Dictyostelium discoideum, a model organism for the study of collective cell migration, extracellular cyclic adenosine 3’,5’-monophosphate (cAMP) acts as a diffusible chemical guidance cue for cell aggregation, which has been thought to be important in multicellular morphogenesis. Here we revealed that the dynamics of cAMP-mediated signaling showed a transition from propagating waves to steady state during cell development. Live-cell imaging of cytosolic cAMP levels revealed that their oscillation and propagation in cell populations were obvious for cell aggregation and mound formation stages, but they gradually disappeared when multicellular slugs started to migrate. A similar transition of signaling dynamics occurred with phosphatidylinositol 3,4,5-trisphosphate signaling, which is upstream of the cAMP signal pathway. This transition was programmed with concomitant developmental progression. We propose a new model in which cAMP oscillation and propagation between cells, which are important at the unicellular stage, are unessential for collective cell migration at the multicellular stage.In  Dictyostelium discoideum. This study suggests that cAMP oscillation is not required for collective cell migration despite its importance at a unicellular stage.Hidenori Hashimura et al. show that cAMP-mediated signaling changes from propagating waves to a steady state at a multicellular stage of amoeba  It is organized by integrated physical and chemical guidance cues between cells, such as cell–cell adhesion and diffusible factor-mediated signaling, which are integrated and act in parallel3. The social amoeba Dictyostelium discoideum is a model organism for the study of collective cell migration because of its morphogenesis and simple cell–cell interactions via diffusible chemical signals4. Dictyostelium cells grow as unicellular organisms at the vegetative stage, but undergo transitions from a unicellular to multicellular organism by aggregation upon starvation. During aggregation, starved cells typically move towards the aggregation center to form one multicellular aggregate. This coordinated migration is achieved by the self-organization of cAMP gradients and by chemotaxis to extracellular cAMP5. When Dictyostelium cells sense extracellular cAMP signals, cAMP receptors activate PI3-kinases through G proteins to produce phosphatidylinositol 3,4,5-trisphosphate (PIP3) transiently on the plasma membrane of the cell front, leading to the transient localization of cytosolic regulator of adenylyl cyclase (CRAC) to the membrane via the Pleckstin Homolog (PH) domain that binds to PIP3, activating adenylyl cyclase7. The Dictyostelium cell has three subtypes of adenylyl cyclase , but only ACA is activated by external cAMP signals8. cAMP is synthesized by ACA in response to external cAMP signals and secreted to induce neighboring cells to similarly produce cAMP. Simultaneously, the transient accumulation of PIP3 at the cell front in response to external cAMP also induces actin polymerization and pseudopod formation, resulting in chemotactic migration9. These reactions finally cause the propagation of cAMP signals as travelling waves called cAMP relay, resulting in chemotactic migration toward the aggregation center. That is, the correlative migrations of multiple cells are mediated by a single diffusible chemical factor, extracellular cAMP.Collective cell migration is ubiquitous in multicellular organisms and contributes to many organismal phenomena, including morphogenesis, wound healing, and cancer invasion10. Upon aggregation, cells form a stream which flows into a loose mound. Loose mounds become tightly packed (tight mounds) by both secretion of the extracellular matrix and the strengthening of cell–cell contacts. In tight mounds, cells differentiate into prestalk or prespore cells. Prestalk cells are sorted at the top of the mound to form the tip, which elongates and forms the front of a multicellular body (slug) to migrate as a whole. In conventional microscopic observations, optical densities of cell populations during chemotactic aggregation describe synchronous changes in cell shapes and act as an index of cAMP relay11. These optical density waves have been detected in streams, mounds, and slugs, giving evidence of cAMP relay