The high-resolution δ13C profiles of samples from the inner shell layer on cross-sections fall within similar narrow ranges and display no regular variations or trends, such as an ontogenetic trend or abrupt short-term drops likely to be related to reproductive activity. This suggests that the calcification site of this species is not likely affected by photosynthetic CO2 uptake or CO2 incorporation during respiration. The δ18O profiles show distinct seasonal cycles. The intraspecific variability in the δ18O values is small in parts of the shell precipitated in the adult stage, but is not negligible in the juvenile and senescent stages. The differences in the monthly and seasonally resolved δ18O values among shells are less than 0.51‰ and 0.76‰, respectively. The shell δ18O values are nearly identical or close to the δ18O values for aragonite precipitated in oxygen isotope equilibrium with ambient seawater (δ18OEA). The largest differences between the shell δ18O and δ18OEA values calculated from the monthly and seasonally resolved data correspond to an overestimate of the seawater temperature by as much as 1.7°C and 2.3°C, respectively. However, these differences are smaller in the adult stage (<0.25‰) than in the other stages. This small difference allows an accurate reconstruction of the seawater temperature with an error of <1.1°C. Consequently, we recommend that multiple shell records be obtained because of the non-negligible intraspecific variations in their δ18O values. Growth banding, composed of alternating narrow white bands and wide light-grey bands, is discernible on cross-sections of the inner shell layer. The δ18Oshell data indicate that they were formed in winter and the other seasons, respectively.We report the carbon (δ Marine carbonate-secreting organisms, such as corals and mollusks, are sensitive to the ambient environment and preserve various types of environmental information in their skeleton in the form of physical  or geochemical variations –3. Of thsecreted –7. Numersecreted , corals secreted –14, mollsecreted –19, and secreted –24.18O values) records in the inner shell layer are more suitable for paleoenvironmental reconstruction than those of the outer shell layers or hinge, so the former have been used for paleoenvironmental studies [The tridacnids  are som studies , 35–38. 18O values as reliable paleoenvironmental proxies. One of the most critical issues is that previous studies of tridacnid δ18O values predominantly dealt with isotopic data from a single shell of a single taxon, so neither the inter- nor intraspecific variations were fully considered [13Cshell) and oxygen (δ18Oshell) isotope ratios of samples from the inner shell layer on cross-sections of modern Tridacna derasa  [18Oshell profiles of this species with each other and with oceanographic data around Ishigaki-jima  . The stug, 1798) . We thenima Figs . In thisThe Ryukyu Islands are located to the southwest of mainland Japan and are composed of several tens of islands and islets . These iIshigaki-jima is located in the southwestern part of the Ryukyus  [T. derasa reaches 50–60 cm. They live byssally anchored at a juvenile stage, but when approaching maturity their byssus glands atrophy, and the adult clams take up unattached existence on coral rubble or hard packed sand [T. derasa and T. gigas-T. mbalavuana [There are currently eight described species within the genus re, 1797 , 44. Theked sand . PhylogeT. derasa shells, designated KTd-1, KTd-4, and KTd-5  for the δ13C and δ18O analyses, based on replicate measurements of the laboratory reference sample , respectively. The correlations between the δ13Cshell and δ18Oshell values were evaluated with a reduced major axis regression technique [Stable isotope analyses of the shell aragonite were performed with a Thermo Scientific DeltaV Advantage mass spectrometer, coupled with a ThermoQuest Kiel III Carbonate Device, at the Graduate School of Science, Tohoku University, Sendai, Japan. The δ , were ±echnique , the sig18Oshell profiles clearly showing seasonal cycles were converted to time series for better interpretation and comparison with those of aragonite precipitated in oxygen isotope equilibrium with ambient seawater (equilibrium aragonite), because it is well known that the aragonitic shells of tridacnids are generally precipitated in oxygen isotope equilibrium with ambient seawater [18Oshell values, represented by the number of isotope data per year, varied from ~19 days to ~4 months per sample. Therefore, we converted the distance domain δ18Oshell profiles to time series with peak-to-peak matching  with the equilibrium aragonite δ18O profiles using AnalySeries software [18Oshell values were assigned to February and August, respectively. The monthly and seasonally resolved shell δ18O profiles were then calculated on the assumption of constant growth between each shell δ18O peak. The δ13Cshell time series were generated simultaneously.The distance domain δseawater , 32. Thesoftware . The tim13Cshell profiles of KTd-1, KTd-4, and KTd-5 showed no distinct cycles  = 0.17‰), from 0.89‰ to 1.81‰ , and from 1.19‰ to 2.21‰ , respectively , from –2.06‰ to –0.24‰ , and from –2.53‰ to –0.12‰ , respectively  using previously published δ13C values of dissolved inorganic carbon (δ13CDIC) and δ18Osw, respectively, of seawater samples collected around Okinawa-jima [13CDIC values at Kabira, where T. derasa grew, were assumed to be 1.1‰–1.6‰. Because the pH of the surface seawater at Ishigaki-jima ranged from 7.9 to 8.0, it was assumed that the δ13CHCO3_ values for this seawater were ~0.2‰ greater than the δ13CDIC values [13CEA values calculated using the δ13CDIC values (1.1‰–1.6‰) and the aragonite HCO3–-enrichment factor  should 18OEA time series were calculated using the IGOSS SST data [18Osw values at Ishigaki Port, ~14 km south of the T. derasa growth site, during the period from December 1997 to May 2004, measured by Abe et al. (2009) [The δSST data  and the . (2009)  and base. (2009) :103lnαa18Osw values [18OEA values were calculated, and range from –0.31‰ to –2.16‰ , which demonstrated distinct seasonal cycles -bearing giant clam, its metabolic activity is expected to be closely related to organic carbon production by zooxanthellan photosynthesis, which is considered to show seasonal cycles corresponding to those of solar radiation. However, the δ13Cshell values show no seasonal cycles  CO2 becomes available to them, whereas the amount required for shell formation decreases, resulting in the incorporation of more metabolic carbon (12C-enriched) into their shells. However, ontogenetic increases in δ13C values are known from non-zooxanthellate and zooxanthellate (tridacnid) bivalve shells, indicating that the model proposed by Lorrain et al. (2004) [The δidacnids , 38, 55.bivalves –60. Base. (2004) , 53.Mytilus edulis is reflected by more negative δ13Cshell values, although the δ13CDIC is generally becoming more positive, which is explained by higher metabolic rates just after spawning, as energy lost during spawning is restored [13Cshell values in M. edulis as being a result of increased respiration associated with periods of higher food availability. However, no such negative peaks have been found in the δ13C profiles of T. derasa or other tridacnid shells [The spawning period restored . Vander restored  also repd shells , 38, 55.13CDIC values relating to phytoplankton productivity and large-scale ocean dynamics are possible causes of ontogenetic trends of δ13C values from long-lived bivalve shells [13Cshell profiles of T. derasa are not long enough to discuss such relationships.It was shown that decadal variability of δe shells . However13Cshell values, characterized by the absence of seasonal cycles, ontogenetic decreases, and abrupt short-term drops in δ13Cshell that are attributable to reproductive activity, suggests that the calcification site of this species is not affected by CO2 uptake resulting from photosynthesis or the incorporation of CO2 from respiration. This is common in other tridacnid species [13Cshell and δ18Oshell values show weak or no significant correlations . At present, we have no explanation why the δ13Cshell values are so low relative to the δ13CEA values. Possible causative factors include the pH at the calcification site, as pH is known to affect the δ13C and δ18O values of skeletal carbonates [The δrbonates , 56, 65.18Oshell profiles are characterized by a series of regular seasonal cycles of varying amplitudes and frequencies. The amplitude of the cycles in the δ18Oshell profiles ranges from 1.03‰ to 1.90‰ for KTd-1, from 1.19‰ to 1.73‰ for KTd-4, and from 1.16‰ to 2.04‰ for KTd-5. Because the seasonal amplitude of the δ18Osw values is <0.2‰ , the contribution of δ18Osw to the δ18Oshell variations is less than <19%. Therefore, the cycles of the δ18Oshell profiles correspond predominantly to seasonal changes in seawater temperature. δ18Oshell profiles characterized by distinct seasonal cycles have previously been reported for the inner shell layers of tridacnids [The δidacnids , 54, 66.18O values, defined as the δ18Oshell values minus the δ18OEA values, are not negligible, ranging from –0.25‰ to 0.00‰ , from –0.12‰ to 0.15‰ , and from –0.38‰ to 0.00‰ , if we calculate them using the δ18Oshell and δ18OEA values for the coolest and warmest months in the monthly resolved data from the shell portion in which 12 or more samples for isotope analysis were collected   for all monthly resolved data  are used, the slopes and intercepts of the regression lines range from 0.99 to 1.05 and from –0.22 to –0.03, respectively, and the cross-correlation coefficients are 0.91–0.97 (p < 0.05) (18OEA estimation and sampling errors), these results suggest that the seawater temperatures reconstructed from the δ18Oshell values are largely the same as the actual temperatures.The aragonitic shells of tridacnids are known to be precipitated in oxygen isotope equilibrium with ambient seawater , 32. Howollected . The dif n = 29) . These d profile . The smaved data . If annu < 0.05) . Taking 18Oshell values was relatively small in the period between 1997 and 2006 but relatively large in the periods between 1995 and 1997  and between 2006 and 2009 (0.76‰ between KTd-1 and KTd-4/5)  . These dT. derasa (and other tridacnids) have potential advantages over the skeletal carbonates of other organisms, such as brachiopods and corals, because they are precipitated very close to oxygen isotope equilibrium with ambient seawater at least along the axis of maximum growth, which allows the δ18Oshell values from any part of the inner shell layer to be converted directly to seawater temperature if the contribution of δ18Osw is negligible. The secondary shell layer of brachiopod shells is believed to be precipitated in carbon and oxygen isotope equilibrium with ambient seawater. However, recent investigations have shown that the δ13C and δ18O values of modern brachiopod shells may be partly or wholly outside the range of those values for equilibrium calcite, which is attributed, at least in part, to kinetic and metabolic isotope fractionation effects [13C and δ18O values of coral skeletons deviate significantly from those of equilibrium aragonite because of the effects of kinetic and metabolic isotope fractionation [18Oshell values for the adult growth stages of T. derasa shells, with little intraspecific variability, are in good agreement with the δ18OEA values. This relationship can be used to generate high-resolution δ18Oshell time series of seawater temperatures and δ18Osw in coral reef environments. It should also be noted that the use of multiple shell samples provides a more reliable reconstruction of seawater temperatures, with an error of <1.1°C.The shells of  effects , 70–73 o effects . It is wionation . Our stu18Oshell allowed the construction of growth curves for the tridacnid shells studied. A comparison of the δ18Oshell and δ18OEA profiles indicated life spans of 10, 17, and 16 years for KTd-1, KTd-4, and KTd-5, respectively (18Oshell maxima (= seawater temperature minima), which was measured perpendicular to growth lines/bands, was 5.0–15.5 mm/year during the juvenile to adult stages, and decreased to 1.0–7.2 mm/year during the senescent stage. The growth curves representing shell thickening  correlated with the white growth bands. This indicates