciences and Dako Cytomation). Estimation of platelet antibodies was performed conventionally using enzyme-linked immunosorbent assay (ELISA)-based assays, which give the measure of antibodies as PAIgG (ng/10d assays. The specd assays. and 1b.GT patients who receive repeated platelet transfusions are known to show the presence of antiplatelet antibodies directed against the glycoprotein receptors.–3 Howeve9In conclusion, it may be said that severe GT patients may spontaneously develop antiplatelet antibodies directed to the GPIIb/IIIa complex and that their platelet counts remain normal, in contrast to what happens to many patients with chronic or acute ITP where antibodies may be directed to the GPIIb/IIIa complex. In case of ITP, this antibody is considered as an autoantibody whereas in GT patients, this is an alloantibody in nature. Whether antigenic mimicry to some bacterial or viral peptides, which are common in our environment, is the cause for this finding needs to be determined. It also makes it plausible to face a patient of severe GT who may show primary refractoriness to platelet transfusion, although we did not find any reference to that effect in the English literature.