at these stages too13. Cell sorting to the tip of the mound also can be explained by cAMP relay. There is a difference in the response of chemotaxis toward cAMP between prespore and prestalk cells in mounds, resulting in cAMP relay guiding the sorting of prestalk cells to the tip of the mound15. Cells dissociated from slugs produce cAMP upon extracellular cAMP stimulation16 and show chemotactic movement toward cAMP17, indicating that slug cells have the ability of cAMP relay and chemotaxis toward cAMP. Furthermore, cAMP microinjection in slugs causes chemotactic attraction of some cells in the population and perturbation of the optical density wave propagation18. These observations suggest that cAMP signals control cell movement in slugs. Thus, cAMP relay is regarded as an essential mechanism for organized collective cell migration, such as cell sorting and multicellular movement, in Dictyostelium cells.It has been argued that cAMP relay is also essential for the organization of collective cell migration during developmental events following the aggregationDictyostelium, some observations suggest that the role of cAMP relay in slugs is controversial. acaA-null cells, which lack the ability of cAMP relay, normally cannot aggregate and form multicellular bodies, but the phenotypes of the mutant are rescued by constitutive activation of PKA, which is downstream of the cAMP signaling pathway, implying that Dictyostelium cells have developmental ability without cAMP oscillation19. Furthermore, cAMP signals in mounds and slugs have not been investigated, whereas the cAMP relay during cell aggregation has been directly verified by live imaging of cAMP signals using sophisticated cAMP-sensitive fluorescent probes, which has revealed that intracellular and extracellular cAMP levels show synchronous oscillations in cell populations and that propagation of the oscillations changes between cells21. Therefore, no clear evidence exists for cAMP relay organizing collective cell migration at multicellular stages. In this study, we investigated the dynamics of cAMP signals through the development of Dictyostelium cells by visualizing the changes in cytosolic cAMP levels ([cAMP]i), which reflect the response to cAMP relay. Our live-imaging approaches demonstrated the role of cAMP relay during aggregation and mound stages. Surprisingly, we found that [cAMP]i oscillation and its propagation, which is an index of cAMP relay, gradually decreased and disappeared after slug formation. This result indicates a dramatic transition of cAMP signaling dynamics during the development of Dictyostelium cells and the possibility that oscillatory cAMP signaling is not essential for collective cell migration in slugs, which challenges the traditional view about the role of cAMP relay in the organization of collective cell migration.In spite of these traditional views of cAMP relay for the coordination of collective cell migration in Dictyostelium cells, we monitored [cAMP]i by using a cAMP indicator, Flamindo222. The binding of cAMP to the probe causes a decrease in the fluorescence intensity of the sensor. It has been reported that Flamindo2 can detect [cAMP]i changes in aggregating Dictyostelium cell populations23. We confirmed that Flamindo2 was stably expressed in Dictyostelium cells with no obvious defects in the developmental progression 25. It has been shown using biochemical assays and FRET-based imaging analyses that the peak of ACA activity occurs 60–120 s after external cAMP stimulation27. Considering Flamindo2 binds not only to cAMP but also to cGMP but with lower affinity22, our results suggest that the first peak is an effect of [cGMP]i elevation and that the second peak was due to only the increase in [cAMP]i. This conclusion is also supported by gc- cells, which lack guanylyl cyclases (gca and sgcA) and have no cGMP production ability28, showing Flamindo2 signal responses with only one peak at 90 s after the stimulation  and moved rotationally, while prespore cells made up ~80% of slugs (posterior) and moved straight . GBF regulates late-development gene expression, and gbfA-null cells undergo developmental arrest at the loose mound stage without tip formation due to the lack of post-aggregative and cell-type specific genes35 , acaA−57, gc− (gca−/sgcA−)58, and gbfA−. Ax2 cells were provided from the Dr. Guenther Gerisch laboratory (Max Planck Institute of Biochemistry) by Dr. Kei Inouye (Kyoto University). acaA− cells were kindly provided by Dr. Satomi Matsuoka (RIKEN). gc− cells were laboratory stocks. gbfA− cells were obtained from the NBRP-nenkin Stock Center. Cells were grown axenically in HL5 medium  in culture dishes or shaking flasks at 21 °C. Transformants were maintained at 20 μg ml−1 G418 or 10 μg ml−1 BlastcidinS.The Akt/PKB-GFP59, pHK12neo_Citrine, and pHK12neo_Flamindo2. pBIG_PHAkt/PKB-GFP is from a laboratory stock. pEcmAO-RFPmars plasmid was obtained from the dictyBase Stock Center. pHistone2B-RFP plasmid was kindly provided by Dr. Tetsuya Muramoto (Toho University). pHK12neo_Citrine was constructed by the insertion of Citrine fragments into the BglII and SpeI sites of pHK12neo by the In-Fusion technique (Clontech laboratories Inc.). pHK12neo_Flamindo2 was constructed by the insertion of synthesized Flamindo2 fragments (GenScript) into the BglII and SpeI sites of pHK12neo. The codon usages of the Flamindo2 sequence were optimized to those of D. discoideum for efficient protein expression in Dictyostelium cells. Vectors except for pEcmAO-RFPmars allow constitutive expression of the proteins in cells under the act15 promoter. The wild-type strain and mutant cells were transformed with ~1.5 μg plasmid by electroporation60, and transformants were selected with G418 and BlastcidinS at a final concentration of 20 μg ml−1 and 10 μg ml−1, respectively.The plasmids used in this study are as follows: pEcmAO-RFPmars, pHistone2B-RFP, pBIG_PH5 cells were blotted onto a polyvinylidene difluoride membrane and reacted with a polyclonal anti-GFP antibody  diluted 1:1000 in blocking solution (1% skim milk in TBS-T). Signals were visualized by the chemiluminescence of reactions with HRP substrate , and images were acquired with ChemiDocTM XRS (BioRad).Cells were developed on a cellulose membrane filter (see the Method subsection titled ‘Verification of proper function of Flamindo2 as the cAMP sensor at the slug stage’) and harvested at 5 and 12 h after starvation. Vegetative and developed cells were lysed by 4 × SDS sample buffer  and boiled at 95 °C for 5 min. Proteins from 4 × 1061. In the sequential time-lapse images, the positions of each cells at the first frame were set manually from the fluorescence of Histone-RFP and movements of each cells were tracked automatically. Changes in Flamindo2 signals at the individual cell level were estimated by measuring the mean intensities of Flamindo2 in the regions (~3–5 μm2 regions) positioned on the cytosol of the tracked cells at each time point. Cell velocity was calculated by dividing the displacement between two sequential frames by the interval time, and the unit of velocity was converted to μm per min. The period of an oscillation was calculated by averaging the difference between the peaks of the oscillation  or defined as the first largest peak at t > 0 of the autocorrelation function of the PHAkt/PKB-GFP translocation data. Data with at least three peaks in the oscillation were used for the analysis. In general, the fluorescence intensities of Flamindo2 were normalized with values at t = 0.In all experiments, cells were observed at 22 °C. Confocal images including a series of Z-stacks were taken by a confocal laser microscope  with an objective  and oil immersion lenses  or an inverted microscope  equipped with a CSU-W1 confocal scanner unit (Yokogawa), two sCMOS cameras  and objective lenses . Flamindo2, GFP and Citrine were excited by a 488 nm solid-state CW laser, and mRFPmars was observed using a 561 nm solid-state laser. Epifluorescence imaging was taken by using an inverted epifluorescence microscope  equipped with a 130 W mercury lamp system , sCMOS cameras  and objective lenses . Flamindo2 and tetramemethylrhodamine-maleimide  were observed using fluorescence mirror units U-FGFP  and U-FMCHE , respectively. All images were processed and analyzed by Fiji and R software. For cell tracking, laboratory-made software was used6 cells ml−1) and washed three times in KK2 phosphate buffer . In this study, two methods were used to observe development on agar. To observe early aggregation, cells