that the white bands were predominantly formed during winter, which is consistent with previous findings [Clear growth banding, composed of alternating wide light-grey bands and narrow white bands, is discernible on cross-sections of the inner shell layer Figs –5. Diffefindings , 36, 74.13Cshell and δ18Oshell values of samples from the inner shell layers, taken from cross-sections, of three modern T. derasa shells from Ishigaki-jima, Ryukyu Islands, southwestern Japan. The results can be summarized as follows.We investigated the intraspecific variations in the δ13Cshell profiles of the samples fall into a relatively narrow range and show no seasonal cycles, ontogenetic decreases, or abrupt short-term drops that might be related to reproductive activity. These observations suggest that the calcification site of T. derasa is unlikely to be affected by CO2 uptake or influx caused by photosynthesis or respiration, respectively. The δ13Cshell values and the δ18Oshell values show no or very weak positive correlations, indicating no or little kinetic isotope fractionation during the carbonate precipitation of the T. derasa shells.The δ18Oshell profiles are characterized by distinct cycles corresponding to seasonal changes in seawater temperature. The δ18Oshell values are usually greater than the δ18OEA values by up to 0.38‰ and 0.52‰ when calculated from monthly and seasonally resolved data, respectively. These differences lead to seawater temperature to be overestimated by 1.7°C and 2.3°C, respectively. However, these differences are small (<0.25‰) in the parts of the shell that formed in the adult stage, which allows the reconstruction of accurate seawater temperatures with an error of <1.1°C. Therefore, the high-temporal-resolution δ18Oshell data from the adult stage are most suitable for paleoenvironmental reconstructions using T. derasa. However, the maximum intraspecific differences in the monthly and seasonally resolved δ18Oshell values are as large as 0.51‰ and 0.76‰, corresponding to differences in water temperature of 2.2°C and 3.3°C, respectively. This result suggests that multiple δ18Oshell records should be used to reconstruct seawater temperatures, because although the intraspecific variations in the δ18O values are not negligible, especially during the juvenile and senescent stages. If multiple data are collected, the reconstructed seawater temperatures are expected to be very close to the actual temperatures.The δ18Oshell data indicate that the narrow white bands and the wide light-grey bands were formed in winter and the other seasons, respectively Click here for additional data file.


Chlamydomonas reinhardtii, on microparticles. Macro- and microscopic experiments reveal that microorganism-colloid interactions are dominated by rare close encounters leading to large displacements through direct entrainment. Simulations and theoretical modelling show that the ensuing particle dynamics can be understood in terms of a simple jump-diffusion process, combining standard diffusion with Poisson-distributed jumps. This heterogeneous dynamics is likely to depend on generic features of the near-field of swimming microorganisms with front-mounted flagella.The incessant activity of swimming microorganisms has a direct physical effect on surrounding microscopic objects, leading to enhanced diffusion far beyond the level of Brownian motion with possible influences on the spatial distribution of non-motile planktonic species and particulate drifters. Here we study in detail the effect of eukaryotic flagellates, represented by the green microalga  Passive particles surrounded by swimming protists diffuse tens of times faster than their thermal motion, which might have an impact on microscopic predator-prey interaction in nature. Here, Jeanneret et al. investigate its physical origin and identify direct particle entrainment as the dominant feature. From malarial parasites meandering around densely packed red blood cells and infecting them245Chlamydomonas reinhardtii (CR), often studied as model eukaryotic microswimmer, the pioneering study of Leptos et al.2122282129Microparticles' interactions with the other major class of microorganisms, eukaryotic flagellates, is distinctly less explored. Almost exclusively larger than bacteria (∼10–100 μm), these species probe a new and biologically relevant physical regime, where the microparticles' size is significantly smaller than that of the microorganisms , concentrated by gentle spinning and then resuspended to reach the desired concentration. Polystyrene microparticles (1 μm diameter) were then added to the suspension, and their diffusivity at different CR concentrations (Nc) was measured from three different sets of microfluidic experiments (ηpercoll=(1.5±0.1)ηwater) as reflected in the slower average swimming speed, −1, −1 and −1 for the sedimentation, spreading and tracking experiments respectively. A colloidal Péclet number for tracer/swimmer interactions can be defined as Pe=vL/D0, where v and L(∼10 μm) are CR's characteristic speed and body length, and D0 the Brownian diffusivity of the colloids. We obtain PeS≃2,000, PeCS≃1,500 and PeHS≃1,750, respectively for the three experiments.A full description of the experimental procedures can be found in the Methods and eriments : mappinglg,eff /(106cells ml−1) (slopes α will be expressed in these units throughout the paper). Within the same experiments, however, the diffusivity can also be inferred microscopically from direct short-duration tracking of microparticles' trajectories in bulk (mean tracks duration δttracks=2.6 s), as previously done in ref. Deff=D0+αTNc , is missing a key element. The crucial microscopic insight is provided by long-time tracking of the particles at a range of Nc values. This is achieved here by confining the system within a 26 μm-thick Hele–Shaw cell, which allows us to follow individual colloids for ∼200 s. 21223139αTNc  exponentially distributed with a characteristic length LJ=7.5±0.5 μm, above a threshold value LT=7.5±0.5 μm  but they are rare: as shown in NC≃5 × 106 cells ml−1, the average time between two consecutive jumps is rather long, Jumps are fast , inset, Deff, Deff is linear with cell concentration, with a slope αHS=1.67±0.13 in excellent quantitative agreement with both macroscopic values. This agreement confirms that the properties of the colloids' microscopic dynamics, which includes important but rare jumps, have indeed been probed appropriately.Computing the mean-square displacement from these long colloidal trajectories offers a microscopic estimate of the effective particle diffusivity X(t), Y(t)), asThe agreement between the macroscopic and microscopic diffusivity measurements highlights the importance of entrainment events, and suggests a combination of jumps and (far-field-enhanced) diffusion as a plausible minimal model of microparticles' dynamics, at least over the medium-to-long timescales we are interested in. Consequently, we model the two-dimensional projection of the stochastic trajectory of a colloid in the Hele–Shaw experiments,  leading to diffusion with diffusivity DWJ, with a Poisson process for the jumps, dP(t), characterized by the average time interval 〈ΔTJ(Nc)〉 between jumps , produces trajectories very similar to the experimental, reshold   inset, aDeff(Nc) on the concentration of algae. Here we extend to two dimensions the one-dimensional approach developed in ref. x, y) and time t are ρd and ρb, corresponding respectively to particles diffusing with diffusivity DWJ, and to particles moving ballistically with a constant velocity u in the direction φ. Particles switch from diffusion to directed motion and vice versa with constant transition rates λd(=1/〈ΔTJ〉) and λb. At low concentrations, where λd<<λb  which scales with the active flux vNc, as originally predicted in ref. Nc increases and 〈ΔTJ〉 becomes closer to Deff plateaus to a The coefficient ulations , inset aDeff=xdDWJ, where xd is the fraction of particles that are in the diffusing state. Estimating xd≃1 within the whole range of cell concentrations probed experimentally. Short timescale tracking of microparticles, then, will inevitably return Deff≃DWJ rather than the full expression in Finally, the short timescale limit of the particles' mean-square displacement returns Proposed theoretically either as a consequence of microparticle capture within ‘wake bubbles'vNc, already introduced in the bacterial context1617Here we show experimentally not only that entrainment of microparticles by microorganisms exists but also that these rare but large events can dominate particle dynamics, leading in the present case to a diffusivity more than 40 × larger than previously reported. Simulation and analytical results support a jump-diffusion process as a good minimal model for the medium-to-long timescales dynamics of the colloids. The simplified continuum model we discuss provides a theoretical support for the observed dependence of the experimental diffusivities on the active flux Although we cannot yet pinpoint the specific mechanism leading to entrainment, the structure of the near-field flow is likely to play a crucial role. The entrainment we observe requires almost head-on collisions, which is likely to be facilitated by the type of stagnation point found (on average) in front of the cell37521Chlamydomonas: entrainment is then likely to be a generic feature of this whole class of microorganisms. Several of these species are predators2Eukaryotic microswimmers with multiple front-mounted flagella will share much of the near-field flow structure found in −2 s−1, OSRAM Fluora). Cells were harvested at ∼5 × 106cells ml−1 in the exponentially growing phase, then centrifuged at 800 r.p.m. for 10 min and the supernatants replaced by DI-water (sedimentation experiment) or by a Percoll solution  already containing the desired concentration of PS colloids .Cultures of CR strain CC125 were grown axenically in a Tris-Acetate-Phosphate medium4 images were recorded at 25 f.p.s.  using a 20 × phase contrast objective (Nikon LWD ADL 20 × F). Particles trajectories were then digitized using a standard Matlab particle tracking algorithm (The code can be downloaded at http://people.umass.edu/kilfoil/downloads.html). Finally the spreading dynamics of the band of colloids was probed by recording the system for a few hours at × 10 magnification (Nikon ADL 10 × I) and low frame rate (0.5 f.p.s.) using a Nikon D5000 DSLR camera. The colloids were then featured using the same Matlab particle tracking algorithm in order to reconstruct the density profiles. In all experiments, the concentration of algae was determined in situ by imaging the system under dark-field illumination at low magnification (objective Nikon CFI Plan Achromat UW 2 ×).The Percoll solution (38.5% vol/vol) was made to density-match the beads for the Hele–Shaw and spreading experiments, while preserving the Newtonian nature of the flowsδt=0.004 s, which is 10 times smaller than the acquisition period in the Hele–Shaw experiment. At each time step a random walk with diffusivity DWJ is performed. To simulate the Poisson process, we draw at each time step a random number in the open interval ]0;1, then we perform a jump that lasts PDF(L), along the direction corresponding to θ, which is uniformly distributed in [−π, π[. This technique allows to simulate accurately the Poisson processThe parameters of the simulation correspond to the red lines in The authors declare that the data supporting the findings of this study are available on request.How to cite this article: Jeanneret, R. et al. Entrainment dominates the interaction of microalgae with micron-sized objects. Nat. Commun. 7:12518 doi: 10.1038/ncomms12518 (2016).Supplementary Figure 1-9, Supplementary Notes 1-5, Supplementary Methods and Supplementary ReferencesDynamics of a single microparticle in the Hele-Shaw experiment taken at 30x magnification, phase contrast, 25fps. The colour shows the instantaneous velocity of the bead (frame-to-frame displacement multiplied by frame rate). Colour code is the same used in Fig 2a of the main text.Example of an entrainment event during the sedimentation experiment. 60x magnification, bright field, 10fps.High speed movie of an entrainment event in the Hele-Shaw experiment recorded at 500fps, 100x magnification, bright field. Slowed down 17x.