were plated on the entire surface of 2% water agar plates (2% w/v Difco Bacto-agar in ultrapure water) at a density of 5–7 × 105 cells cm−2 and incubated at 21 °C. To observe the mound and slug stages, 5 μl of cell suspension at a density of 2–4 × 107 cells ml−1 was deposited on 1.5% water agar and incubated at 21 °C for 6–15 h. To image development, a method described previously62 was used. Here, a piece of agar was cut out and placed upside down on a 35-mm glass bottom dish  directly or on a spacer  attached to the dish. The spacer was filled with liquid paraffin  to avoid light scattering. To prevent desiccation during the observation, wet paper was placed in the dish and the agar piece was covered with liquid paraffin. In this condition, cells and multicellular bodies could move freely and develop normally for more than 12 h under the microscope. The Z series of fluorescence images was taken by the confocal microscope at 10–30 s intervals.To induce starvation and development, cells were harvested during the exponential phase  scroll movement of the slug and thickness of the tissue make it difficult to follow individual cell movements in normal slugs. One microlitre of cell suspension at a density of 4 × 107 cells ml−1 was deposited on 2% water agar plates together with 2 μl liquid paraffin. A coverslip was placed over the suspension, which was incubated at 22 °C for more than 15 h. The Z series of the fluorescent images was acquired at 15-s intervals for 20–30 min by the confocal microscope. The 2D slug, which has only few (~4) cell layers and thus enables us to follow cell movement easily, showed similar properties with normal slugs with respect to cell movement and proportion of cell types64. We observed periodic cell movement with no obvious [cAMP]i oscillation in both normal slugs and 2D slugs  at a density of 5 × 105 cells ml−1 for 1 h and incubated for a subsequent 5 h in the presence of 100 nM cAMP pulses given at 6-min intervals. Cells were then washed three times with 1 ml DB and resuspended in DB at a density of 106 cells ml−1. Forty microlitre of the cell suspension was dropped onto a glass bottom dish. Cells were stimulated by adding 160 μl cAMP solution (the target concentration of cAMP) to the cell droplet. In caffeine experiments, cells were exposed to 4 mM caffeine for 30 min on a glass bottom dish before cAMP stimulation. Fluorescent images were acquired by the confocal microscope at 5-s intervals during stimulation. Averaged fluorescence intensities of Flamindo2 or Citrine in 5 μm2 regions positioned within the cytosol were measured at each time point.Cells expressing Flamindo2 or Citrine were starved in 1 ml of developmental buffer , protein amount per cell  and cell volume (0.43 pl cell−138). For example,To confirm that Flamindo2, which has an EC−1) × 7 × 10–8 (mg cell−1)/0.43 (pl cell−1) = 0.32 μM2  at a density of 5 × 105 cells cm−2 and incubated at 21 °C for 12 h to allow slug formation. The slugs were harvested in DB and dissociated into single cells by repeated passages through a 25 G needle (Terumo) with a 1 ml syringe on ice65. Slug-disaggregated cells were resuspended in DB at a density of 106 cells ml−1, and 40 μl of the suspension was deposited onto a 12-mm glass bottom dish. cAMP stimulation and caffeine treatment were performed as described in the Method subsection titled ‘Verification Flamindo2 as an indicator of [cAMP]i changes in Dictyotelium cells at the unicellular phase’. Fluorescent images were acquired by the confocal microscope at 5-s intervals during stimulation. Averaged fluorescence intensities of Flamindo2 in 5 μm2 regions positioned within the cytosol were measured at each time point. Cell types of slug-disaggregated cells were distinguished by the intensity of ecmAO::mRFPmars.Cells expressing Flamindo2 and 13. Fluorescent and DIC images were acquired at 15-s intervals by the IX83 epifluorescence microscope.We also performed the cAMP stimulation test on intact slugs by cAMP microinjection into water agar Fig. . Cells wAKT/PKB-GFP were washed and deposited on a cellulose membrane filter at a density of 5 × 105 cells cm−2 and incubated at 21 °C for 12 h to allow slug formation, or 5 μl of cell suspension at a density of 4 × 107 cells ml−1 was deposited on 2% water agar and incubated at 21 °C for 6 h to allow loose mound formation. Mechanical dissociation of cells from loose mounds or slugs was performed as described in the Method subsection titled ‘Verification of proper function of Flamindo2 as the cAMP sensor at the slug stage’. Forty microlitre of the cell suspension  was deposited onto a 12-mm glass bottom dish and allowed to settle for 5 min (loose mound cells) or 20 min (slug cells). Cells were stimulated by 10 μM cAMP as described in the Method subsection titled ‘Verification Flamindo2 as an indicator of [cAMP]i changes in Dictyotelium cells at the unicellular phase’. Fluorescent images were acquired by the confocal microscope at 5-s intervals during stimulation. Averaged fluorescence intensities of PHAKT/PKB-GFP in 4 μm2 regions positioned within the cytosol were measured at each time point.Cells expressing PHAKT/PKB-GFP by cAMP microinjection into water agar as shown in Fig. AKT/PKB-GFP in slugs with high magnification, slugs were formed on thin agar covered directly on the glass bottom dish. Here, 100 μl of melting agar was poured into a well of 12-mm glass bottom dish and then 65 μl of agar was sucked up, resulting in the formation of a uniform thin agar layer on the glass. Five microlire of cell suspension at a density of 4 × 107 cells ml−1 was deposited at the center of the thin agar layer and incubated at 21 °C for 12 h to allow for slug formation. The manipulation of cAMP microinjection was performed as described in the Method subsection titled ‘Verification of proper function of Flamindo2 as the cAMP sensor at the slug stage’.In addition, we performed the cAMP stimulation test on intact slugs expressing PH32 was applied with some modification. acaA-null cells expressing Flamindo2 were washed and suspended in 1 ml DB at a density of 6 × 106 cells ml−1. The cells were then incubated on a 35-mm plastic dish (Iwaki) for 4 h and stimulated with 30 nM cAMP pulses at 6-min intervals. Cells were then washed and suspended in 1 ml DB again and subsequently incubated on a 35-mm plastic dish or a 35-mm glass bottom dish  for more than 12 h with 30 μM cAMP pulses at 60-min intervals. After terminating the exogenous cAMP pulse treatment to induce multicellular formation, acaA-null cells were washed and resuspended in DB at a density of about 4 × 107 cells ml−1, and 5 μl of cell suspension was deposited on 2% water agar plates because a submerged culture inhibits the multicellular development of even wild-type cells. Time-lapse images during the development were acquired at 30-s intervals by the confocal microscope. We confirmed that the clumps of acaA-null cells could not synthesize cAMP in response to external cAMP signals by monitoring Flamindo2 signals  attached to a glass bottom dish. The spacer was filled with liquid paraffin to avoid light scattering. A piece of 2% water agar with or without 4 mM caffeine was then put on the filter (Supplementary Fig. 66, the migration distance for 30 min divided by each slug length was regarded as the migration rate.Cells expressing Citrine were washed, and 5 μl of cell suspension at a density of 4 × 10Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6


These observations suggest a transient but widespread presence of marine euxinia in an ocean characterized by redox stratification, high bioproductivity, and high-fluxes of sulfate from chemical weathering before the deposition of the cap carbonate.Termination of the terminal Cryogenian Marinoan snowball Earth glaciation (~650–635 Ma) is associated with the worldwide deposition of a cap carbonate. Modeling studies suggest that, during and immediately following deglaciation, the ocean may have experienced a rapid rise in pH and physical stratification followed by oceanic overturn. Testing these predictions requires the establishment of a high-resolution sequence of events within sedimentary records. Here we report the conspicuous occurrence of pyrite concretions in the topmost Nantuo Formation (South China) that was deposited in the Marinoan glacial deposits. Sedimentary facies and sulfur isotope data indicate pyrite precipitation in the sediments with H The termination of the Marinoan snowball Earth event marks one of the most drastic transitions in Earth history, but the oceanic