People with an autistic spectrum disorder (ASD) display a variety of characteristic behavioral traits, including impaired social interaction, communication difficulties and repetitive behavior. This complex neurodevelopment disorder is known to be associated with a combination of genetic and environmental factors. Neurexins and neuroligins play a key role in synaptogenesis and neurexin-neuroligin adhesion is one of several processes that have been implicated in autism spectrum disorders.In this report we describe the manual annotation of a selection of gene products known to be associated with autism and/or the neurexin-neuroligin-SHANK complex and demonstrate how a focused annotation approach leads to the creation of more descriptive Gene Ontology (GO) terms, as well as an increase in both the number of gene product annotations and their granularity, thus improving the data available in the GO database.The manual annotations we describe will impact on the functional analysis of a variety of future autism-relevant datasets. Comprehensive gene annotation is an essential aspect of genomic and proteomic studies, as the quality of gene annotations incorporated into statistical analysis tools affects the effective interpretation of data obtained through genome wide association studies, next generation sequencing, proteomic and transcriptomic datasets.The online version of this article (doi:10.1186/s12859-015-0622-0) contains supplementary material, which is available to authorized users. Summarising the known role of gene products from published papers to populate the GO database, a process known as annotation, allows researchers to have access to information on the role of individual proteins and protein families in the form of controlled vocabulary terms [The Gene Ontology  is a complex neurodevelopmental condition known to be associated with a combination of genetic  and envide novo losses or gain mutations and linkage studies [There is strong evidence for multiple genetic factors determining the development of ASD. To date, around 40 genomic loci and over 100 genes have been associated with ASD based on rare variant approaches. These approaches have identified sequence and copy number variants as well as chromosomal rearrangements . These g studies , 18. AltSHANK gene families have only been associated with a few percent of ASD cases [Synaptogenesis, chromatin re-modeling, morphogenic developmental processes, calcium homeostasis and mitochondrial function have all been identified as potentially contributing to ASD . In thisSD cases .Neurexins (NRXNs) and neuroligins (NLGNs) are single-pass transmembrane proteins that play a key role in synaptogenesis, a process which occurs before birth and continues into adulthood . In the NLGN [NRXN [NLGN gene family are associated directly with synapse assembly [NLGN3, NLGN4X and NLGN4Y, have been identified in autistic patients [NRXN gene family in humans: NRXN1, NRXN2, NRXN3 [NRXN4 genes known as CNTNAP1 and CNTNAP2 (Contactin-associated protein 1 and 2 respectively) [NRXN1-3 encode alpha and beta isoforms which have identical C-terminal transmembrane regions and cytoplasmic tails. However, alternative promoter use leads to the isoforms having different N-terminal extracellular sequences, the α-isoforms having much longer extracellular domains than the β-isoforms [CNTNAP1 and CNTNAP2 are involved in non-synaptic neurogenic processes [. Mutations associated with ASD are found in both alpha and beta NRXN1 [NRXN2 [NRXN3 [CNTNAP2 [ASD-associated variants have been identified in members of both the NLGN  and NRXNGN [NRXN , 26 geneassembly –29, mutapatients , 30–32. 2, NRXN3  and two ctively) . NRXN1-3isoforms , whereasrocesses , 37. Mutta NRXN1 , NRXN2 [1 [NRXN2 , NRXN3 [2 [NRXN3  and CNTN[CNTNAP2 . Further[CNTNAP2 –44. Neur[CNTNAP2 .http://www.geneontology.org/cgi-bin/references.cgi#GO_REF:0000024) [Comprehensive manual annotation of the NRXN, NLGN, SHANK and DLG4 proteins identified several GO domains that are associated with the majority of these proteins, including behavior, synaptogenesis and neurogenesis. During the annotation process we identified a lack of GO terms which could describe the role of NRXNs and NLGNs in the process of synaptogenesis; to fill this void, new synapse assembly GO terms were created. These new GO terms were then associated with the NRXN, NLGN, SHANK and DLG4 proteins, when there was supporting experimental evidence. Furthermore, confirmed orthology predictions between the human, rat and mouse ASD-associated proteins supported the propagation of GO annotations from each protein to the orthologous proteins in these species (http://www.ncbi.nlm.nih.gov/pubmed) was used to locate papers that contained experimental data describing each member of the NRXN, NLGN, SHANK or DLG4 protein families. The searches were performed, during April to November 2011, using the following gene names and symbols: neurexin, neuroligin, NRXN*, NLGN*, SHANK1, SHANK2, SHANK3, CASPR, DLG4, DLG-4, PSD95, PSD-95. This search retrieved over 2000 papers. Consequently, we undertook a PubMed search with each individual gene symbol or name and additional filters, in order to provide a comprehensive coverage of the role of these proteins with respect to autism. The following filters were applied to each of the symbol and name searches: ‘AND autism/ASD’, ‘AND synaptogenesis/synapse assembly’, ‘AND autism/ASD AND synaptogenesis/synapse assembly’. The number of papers available that described each gene influenced the number of filters applied. The decision about which paper to annotate was then based on whether: 1) new information would be added to the current GO annotation data associated with the protein; 2) it was possible to identify the species the protein or transfected cDNA construct was derived from. Only papers that met both criteria were annotated. The choice of papers annotated was therefore influenced by the information captured in previously annotated papers. In total, 66 papers with experimental data that were relevant to ASD or synaptogenesis were originally selected for annotation. However, following the identification of an association of NLGN4Y, NRXN2 and NRXN3 alpha and beta variants with ASD [The PubMed database  ortholog prediction tool (HCOP). HCOP compiles data from 11 different orthology prediction tools, including EnsemblCompara, Homologene and Inparanoid [http://blast.ncbi.nlm.nih.gov/Blast.cgi), to confirm high homology. In all cases there was greater than 89 % amino acid sequence identity between these aligned mammalian orthologs.Orthologous proteins were identified for the NRXN, NLGN, SHANK and DLG4 protein families between the human, mouse and rat species, using the HUGO Gene Nomenclature Committee (paranoid . The prehttp://www.ebi.ac.uk/QuickGO) was used to identify the most specific GO terms to ‘capture’ the experimental data presented in each paper, and a consistent annotation approach was undertaken [www.geneontology.org/GO.evidence.shtml) [Manual GO annotation requires a GO curator to read publications and convert the data presented into an annotation. Each annotation includes the protein identifier, a GO term, an evidence code, and a reference . During dertaken . Evidence.shtml) . To compe.shtml) .molecular function of each protein, the biological processes these proteins contribute to, and their intracellular location  or ISS. Furthermore, many of these cellular component manual annotations describe the neuron-relevant localisation of these proteins, such as ‘excitatory synapse’, ‘postsynaptic membrane’, and ‘dendrite’  is applied. This inference is based on the immunofluorescence analysis of NRXN1-α transfected COS cells [cell surface’ annotation, along with the knowledge that this protein contains a transmembrane domain and is therefore likely to be located in the plasma membrane.NRXNs and NLGNs contain a single transmembrane region, and their involvement in cell adhesion suggests they are located in the plasma membrane. Experimental evidence supports the association of the GO terms ‘OS cells , which sexcitatory synapse’ [dendrite’ [juxtaparanode region of axon’ and ‘paranode region of axon’ [The NRXN, NLGN, SHANK and DLG4 proteins play a key role in synapse assembly and there is considerable experimental evidence to support GO annotations describing the localization of these proteins to neuronal-specific components, such as ‘synapse’ , 48–52 aendrite’ , 53, 54.of axon’ , 37  with the NLGN, NRXN, SHANK and DLG4 proteins [(R473C), corresponding to a human variant associated with an autism disorder, showed a decrease in excitatory synaptic transmission for both AMPA receptor and NMDA receptor response, suggesting positive regulation of this process [negative regulation of excitatory postsynaptic membrane potential’ [regulation of inhibitory postsynaptic membrane potential’ (or child terms) with mouse and rat Nlgn2 and Nlgn3 proteins [. Neurons transfected with rat Nlgn3(R471C), corresponding to another ASD associated variant, have a decreased inhibitory postsynaptic membrane potential [Experimental evidence supports the association of the GO term ‘proteins , 51 (Figtential’ . Transfeproteins , 69 (Figotential  are founotential , 51, 69.otential . Howeverotential , which iNRXN [NLGN [SHANK [CNTNAP2 gene are associated with ASD [social behavior’, ‘vocal learning’ and ‘adult behavior’ (Table Impaired communication and social behavior are the key behavioral changes seen in ASD individuals, and variants in the NRXN , 38, NLGXN [NLGN , 71, SHAN [SHANK –44 gene with ASD . Consequr’ Table  and 8.Taet al. [Nlgn2 mutation displayed no preference between either rooms [social behavior’. This simple behavior is similar to that observed in ASD individuals, where there is no eye contact or interest in another person in the room, and this ASD phenotype is also captured using the GO term ‘social behavior’ [Model organisms have been used to investigate the impact of ASD-associated gene mutations on animal behavior. Simple behaviors in model organisms can provide information about the more complex behaviors seen in humans. For example, Hines et al. , measureer rooms . These tehavior’ , 44, 72.Shank [Nlgn4l [Dlg4 [Shank3 heterozygous mice make fewer ultrasonic vocalizations to female mice, compared to their male wild type littermates [vocalization behavior’. Similarly, there is often an impairment of communication in ASD individuals with variants in the NRXN1-α [, NRXN1-β [CNTNAP2 [NLGN4X [SHANK1 [vocalization behavior’ or more specifically ‘vocal learning’. Human proteins are only associated with the GO term ‘vocal learning’ when the authors provide detailed information about a lack of speech or very limited word usage [Vocalization is also impaired in mice and rats carrying defects in Shank , Nlgn4l  [Nlgn4l  or Dlg4 4l [Dlg4  genes. Ftermates . This ex NRXN1-α , NRXN1-β NRXN1-β , CNTNAP2[CNTNAP2 , NLGN4X  [NLGN4X , 28 and  [SHANK1  genes (Trd usage , 43, 72 male courtship behavior’ and ‘olfactory behavior’. Consequently, very few behavioral annotations have been transferred from mouse or rat proteins to the human orthologs (Table regulation of grooming behavior’ with the mouse Shank3, Dlg4 and Nrxn1-α proteins [exploration behavior’ and its child term ‘locomotory exploration behavior’ are associated with the mouse Shank2 [DLG4 and NLGN2 are associated with behavioral traits in human (Table Behavior phenotypes described in mouse or rat cannot be mapped exactly to human behaviors. Furthermore, some of these behavioral traits are not relevant to human, such as ‘gs Table  and 8. Fproteins , 75, 77.e Shank2  and Shane Shank2 , 75, 78 molecular function GO terms associated with the members of the NRXN, NLGN, SHANK and DLG4 gene families capture information about the protein interactions they participate in (Table neuroligin family protein binding’ and ‘scaffold protein binding’ (Table neurexin family protein binding’ [neuroli gin family protein binding’ [scaffold protein binding’ [molecular function annotations were also used to identify the specific classes of receptors bound by the SHANK and DLG4 proteins, such as ‘ionotropic glutamate receptor binding’ [beta-1 adrenergic receptor binding’ [P2Y1 nucleotide receptor binding’ [receptor activity’ [GKAP/Homer scaffold activity’ [The majority of in Table . To provg’ Table . These nbinding’ , 39, 71,binding’  and ‘scabinding’ –81 (Tablbinding’ , ‘beta-1binding’  and ‘P2Ybinding’ . In addictivity’ , 68, whectivity’ , 52, 80 e.g. the peroxisome [e.g. those annotated by the Reference Genome Project [Members of the GO Consortium have undertaken a variety of focused manual annotation approaches to annotate the human proteome. Two large manual annotation efforts include renal and cardiovascular focuses , 84, whiroxisome , or spec Project . It is p Project , 84. FurSince their creation, the 19 new GO terms have been associated with 58 distinct human proteins creating 158 annotations (see Additional file Several groups have now used functional annotation data to identify candidate risk alleles associated with complex multigenic diseases , 89. ConN-methyl-D-aspartate receptor clustering’, ‘neurexin clustering involved in presynaptic membrane assembly’ and ‘presynaptic membrane assembly’, and enable a detailed description of the biological role of the NRXN, NLGN, DLG4 and SHANK proteins in synaptogenesis (see Additional files The annotation data and ontology terms within the GO database have been improved through this focused annotation project. Published experimental and patient data was used to capture the involvement of the NRXN, NLGN, SHANK and DLG4 proteins in synaptogenesis, neurogenesis and the behavioral traits seen in ASD. In order to create descriptive annotations the representation of synaptogenesis in GO was expanded, with the addition of 14 expressive terms within the synapse organization domain (Table molecular function terms annotations. Furthermore, there are limited terms available in the behavioral domain of GO, and the Neurobehavior Ontology [GO is a dynamic database that is always expanding as new annotations are added and new GO terms are created in the ontology. The advantage of a focused annotation approach is that it ensures the immediate use of newly created GO terms for annotations (see Additional file Ontology  may be bNRXN, NLGN and SHANK gene families, and in the DLG4 gene, have the potential to result in impaired synaptic formation and impaired regulation of synaptic transmission; however, not all of these proteins have been associated with ASD [Variants in the with ASD . The qua


MultiCS conditioning is an affective associative learning paradigm, in which affective categories consist of many similar and complex stimuli. Comparing visual processing before and after learning, recent MultiCS conditioning studies using time-sensitive magnetoencephalography (MEG) revealed enhanced activation of prefrontal cortex (PFC) regions towards emotionally paired versus neutral stimuli already during short-latency processing stages . The present study aimed at showing that this rapid differential activation develops as a function of the acquisition and not the extinction of the emotional meaning associated with affectively paired stimuli. MEG data of a MultiCS conditioning study were analyzed with respect to rapid changes in PFC activation towards aversively (electric shock) paired and unpaired faces that occurred during the learning of stimulus-reinforcer contingencies. Analyses revealed an increased PFC activation towards paired stimuli during 50 to 80 ms already during the acquisition of contingencies, which emerged after a single pairing with the electric shock. Corresponding changes in stimulus valence could be observed in ratings of hedonic valence, although participants did not seem to be aware of contingencies. These results suggest rapid formation and access of emotional stimulus meaning in the PFC as well as a great capacity for adaptive and highly resolving learning in the brain under challenging circumstances. Emotions influence the value ascribed to experiences, they aid in selecting the appropriate action, and they guide behavior towards approaching or avoiding specific situations MultiCS conditioning revealed the involvement of PFC regions in the rapid processing of emotional stimuli Furthermore, recent studies using However, it remains unclear whether the increase in rapid prefrontal activation recorded after learning truly arises due to the access of the emotional value of the CS+. So far, MultiCS conditioning studies have investigated the learning-induced changes in stimulus processing only by comparing CS+/CS− evoked neural activity after versus before learning. Stimulus processing during learning has not yet been examined. Since measurements after learning were based on unreinforced presentations of the previously paired stimuli, enhanced PFC activation could also point towards extinction processes, instead of indicating access of CS+/US representations formed during learning. Indeed, a great number of classical conditioning studies in humans and rodents e.g.,  point toTherefore, we analyzed the acquisition phase and reanalyzed the pre- and post-learning phases of one MultiCS conditioning study, in which multiple neutral faces were paired with an aversive electric stimulation to the right or the left hand, while the same number of neutral faces remained unpaired. In this study, whole-head magnetoencephalography (MEG) revealed enhanced activation towards paired vs. unpaired faces in the PFC during 50 to 80 ms after learning  have briefly been included in a review about MultiCS conditioning SD = 2.9), and normal or corrected-to-normal vision. Before participation, all individuals received written and oral information about the procedure and gave informed written consent to the protocol approved by the ethics committee of the Medical Faculty, University of Muenster (2007-563-f-S). Participants received 30 € for participation.Forty-eight healthy right-handed individuals  took part in the study. All participants were native German speakers, had a mean age of 24.5 years  with neutral expression from frontal view were used as conditioned stimuli (CSs). Faces were taken from the Karolinska Directed Emotional Faces archive highly unpleasant, but not painful. The US was delivered using a Grass Instruments S-88 dual-channel square-pulse stimulator with an Isolation Unit SIU7 .An electric shock consisting of a 300 ms train of 0.5 ms pulses at a rate of 64 Hz served as unconditioned stimulus (US). For half of the CS+ within each block, the US was delivered to the tip of the right index finger, for the other half, to the left index finger. Shock intensity was set for every participant individually to be The experiment consisted of three subjective  and behavioral tests administered before and after MultiCS conditioning as well as of a measurement of neuronal activation conducted during MultiCS conditioning . The subunpleasant) to +300 (pleasant) for hedonic valence and from −300  to +300 (arousing) for emotional arousal. Before each rating, participants viewed each face for 900 ms, preceded by a fixation cross, shown for 600 ms. For practice, they completed three test trials.Prior to conditioning, participants completed subjective ratings of hedonic valence and emotional arousal for all CSs, using a computerized version of the Self-Assessment-Manikin (SAM) scale not perceptible), 2 (slightly perceptible), 3 , 4 (slightly