response remains unclear. Here, the authors’ integrated analysis demonstrates that the ocean experienced transient but widespread euxinia following this Snowball Earth event. Climatic and geochemical models show that the termination of a snowball Earth glaciation requires a high level of atmospheric CO2 (pCO2 > 0.2 bar)12 in order to overcome the high albedo from global ice cover. Such a high pCO2 atmosphere will result in a rapid meltdown of glaciers13 and an extreme greenhouse climate10. Rapid deglaciation may lead to the shutdown of thermohaline circulation and the intensification of ocean stratification15, with warmer and dysoxic freshwater in the surface ocean overlying cold and anoxic seawater in the deep15.The Sturtian (~717–660 Ma) and Marinoan (~650–635 Ma) snowball Earth events in the Cryogenian Period19. Globally, sedimentary sequences during deglaciation are mostly manifested as a cap carbonate sharply overlying glacial diamictites21. Although the cap carbonate is often assumed to have been deposited immediately after the onset of deglaciation, a window of time is required to allow atmospheric pCO2 drawndown and seawater alkalinity buildup20. This necessarily means that deglaciation and associated continental weathering must have started before the initiation of cap carbonate deposition. This time lag is supported by recent work exploring Mg isotopes within post-Marinoan sequences20 and may be on the order of 105 years9.Such a catastrophic end of a snowball Earth glaciation is expected to drive drastic perturbations in ocean chemistry22. Here, we report the occurrence of abundant pyrite concretions from the topmost Nantuo Formation24, a sedimentary sequence deposited during the terminal Cryogenian Marinoan glaciation in the Yangtze Block of South China , but δ34Spy may vary substantially between different concretions or between different sections. In basinal sections, concretions display a smaller range of variation in δ34Spy . Concretions from the slope sections have higher δ34Spy than those from the basinal sections, and δ34Spy values display a wider range of variation [Huakoushan  and Bahuang ]. δ34S values of disseminated pyrite extracted from gravelly siltstone in the top Nantuo Formation shows greater fluctuation  in the Taoying section but limited variation  in the Bahuang section. In the outer shelf sections, pyrite concretions from the gravelly siltstone layers have the lowest δ34Spy values . Δ33S (defined as Δ33S = δ33S − 1000([1 + δ34S/1000]0.515 − 1)) also exhibits a wide but overlapping range of variation among different concretions or among different facies   and variable Δ33S values are not characteristics of hydrothermal pyrite27  of +30‰31 and biological fractionation (ΔDSR) in DSR at 40‰32, our modeling results indicate a maximum δ34Spy value of +8‰  in sediment porewater, and DSR is sustained by sulfate diffusion from seawater+8‰ Fig. , and the34S-enriched) pyrite could be generated by DSR in a closed porewater system , which can be simulated by a Rayleigh distillation model 33. Our modeling results indicate that precipitation of 34S-enriched pyrite in the basin and slope sections was driven by a higher degree of sulfate reduction than in outer shelf settings  and seawater sulfate45. We hypothesize that, during the deglaciation of the Marinoan glaciation, enhanced continental weathering20 could have delivered abundant nutrients and sulfate into the ocean. Because the deglacial continental weathering may predate oceanic euxinia20 due to the delayed recovery of productivity in acidified seawater, both nutrients and sulfate might be accumulated in seawater from riverine inputs. Once surface ocean productivity was resumed at high levels, high nutrient content could sustain high productivity, while DSR in water column would be fueled by sufficient supplies of organic matter and sulfate, driving oceanic euxinia.Abundant pyrite concretions in the top Nantuo Formation imply the development of oceanic euxinia before the precipitation of cap carbonate17, and high nutrient fluxes from the continents. This is also an interval characterized by redox stratified oceans where a large quantity of H2S was scavenged by Fe(II) in the glacial diamictite sediments. Importantly, this is also an interval