unpleasant), 5 , and 6 (painful). Stimulus intensity was steadily increased until participants described it as being highly unpleasant, but not painful. After the first and the second acquisition run, participants retrospectively evaluated the shock intensity on the visual rating scale to avoid habituation effects to the US. If necessary, stimulus intensity was again increased up to a highly unpleasant, but not painful self-assessment before the next learning run. The learning phase was carried out in three consecutive runs. During each run, participants were shown all CSs once, with a presentation time of 900 ms and an ISI of 1500±500 ms. One half of the CSs was now paired with an electric shock (CS+), while the other half remained unpaired (CS−). For half of these 52 CS+, the electric shock was applied to the right index finger (CS+right), for the other half to the left index finger (CS+left). The electric stimulation appeared at 650 or 700 ms after picture onset. We used 100% contingent pairings, so that each CS+ was paired three times with the US . All CSs were presented in a randomized order, with the only restriction that not more than three stimuli of the same condition followed each other. After the learning phase, stimulation electrodes were removed and participants completed the post-learning phase which, except for the differing stimulus randomization, was identical to the pre-learning phase. Before the pre- and the post-learning phases, participants were informed that they would see several different faces, but not receive any electric stimulation. To substantiate the subject’s confidence in this information, stimulation electrodes were attached after the pre- and detached before the post-learning phases. Before the learning phase, participants were informed that they would see several different faces and receive electric stimulation. They were, however, not informed about the presence of contingencies between the presentation of a face and the occurrence of an US.In preparation of the MEG measurement, information about the individual head shape was acquired using a Polhemus 3Space Fasttrack. Subsequently, participants were comfortably seated in the MEG scanner and completed the pre-learning phase of MultiCS conditioning . During Following the MEG measurement, participants repeated the valence and arousal SAM-ratings of all CSs and completed an affective priming task as well as a CS-US matching task (more detail provided below). The order of the SAM-rating and the affective priming task was balanced across participants to counteract systematic influences of extinction. The CS-US matching task was always performed last.congruent  or incongruent  with respect to the acquired hedonic valence. Participants were instructed to categorize the targets quickly and accurately as positive or negative, irrespective of the preceding face. For practice, they completed 15 test trials.The affective priming task consisted of 104 trials. On each trial, a fixation cross was shown for 200 ms and, then, a CS+ or CS− was presented for 200 ms. CS offset was immediately followed by a target word, to which participants made a binary forced choice valence decision . Half of the 52 target words were positive and half negative adjectives, matched in length, frequency, and arousal  of the 104 CS faces were pseudo-randomly selected for the task. Participants first viewed the CSs for 600 ms, preceded by a fixation cross shown for 2000 ms. Then, they indicated for each face whether it had been or had not been paired with an electric shock during conditioning (rating of stimulus category) and whether the electric shock had been applied to the right or the left hand (rating of shock location). For CS− categorized faces, participants were asked to guess a potential shock location under the prediction that they might have falsely categorized the face as unpaired. For practice, they completed three test trials.The CS-US matching task assessed whether participants were aware of the stimulus category (CS+ vs. CS−) and the shock location (left vs. right hand). Forty .To assess whether conditioning changed evaluative judgments to aversively paired and unpaired faces, we compared the SAM-ratings acquired after to the ones acquired before MultiCS conditioning. Repeated-measures ANOVAs with the factors SESSION  and CS-TYPE  were calculated across the hedonic valence and emotional arousal ratings.Trials with incorrect responses or reaction times (RTs) above or below two standard deviations of the individual mean were excluded from analysis, resulting in a rejection of 2.6% and 4.5% of the trials, respectively. In the present analysis, a different, more stringent criterion for exclusion of outliers was used as in the review article about MultiCS conditioning right/CS+left) - although instructed otherwise - were excluded from analyses. The level of awareness concerning stimulus category (CS+ vs. CS−) and shock location (CS+right vs. CS+left) was then assessed for each individual by calculating the sensitivity measure d' .Pre-learning vs. post-learning. CS+pre: 146.4±6.3; CS−pre: 147±7; CS+post: 145.2±6.1; CS−post: 145±6.1; F = 1.1, p = .349. Learning. CS+run1∶48.2±3.1; CS−run1∶47.9±3.5; CS+run2∶48.6±3.1; CS−run2∶48.7±3.3; CS+run3∶48.5±3.1; CS−run3∶49.3±2.5; F = 1.22, p = .301]. Epochs were averaged in correspondence to the conditions. On the basis of the averaged responses, cortical sources of the event-related magnetic fields were calculated using the L2-Minimum-Norm-Estimates (L2-MNE) method k of 0.2 was applied across all participants and conditions.Visual evoked magnetic fields (VEMFs) were acquired using a 275 MEG whole-head sensor system  with first-order axial SQUID gradiometers. The individual head position in the MEG scanner was tracked by three landmark coils placed on the two ear canals and the nasion. The individual head coordinate system was determined by a Polhemus 3Space Fasttrack which measured individual head shape information. VEMFs were recorded continuously in a frequency range between 0 and 300 Hz using a sampling rate of 1200 Hz. Offline, the recordings were sampled down to 600 Hz and filtered as to only include responses in a frequency range between 0.2 and 148 Hz. Filtering was conducted using zero-phase (forward and backward) Butterworth high-pass (second order) and low-pass (fourth order) filters. Epochs of 1000 ms duration  were extracted, aligned, and baseline-corrected using a 150 ms pre-stimulus interval as baseline which ranged from 150 ms before picture presentation to 0 ms . Single trials were edited and artifacts were corrected following the method for statistical control of artifacts in high-density EEG/MEG data proposed by Taking into account the individual sensor positions, topographies displaying the direction-independent current dipole activation were calculated for each participant, experimental condition, and time-point. The subsequent analysis consisted of two steps:F-values for the main effects of SESSION and CS-TYPE and the interaction of SESSION × CS-TYPE for each dipole. Monte Carlo simulations of identical analyses based on 1000 random permutations of the complete data set of subjects and experimental conditions were conducted . We calculated first-level cluster statistics of a minimum of five neighboring dipoles achieving an alpha-level p = .05. Direction of effects within source regions surpassing a cluster-mass alpha-level of p = .05 were further explored using a continuative parametric SESSION × CS-TYPE ANOVA and post hoc paired samples t-tests.We investigated whether the SESSION × CS-TYPE interaction as reported in F-statistics, continuative parametric statistics, and post hoc paired samples t-tests were calculated corresponding to the above described procedure.We investigated whether the differential CS processing expressed by a SESSION × CS-TYPE interaction was a function of extinction  or a function of associative learning . Therefore, a repeated-measures ANOVA including the factors RUN  and CS-TYPE  was calculated across the neural activation during the learning phase and again for all modeled neural generators at the a priori defined time interval of interest. Non-parametric cluster-level Preprocessing and analysis of MEG data was carried out using the Matlab-based EMEGS software Greenhouse-Geisser corrected significance values are reported for all repeated-measures analyses, in which the assumption of sphericity was violated.F = 5.13, p = .028. Post-hoc t-tests showed that CS- stimuli were rated as more pleasant (by trend) after as compared to before conditioning , t(47) = 1.98, p = .054, and that shock associated CS+ faces were rated as less pleasant (by trend) than CS− stimuli in the post-conditioning phase , t(47) = −1.83, p = .073. For comparison, an ANOVA on z-transformed valence ratings yielded also a significant SESSION × CS-TYPE interaction, F = 6.72, p = .013. Ratings of emotional arousal did not differ across SESSION or CS-TYPE, and there was no significant interaction .The repeated-measures ANOVA on the ratings of hedonic valence yielded a significant SESSION × CS-TYPE interaction , F = 2.12, p = .152, nor an interaction of CONGRUENCY with any other factor  = 76.83, p<.001, and REPETITION, F = 112.47, p<.001, as RTs were faster in trials in which target words were positive or presented for the second time.The repeated-measures ANOVA did yield neither the expected main effect of CONGRUENCY, d' for the stimulus category nor d' for the shock location differed significantly from zero , st(47) = 1.10 and 0.08, sp = .275 and.937, indicating a lack of detectable contingency awareness for both stimulus category and shock location  = 4.66, p = .036. Indeed, CS+ activation increased from the pre-  to the post-learning phase , t(47) = 2.05, p = .046, and was enhanced (by trend) in comparison to CS− activation  after learning (t(47) = 1.93, p = .059. CS+ and CS− activation  did not differ before learning, t(47) = −1.19, p = .242.Analysis revealed a significant SESSION × CS-TYPE interaction in the right inferior frontal PFC during 50 to 80 ms , F =F = 6.54, p = .014, but the SESSION × CS-TYPE × HEMISPHERE interaction was not significant, F = 0.43, p = .516. Thus, although first-level cluster statistics revealed a two-way interaction only in the right inferior frontal PFC, continuative analyses suggest that effects were not lateralized to this region.To explore whether effects were lateralized to the right inferior frontal PFC, we selected a left-hemispheric dipole group, homologous to the original right-hemispheric group, and calculated an ANOVA with the factors SESSION, CS-TYPE, and HEMISPHERE  across activation in both clusters during 50 to 80 ms  = 5.69, p = .006. The interaction was mainly driven by a linear effect of the factor RUN on CS+ activation, F = 5.42, p = .024, and (by trend) by a quadratic effect of RUN on CS− activation  = 3.88, p = .055. Indeed, CS+ activation increased from the first  to the third run , t(47) = 2.33, p = .024, and CS− activation decreased from the first  to the second run , t(47) = −2.18, p = .034. Importantly, CS+ and CS− activation did not differ during the first learning run , t(47) = −1.54, p = .130, but CS+/CS− differentiation was already enhanced after a single CS+/US pairing , t(47) = 3.34, p = .002, and remained enhanced after two CS+/US pairings , t(47) = 2.35, p = .023.During the learning phase, we observed a RUN × CS-TYPE interaction again in the right inferior frontal PFC during 50 to 80 ms after CS onset , F = 4.14, p = .022, but no RUN × CS-TYPE × HEMISPHERE interaction, F = 1.06, p = .348, emerged. Thus, effects cannot be considered as being lateralized to the right PFC.Consistent with the pre−/post-learning comparison, we investigated whether learning effects were lateralized to the right hemisphere, by selecting a left-hemispheric dipole group, homologous to the right prefrontal group, and computing an ANOVA with the factors RUN, CS-TYPE, and HEMISPHERE across activation in both clusters during 50 to 80 ms. A significant RUN × CS-TYPE, Analysis returned significant RUN × CS-TYPE interactions during the 50 to 80 ms time interval also in the right occipital and left middle temporal lobe. Since this study focused on early prefrontal activation during conditioning, these additional effects were not further explored.Previous MultiCS conditioning studies reported enhanced PFC activation towards aversively paired vs. unpaired faces during 50 to 80 ms after as compared to before learning Unpaired faces were rated as more pleasant after compared to before learning, whereas pleasantness ratings of aversively paired faces did not change across sessions. This finding is in line with previous MultiCS conditioning studies in vision The affective priming paradigm, with CS+ and CS− faces as primes to positive and negative words, did not show faster RTs in congruent compared to incongruent trials. This finding does not correspond to results of a previous MultiCS conditioning study in which an affective priming effect to emotional words was observed after the presentation of aversively paired (electric shock) and unpaired click-tones Taken together, the findings suggest that MultiCS conditioning modifies behavior even under challenging conditions, which points to a great ability in humans to quickly acquire, preserve, and act on contingencies present in the environment. However, there are limits as to what extent such rather transient learning can influence action tendencies in an affective priming setting. As the third task failed to indicate awareness of CS+/US contingencies, the fast and highly resolving learning most likely occurred in the absence of contingency awareness.The PFC was differentially activated by aversively paired and unpaired stimuli already during the learning phase of MultiCS conditioning when a CS+/US association was being established. Importantly, this differential PFC activation during fear learning resembles in location and time of occurrence the differential PFC activation after fear learning as observed in the present and in previous MultiCS conditioning studies in vision The finding that aversive conditioning influences PFC activation during very early processing stages matches the results reported in visuo-olfactory MultiCS conditioning With respect to rapid PFC activation, we would like to note that differential processing observed in MultiCS conditioning matches findings of several studies investigating different types of learning other than classical conditioning. Using stimuli with targets at trained or untrained locations, it was recently shown that mere perceptual experience can modulate the C1 component as early as 44 ms after picture onset Analysis of the learning phase indicated that one learning trial was sufficient to evoke differential PFC activation. Single-trial learning in classical conditioning is a quite controversial phenomenon. The most compelling evidence arguing for the existence of single-trial learning comes from studies that demonstrate the acquisition of taste aversion in rodents and humans after only one bad experience with certain foods Apart from classical conditioning, single-trial acquisition is also known for learning of novel stimuli. For instance, Rutishauser and coworkers Importantly, increased firing rates in amgydala neurons or enhanced activity within the inferior lateral PFC – which is closely connected to the amygdala e.g.,  - seem tAt last, we would like to point out that the present study has limited informative value with regard to revealing the exact spatial location and regional extent of the differential PFC activation. This limitation arises as consequence upon the choice of the inverse method used here. Assuming that our target region in the inferior frontal cortex is just one of multiple nodes in a distributed and simultaneously active network, we chose a distributed source reconstruction with Minimum Norm Estimate. This method approximates the spatial location of cortical generators and allocates these generators to larger cortical structures fairly well, but identification of distinct Brodman areas would - with respect to the approximated norm - in this special case pretend false accuracy. Furthermore, the Minimum Norm method puts specific emphasis on distributed cortical sources which is why inferences about the regional extent of the underlying neuronal activity cannot be made.MultiCS conditioning is a challenging and highly resolving learning paradigm. It induces changes in subjective ratings towards the conditioned stimuli that are typically observed during classical conditioning. It is accompanied by rapid neuronal plasticity in the PFC after only a few learning trials, visible in enhanced neuronal activation towards paired stimuli at short-latency processing stages during and after learning. Due to the multitude of stimuli in one condition (paired vs. unpaired), it offers a high signal-to-noise ratio and is thus well suited for EEG/MEG research. Moreover, it seems to be an ideal method for visualizing fast implicit learning processes in the brain. Since it encompasses a relatively weak and ambiguous learning situation in which CS+/US associations are not clear, it could aid in uncovering individual differences between patient and control populations as well as subclinical risk- and non-risk groups Dataset S1Dataset underlying analysis of subjective ratings of valence and arousal. The spreadsheet contains original (“Response_mean”) and z-transformed (“zscores_mean”) ratings of valence  and arousal (“aro”) for all 48 participants aggregated by session (“pre”/“post”) and CS-type (“plus”/“minus”).(XLSX)Click here for additional data file.Dataset S2Dataset underlying analysis of the affective priming task. The spreadsheet contains original (“RT_mean”) and inverted (“ONEdivbyRT_mean”) reaction times for all 48 participants aggregated by congruency (“congr”/“incongr”), target valence (“pos”/“neg”), and repetition (“first”/“sec”).(XLSX)Click here for additional data file.Dataset S3Dataset underlying analysis of the CS-US matching task (stimulus category). The spreadsheet contains the total number of hits (“hit”), misses (“miss”), false alarms , correct rejections (“correctrejection”), signal trials , and noise trials  for all 48 participants. It additionally displays the hit (“hitrate”) and false alarm rates , the Z values for the hit (“z_hitrate”) and false alarm rates , and the final d' value (“dprime”).(XLSX)Click here for additional data file.Dataset S4Dataset underlying analysis of the CS-US matching task (shock location). Labeling of variables as in S3, except that d' is labeled as “dprime_shockLocation”.(XLSX)Click here for additional data file.Dataset S5Dataset underlying analysis of neuronal activation during the post- versus pre-learning phase. The spreadsheet contains the neuronal activation of all 48 subjects in the right (“Right”) and homologous left-hemispheric (“Left”) dipole groups for the different sessions (“pre”/“post”) and CS-types (“plus”/“minus”) averaged across the 50 to 80 ms time interval.(XLSX)Click here for additional data file.Dataset S6Dataset underlying analysis of neuronal activation during the learning phase. The spreadsheet contains the neuronal activation of all 48 subjects in the right (“Right”) and homologous left-hemispheric (“Left”) dipole groups for the different runs (“R1”/“R2”/“R3”) and CS-types (“plus”/“minus”).(XLSX)Click here for additional data file.