prior to the deposition of the well-known cap carbonates, a time when seawater pH values or alkalinity were still below the threshold of carbonate precipitation or pCO2 levels were sufficiently high in the atmosphere46. The current study extends previous efforts to reconstruct from sedimentary records the sequence of events in the aftermath of the Marinoan meltdown47 in order to test the predictions of snowball Earth glaciations2.The pyrite-rich interval in the topmost Nantuo Formation marks a brief yet widespread euxinic condition in the aftermath of the Marinoan glaciation immediately before cap carbonate precipitation. It is an interval with rising sulfate levelsPyrite concretions were first split by a rock saw and then slabs were polished before sampling. Pyrite samples were collected by a hand-held micromill from the polished slabs. To capture the isotopic variations within a single pyrite concretion, multiple samples were sequentially collected from rim to core. Sulfur isotopic compositions of pyrite were measured at the Oxy-Anion Stable Isotope Consortium in Louisiana State University, at the EPS Stable Isotope Laboratory in McGill University, and at the Geochemistry laboratory in University of Maryland.34S analysis, about 0.05 mg pyrite-rich powder was mixed with 1–2 mg V2O5, and was analyzed for S isotopic compositions on an Isoprime 100 gas source mass spectrometer coupled with a Vario Microcube Elemental Analyzer. Sulfur isotopic compositions are expressed in standard δ-notation as permil (‰) deviations from the Vienna-Canyon Diablo Troilite standard. The analytical error is <0.2‰ based on replicate analyses of samples and laboratory standards. Samples were calibrated on two internal standards: LSU-Ag2S-1: −4.3‰; LSU-Ag2S-2: +20.2‰.For δ2S(g) via the chromium reduction method. H2S gas was then carried through a N2 gas stream to a Zn acetate solution where it precipitated as ZnS. ZnS was then converted to Ag2S through addition of AgNO3. Samples were then filtered and dried at 80 °C. Ag2S samples were converted to SF6(g) through reaction with F2(g) in heated Ni bombs. Generated gas was then purified through a series of cryo-focusing steps followed by gas chromatography. The purified samples were then analyzed on a Thermo MAT-253 on dual-inlet mode. The total error on the entire analytical procedure is less than (1σ) 0.1‰ for δ34S and 0.01‰ for ∆33S.Multiple sulfur isotope analyses were conducted by converting pyrite samples to HSedimentary or authigenic pyrite can precipitate either from sediment porewater or water column. In sediment porewater, pyrite formation can occur in a closed or an open porewater system. For a closed system, DSR occurs within sediment porewater with no additional sulfate supply from seawater. In contrast, open system DSR refers to sulfate supply via diffusion from the overlying seawater and pyrite formation in sediment porewater  or pyrite  can be described as:Rayleigh distillation model. The Rayleigh distillation model can be expressed as follows:Mpy) formation can be calculated by:ρpy is the density of pyrite (4.9 g cm–3), Mr is the relative molecular mass of pyrite, [SO4] is seawater/porewater sulfate concentration (mol L–1).The amount of pyrite , s is the sedimentation rate (cm ky−1), R is the first-order rate constant during DSR.1D-DAR model. The 1D-DAR model can be applied to quantify the geochemical profiles in sediment porewater. The DAR model describes three physio-chemical processes: molecular diffusion, advection, and chemical reaction. During DSR in porewater, sulfate is supplied from seawater by diffusion. The diffusion is driven by the sulfate concentration gradient between seawater and sediment porewater, which is generated by sulfate consumption in sediment porewater by DSR. To model sulfur isotopic profiles of porewater, we treat 34S and 32S. Assuming a steady state , the solution of Eq.  starting from the upper bound of DSR zone (i = 0). Assuming no sulfur isotope fractionation during pyrite precipitation between H2S and pyrite, δ34Spy at depth z can be calculated by:Pyrite precipitation within sediment is a continuous process within the DSR zone with the lower bound marked by the depletion of porewater sulfate. To calculate the instantaneous sulfur isotope of pyrite