Pseudomonas aeruginosa. We find that the coexistence of cooperators and defectors that produce and exploit, respectively, the iron-scavenging siderophore pyoverdine, is stabilized by the presence of loners with an independent iron-uptake mechanism. Our results establish the loner effect as a simple and general driver of cooperation and diversity in environments that would otherwise favour defection and the erosion of diversity.Cooperation and diversity abound in nature despite cooperators risking exploitation from defectors and superior competitors displacing weaker ones. Understanding the persistence of cooperation and diversity is therefore a major problem for evolutionary ecology, especially in the context of well-mixed populations, where the potential for exploitation and displacement is greatest. Here, we demonstrate that a ‘loner effect’, described by economic game theorists, can maintain cooperation and diversity in real-world biological settings. We use mathematical models of public-good-producing bacteria to show that the presence of a loner strain, which produces an independent but relatively inefficient good, can lead to rock–paper–scissor dynamics, whereby cooperators outcompete loners, defectors outcompete cooperators and loners outcompete defectors. These model predictions are supported by our observations of evolutionary dynamics in well-mixed experimental communities of the bacterium  The ‘Beyond evolutionary ecology, economic game theorists have proposed a simple mechanism—the ‘loner effect’—that can maintain cooperation and diversity, even in the absence of spatial genetic structure . The idePseudomonas aeruginosa. This bacterium produces pyoverdine, a diffusible siderophore that is released into the extracellular environment where it can solubilize iron for the producer cell and for other cells in the neighbourhood /[1 + xC + xD]} + (1 − α){[pC]/[pC + pD]}) − c, where xC and xD are the multinomially distributed numbers of (other) cooperators and defectors in its neighbourhood, respectively, and c is the cost of siderophore production . Accordingly, a defector's pay-off is PD = (1 − γ)(α{[xC]/[1 + xC + xD]} + (1 − α){[pC]/[pC + pD]}) and a loner's pay-off is PL = b − c, where c < b < 1 describes the relative efficiency of the loner's siderophore. We also extend the basic model to consider the evolution of a ‘loner-defector’ (LD) strategy, which exploits the loner's siderophore and occurs with frequency pLD. This yields pay-offs PC = γ + (1 − γ)(α{[1 + xC]/[1 + xC + xD]} + (1−α) {[pC]/[pC + pD]}) − c, PD = (1−γ)(α{[xC]/[1 + xC + xD]}+(1−α){[pC]/[pC + pD]}), PL = γb + (1−γ)(α{[1 + xL]/[1 + xL + xLD]} + (1 − α){[pL]/[pL + pLD]})b − c and PLD = (1 − γ)(α{[xL]/[1 + xL + xLD]} + (1 − α){[pL]/[pL + pLD]})b.We consider that a siderophore-producing cell retains a fraction & Rainey  and Kümm& Rainey ) and relmS of a strain adopting strategy S ∈ {C, D, L, LD} and use this to calculate each strain's change in frequency over time, dpS/dt. Specifically, the growth rate of strain S is given by ψ = ([n − 1]!/[xC!xD!xL!xLD!])(pCxCpDxDpLxLpLDxLD) is the multinomial probability distribution function. In addition to these growth pay-offs, we assume that strain frequencies may change by non-selective processes , occurring at rate μ, hereafter described as the ‘mutation rate’. For simplicity, and without affecting the results, we consider that mutations change a particular strain type into the type that can directly outcompete it  /[q1(1 − q2)], where q1 and q2 are, respectively, the initial and final proportions of the bacterial strain of interest [We carried out competition experiments by inoculating 7.5 µl of both competing strains at an OD  of 1 into 1.485 ml of iron-limited media consisting of casamino acids (CAA) medium . This shows that strain dynamics should not be influenced by the fluorescent markers.To first check whether the fluorescent markers affected strain fitness, we competed our marked strains against their unmarked ancestral variants in iron-limited CAA medium. We could not detect any significant difference in relative growth between marked and unmarked strains (relative fitness of marked strain did not differ from P. aeruginosa producing a different pyoverdine type can act as loners (i.e. can outcompete defectors but get outcompeted by cooperators). To do this, we first performed pairwise competition experiments between the six putative loner strains against PAO1 and PAO1ΔpvdD pchEF (four replicates per competition). These assays identified strain ATCC 013 as a potential loner . In order to confirm that these competitive differences were in fact due to pyoverdine, and not just based on other competitive factors affecting growth rate, we repeated the competition experiments in CAA medium supplemented with 20 µM FeSO4. In this iron-replete environment, the production of pyoverdine is not required, and therefore we expect the loner effect to disappear. In particular, the defector and cooperator strain become phenotypically identical when iron is replete (as none of the strains produces pyoverdine), and consequently the loner should perform similarly against both strains . Furthermore, we used the strain triplet PAO1, PAO1ΔpvdD pchEF and ATCC 013, to test four key assumptions of our model: (i) access to pyoverdine is required for growth; (ii) pyoverdine production is costly (c > 0); (iii) the producer cell retains a proportion of its pyoverdine for its own use (γ > 0) and (iv) the pyoverdine produced by the loner is less efficient than the pyoverdine produced by the cooperator (b < 1). The exact methods for these tests are described in the electronic supplementary material and [Next, we tested whether strains of rial and ,34.(d)n = 12) over 10 days . Separately, we also performed experimental evolution with all two-strain communities  over 5 days  in sixfold replication for each strain combination. Experimental evolution was carried out across relatively short timescales because Pseudomonas strains are known to rapidly adapt to laboratory conditions, and such adaptations could distort the evolutionary dynamics of interest [We used experimental evolution to test our model predictions about strain dynamics and community stability. A primary aim of these experiments was to compare the dynamics and stability of two-strain communities, initially composed of cooperators and defectors only, with that of three-strain communities, initially composed of cooperators, defectors and loners. For this comparison, we performed experimental evolution with replicate communities . To prevent contamination between wells, we filled every second well only (hence 12 of the 24 wells were inoculated). Plates were subsequently placed in a 37°C incubator and shaken at 220 r.p.m for 24 h. Following growth, 150 µl of bacterial culture was transferred to fresh media every 24 h for the duration of the experiment. After transferring to new media, the bacterial competitions were serially diluted and plated on iron-supplemented LB agar. These were incubated for 24 h at 37°C and left at room temperature (20°C) for another 48 h. CFUs were counted using a transilluminator.(e)To assess the emergence of de novo defectors in pyoverdine-producing strains (i.e. PAO1 and ATCC 013) in our evolution experiments, we assayed pyoverdine production in these strains over time. In the 10-day experiments comparing two-strain (cooperator–defector) and three-strain (cooperator–defector–loner) communities, we screened pyoverdine producers at days 5 and 10 of the experiment (120 clones for each community). In the 5-day experiments with two-strain communities , we screened 48 clones from each community for pyoverdine production daily of either PAO1 (in the cooperator versus defector and cooperator versus loner competitions) or ATCC 013 (in the loner versus defector competition). Each bacterial competition was plated on iron-supplemented LB agar, and clones of either PAO1 or ATCC013 were isolated from each replicate. Pyoverdine production was assayed by culturing these clones in a 96-well plate containing 200 µl iron-limited CAA for 24 h at 37°C. Pyoverdine production for each clone was quantified using the RFU/OD measure described above. We defined strains as de novo defectors if their RFU/OD was 75% or less than that of the ancestral wild-type .3.(a)v = 1.60 ± 0.18, one-sample t-test, t11 = 6.57, p < 0.0001), but outcompeted the defector . Crucially, the ATCC 013 loner and the PAO1 cooperator each produce a different, mutually exclusive pyoverdine type [v = 0.58 ± 0.24, one-sample t-test, t7 = 43.4, p = 0.012) and the defector . Moreover, as expected given that the competition between cooperators and defectors was dependent on iron availability, the defector outcompeted the cooperator under iron-limited conditions , but not under iron-replete conditions , where pyoverdine production is switched off. Altogether, these results suggest that ATCC 013 could act as a true loner in a community of PAO1 (cooperator) and PAO1ΔpvdD pchEF (defector) in the iron-limited conditions where pyoverdine is required for growth.We found that one of the six candidate strains, ATCC 013, behaved as a loner—that is, it was outcompeted by the cooperator pvdD pchEF, ATCC 013) to test key assumptions of our model, and found all of them to hold: (i) siderophores are important for growth, as indicated by pyoverdine producers growing significantly better than pyoverdine-deficient defectors ; (ii) the cost c of siderophore production is substantial, as confirmed by our estimate of c = 0.197; (iii) the fraction γ of siderophores that producers retain for themselves is low, as supported by our iron-chelation assay revealing γ = 0.05 and (iv) the cooperator's siderophore is more efficient in delivering iron to the cell than is the loner's siderophore (b < 1), as demonstrated by our iron-chelation assays yielding b = 0.74 for the loner's pyoverdine.We used our strain triplet b < 1), loners will displace defectors (given b > c), and—if siderophores are sufficiently costly (large c)—defectors will replace cooperators. As an illustrative example of the latter result, we derive analytical results for a simple case with n = 2, γ = 0 and α = 1 . In this case, we find that cooperation is an evolutionarily-stable strategy  and defection is an ESS when siderophores are costly (c > 1/2) .In pairwise competition, our model predicts that cooperators will displace loners  when siPseudomonas communities: cooperators displaced loners , loners displaced defectors , and defectors outcompeted cooperators . Even though defectors steadily increased in frequency during the initial phase of the experiment when competed against cooperators, we found that by day 5 their increase had slowed and that this coincided with the emergence of de novo defector mutants  in all replicates . The appearance of de novo defectors in the cooperator background is a well-described phenomenon [In support of these predictions, we found that in experimental evolution with two-strain enomenon ,23, and enomenon . In supp(d)n = 2, γ = 0 and α = 1), we find that if individuals may adopt a loner strategy then, while cooperation remains an ESS when siderophores are cheap (c < 1/2), there is no pure-strategy ESS when siderophores are costly (c > 1/2) and a mixture of all three strategies is maintained. The mixture admits a locally stable equilibrium point at which the pay-offs for the three strategies are equal, but the strain frequencies always cycle around this equilibrium . Thus, the presence of the loner promotes both cooperation and diversity through rock–paper–scissors dynamics. This is because the presence of loners in a cooperator's neighbourhood reduces the number of cells that can use the public good, thus increasing the cooperator's share. When cooperation is rare, loners outcompete defectors and rise to high frequency, which alters the social environment in a way that promotes cooperation. A necessary condition for the promotion of cooperation and maintenance of polymorphism is that loners do not ‘soak up’ the cooperator's public good . Cooperation remains an ESS when siderophores are sufficiently cheap, although larger neighbourhood size tends to inhibit pure cooperation . This is because, in a larger neighbourhood, a focal cooperator would receive a smaller share of the benefit from its own siderophores. By contrast, when siderophores are sufficiently costly (e.g. c > 1/n when γ = 0 and α = 1), all three strategies are maintained in rock–paper–scissors dynamics so long as γ is sufficiently low and α is sufficiently high (figure 2a–c), such that most siderophores are shared as public goods within the producer's neighbourhood . Larger neighbourhood size tends to increase the amplitude of cycles (results not shown), implying that, in a finite population of large social neighbourhoods, cyclical dynamics could be destroyed by the stochastic fixation of a single type.We use a numerical analysis to recover the analytical results above and to confirm that the same qualitative dynamics obtain for more realistic scenarios with d; see also the electronic supplementary material, figure S6d for a time-series plot and figure S8 for dynamics of individual replicates). We further examined whether de novo defectors also emerged in the three-strain communities as previously described for our two-strain communities , and the prevalence of the original cooperator strain  was always approximately six-times higher in three-strain communities than it was in two-strain communities . These comparisons show that de novo defectors are unlikely to affect community stability in our three-strain communities. However, in addition to these de novo defectors, we also observed the evolution of de novo loner-defectors . These are mutants that could potentially exploit the loner's pyoverdine and could therefore undermine community stability in the long term.Next, we used experimental evolution to directly examine community stability and strain dynamics in communities initially composed of cooperators, defectors and loners. In all replicates, we observed coexistence of all three strains and cyclical shifts in strain frequencies, consistent with the rock–paper–scissors dynamics predicted by our model (figure 2munities . We inde(e)n = 2, γ = 0 and α = 1  and numerical analyses for n > 2, γ > 0 and α < 1 reveal that the addition of a loner-defector can result in three possible evolutionary outcomes: (i) when siderophores are cheap (low c), cooperation is strongly favoured, which eliminates the loner-defector; (ii) when siderophores are costly (high c), both defector strategies (D and LD) are favoured and maintained in a stable polymorphism; (iii) when the cost of siderophores is intermediate, the loner-defector is eliminated and the three remaining strains display rock–paper–scissors dynamics .To theoretically explore the effect of de novo loner-defectors on community stability, we analysed the complete four-strategy system in equation (2.1). Our analytical model results for v = 1.65 ± 0.09, one-sample t-test, t5 = 14.74, p < 0.0001). By contrast, we could detect no fitness advantage enjoyed by the loner-defectors over the defector , and all loner-defectors dramatically lost out in competition with the cooperator . Given this highly imbalanced fitness network, where loner-defectors are almost completely displaced by cooperators within 24 h, we predicted that the four-strain system will typically be unstable [figure 3b and see also the electronic supplementary material, figure S9b for a time-series plot and figure S11 for dynamics of individual replicates). Hence, the emergence of loner-defectors does not necessarily eliminate the cooperation and diversity that is maintained by the presence of a loner.To determine which of these outcomes applies to our experimental system, we isolated six independent de novo loner-defectors at random and competed them against the other three strains in the community . We found that the loner-defectors outcompeted the ancestral loner . This prediction was supported by our observations of open-ended evolutionary dynamics in siderophore-producing communities of Pseudomonas. Under well-mixed conditions that favour defectors over cooperators , we found that the presence of a loner strain can stabilize the coexistence of defectors and cooperators . Moreover, this dynamic coexistence of cooperators, defectors and loners was not destroyed by the origin of defectors that exploit the loner , the maintenance of cooperation and diversity requires that cells interact in local neighbourhoods . This istogether . Furtheret al. [et al. [Our study provides a unique example of how rock–paper–scissor dynamics can maintain biological diversity. Our finding that cooperation and diversity are maintained in well-mixed populations sets our study apart from most previous theoretical and empirical work, where rock–paper–scissor dynamics exclusively occurred in genetically structured environments –45. For et al.  showed tet al. . More re [et al.  extendedPseudomonas communities in our study, it is important to note that the role of the loner is potentially very general and may even be enacted by organisms belonging to a different taxonomic group than the cooperator and the defector. In aquatic microbial systems, for example, the loner could be a distinct bacterial species or potentially even a small eukaryote, such as an alga. Moreover, our study suggests that the loner strategy is not limited to cognitively advanced economic agents like humans [Although we have focused on e humans  but can e humans . This hie humans ; metabole humans ,51 and te humans , which cFinally, we propose that public goods cooperation itself can favour the evolution of loners and may therefore promote diversity via a multi-step evolutionary process. This is in fact evidenced by our open-ended evolutionary study in which bacteria were not confined to an artificially minimal strategy set but were instead able to innovate new strategies in a naturalistic way. Similarly, we expect that in a well-mixed population of cooperators, defectors will first arise and enjoy an evolutionary advantage. In turn, these defectors may modify their environment in a way that favours the evolution of loners whose public goods are accessible to neither cooperators nor defectors. The resulting three-strain community can be either: (i) transitive, and reverting to a one-strategy community dominated by a superior loner; (ii) non-transitive, and leading to rock–paper–scissors dynamics, as observed in our study or (iii) open-ended, and evolving into higher-order systems with multiple cooperators and defectors coexisting, at least temporarily . Our res


There have been few reports regarding quetiapine-associated hematological effects other than white-blood-cell alteration. We present the first reported Han-Chinese case that developed leucopenia and thrombocytopenia after taking quetiapine.We present a case of a person with a bipolar I disorder who experienced leucopenia and thrombocytopenia after taking 400 mg/day of quetiapine and 1000 mg/day of valproic acid for three and one-half months.The hematological toxicity abated upon the discontinuation of both drugs. However, due to the intolerable side effects of the replaced antipsychotic , and according to the patient’s preference, we prescribed quetiapine and valproic acid again. There was a recurrence of leucopenia and a decreased platelet count by the sixth day. The adverse effects disappeared soon after we discontinued quetiapine, while keeping valproic acid treatment.Quetiapine-associated leucopenia and thrombocytopenia seems reversible but possibly fatal. Therefore, clinical practitioners should be aware of this adverse reaction. Quetiapine is a commonly-prescribed atypical antipsychotic drug used worldwide for schizophrenia and mood disorders. It is a dibenzothiazepine derivative related to clozapine in chemical structure and pharmacological profile ,2.3/μL) develops in approximately one percent of the subjects treated with quetiapine, and it is resolved upon discontinuation [3/μL) and agranulocytosis (an absolute neutrophil count below 500/μL) may emerge when using quetiapine [Several cases of quetiapine-associated hematologic side effects have been reported. Leucopenia (a white blood cell count below 4.0 × 103/μL) [There have been fewer case reports addressing quetiapine-associated hematological effect of combined alteration of white-blood-cells and platelets. We could find only two reports in previous literature reporting quetiapine-associated leucopenia and thrombocytopenia (a platelet count below 150 × 103/μL) ,3. We wiMr. A, a twenty-three-year-old man, was diagnosed as having a bipolar I disorder at the age of nineteen due to the manifestations of manic symptoms with psychotic features. He had been admitted six times in various hospitals for the management of recurrent manic episodes. He had successively taken lithium, valproic acid, and carbamazepine, though with poor drug compliance after his hospital discharge. He did not have a history of systemic or hematologic diseases.3/μL, an absolute neutrophil count (ANC) of 1.56 × 103/μL, a red blood cell (RBC) count of 4.63 × 106/μL, a hemoglobin (Hb) count of 15.7 g/dL, and a platelet count of 149 × 103/μL.After taking 400 mg/day of quetiapine and 1000 mg/day of valproic acid for four weeks, with no other psychotropic agents, he was transferred to our rehabilitation ward for occupational training. On the first day of admission, routine blood tests revealed a white blood cell (WBC) count of 4.37 × 103/μL, ANC 860 /μL, platelet 81 × 103/μL, while his RBC and Hb remained in the normal ranges. We stopped using valproic acid on Day 2 and closely monitored the clinical conditions. However, the fever persisted; and the laboratory data showed agranulocytosis with thrombocytopenia  on Day 4. Therefore, we immediately discontinued quetiapine. His clinical condition stabilized on Day 6, with the lab data showing the following: WBC 2.84 × 103/uL, ANC 1,107/μL, and platelet 91 × 103/μL. On day 8, there was an obvious improvement of the hematological profile .Unfortunately, after a total of three and one-half months with the above medications, he developed a sudden onset of fever and upper respiratory tract infection (Day 0). Two days later (Day 2), his leucopenia, neutropenia, and thrombocytopenia counts were noted as follows: WBC 1.99 × 103/μL), a decreased neutrophil count , and thrombocytopenia (136 × 103/μL) again on Day 24 without clinical symptoms. On Day 26, we discontinued quetiapine and shifted the medication to 4 mg/day of risperidone, while maintaining the valproic acid treatment. On Day 30, we observed normal white-blood-cell and neutrophil count , and a platelet count closer to normal range (platelet 149 × 103/μL). The patient has not been administered quetiapine since then, and the hematological follow-up examinations have all revealed normal results thereafter.On Day 9, due to an acute worsening of manic and psychotic symptoms, we prescribed 10 mg/day of haloperidol. However, we noticed severe and intolerable extra-pyramidal side effects. Considering the patient’s preference, 200 mg/day of quetiapine and 500 mg/day of valproic acid were administered on Day 18 and were titrated up to 400 mg/day and 1000 mg/day, respectively on Day 20. With a cautious follow-up of the hematological data, we noted the following: leucopenia (WBC 3.12 × 10We have presented a case of a man with a bipolar I disorder who developed leucopenia, neutropenia, and thrombocytopenia after being administered quetiapine and valproic acid. The hematological abnormalities were resolved upon discontinuation of both drugs but re-emerged following the reuse of them. However, the abnormalities abated again after we stopped quetiapine but continued using valproic acid. The temporal relationship between drug exposure and laboratory data suggests direct quetiapine toxicity. In our case, the Naranjo adverse drug-reaction probability scale was seven, which suggested a probable causality.We could find only two reports in previous literature reporting quetiapine-associated leucopenia and thrombocytopenia ,3. Both However, we cannot completely exclude the contributing effect of valproic acid. Two case reports also showed that neutropenia might be induced by a combined use of quetiapine and valproic acid ,6. It ha2-relative-to-DA2 receptor affinity and virtually no affinity for muscarinic receptors [The mechanisms underlying the toxicity remained unclear. Given a resemblance of the chemical structure and similar pharmacological profile, it has been speculated that quetiapine could cause bone-marrow depression similar to clozapine . Both dreceptors . Other meceptors .According to our case report, we do not have adequate information to conclude whether this phenomenon was dose-related or idiosyncratic. A previous case report stated that quetiapine-induced leucopenia might be a possible dose-dependent phenomenon . AccordiWe presented the first case in Han-Chinese of quetiapine-associated leucopenia and thrombocytopenia. Another unique aspect of our case is the reuse of quetiapine in the treatment course, which hasn’t been mentioned in previous case reports. Nevertheless, the contributing effects from the co-administered valproic acid cannot be neglected. We hope our case report can help in accumulating evidence and enhance further investigation to find out clinical or biological factors related to this side effect.Quetiapine-associated leucopenia and thrombocytopenia seem reversible but possibly fatal. Although it has not been a clinical routine yet for a regular check-up of the complete blood cell count in patients administered quetiapine, clinical practitioners should be aware of this adverse reaction.Written, informed consent was obtained from the patient for publication of this case report and any accompanying image. A copy of the written consent is available for review by the editor of this journal.