within the DSR zone, we divide sediments into different depth slices  can expressed as:The depth 2S diffusion from overlying water column. This process can be quantified by the one-dimensional diffusion-reaction (1D-DR) model. This model includes two processes: H2S diffusion and pyrite precipitation. The sulfide diffusion is driven by the H2S concentration gradient between sulfidic seawater and sediment porewater, which is generated by pyrite precipitation in sediment porewater. The 1D-DR model is expressed as:3iS] is the porewater concentration of H34S and H32S, z is the depth below the water-sediment surface, Ds is the vertical diffusivity coefficient of bulk sediment (m2 year−1), R is the first-order rate constant for pyrite formation via H2S reaction with reactive Fe.1D-DR model. Pyrite can precipitate in porewater with HAssuming a steady state, the solution for this equation is,34SH2S profile can be calculated by:δ3iS]std represents the standard of 34S or 32S. The instantaneous δ34Spy can be calculated by:py represents the sulfur isotope fractionation during pyrite precipitation. The δ34Spy at depth z below water-sediment surface can be calculated by:[The cumulative amount of pyrite formation can be calculated by Eq. .33Spy–δ34Spy plot. Assuming all the Nantuo pyrite were formed within ferruginous porewater and pyrite formation was rapid and irreversible. δ34Spy was modeled by a sulfate concentration model37. In a steady state, sulfate concentration at specific depth can be expressed as:z, k is sulfate reduction rate constant, s is the sedimentation rate.Sulfur isotope model for Δ36S, here the equation of total mass of sulfate can be simplified as As the little mass of For sulfur isotope, Eq.  can be rHere, i equals 3 or 4.3iS of porewater sulfate is controlled by the initial seawater sulfate δ3iS, sulfate reduction rate constant k, sedimentation rate s and sulfur isotope fractionation factor (α) for DSR37. Defined α as the ratio of sulfate reduction rate (SRR) of 33S or 34S over the 32S normalized with corresponding isotope concentration:δ33S of porewater sulfate can be calculated:33λ is the slope of terrestrial mass fractionation line.Then, Δ2S sulfur isotopic compositions can be calculated by the ratios of DSR rate at corresponding depth:The instantaneous H3iH2S.Pyrite sulfur isotopic compositions can be calculated by the accumulated δCpy) can be calculated by:Apy is pyrite concretion area in the photo and AT is the total area of host rock in the photo.Pyrite concretion content was determined by using the photograph area proportion method. Pyrite concretion-bearing Nantuo diamictite and siltstone layer were photographed in field. For each field photograph, pyrite concretion content (n is the total number of analyzed field photographs.The average content of pyrite concretion in each section can be calculated by:The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


ATP1A2 gene, encoding the α2 isoform of the Na+/K+-ATPase, predominantly expressed in astrocytes. Differential comorbidities such as epilepsy and psychiatric disorders manifest in patients. Using a mouse model harboring the G301R disease-mutation in the α2 isoform, we set to unravel whether α2+/G301R mice show an increased susceptibility for epilepsy and cortical spreading depression (CSD). We performed in vivo experiments involving cortical application of KCl in awake head-restrained male and female mice of different age groups (adult and aged). Interestingly, α2+/G301R mice indeed showed an increased susceptibility to both CSD and epileptiform activity, closely replicating symptoms in FHM2 patients harboring the G301R and other FHM2-causing mutations. Additionally, this epileptiform activity was superimposed on CSDs. The age-related alteration towards CSD indicates the influence of female sex hormones on migraine pathophysiology. Therefore, the FHM2, α2+/G301R mouse model can be utilized to broaden our understanding of generalized epilepsy and comorbidity hereof in migraine, and may be utilized toward future selection of possible treatment options for migraine.Migraine is a highly prevalent, debilitating, episodic headache disorder affecting roughly 15% of the population. Familial hemiplegic migraine type 2 (FHM2) is a rare subtype of migraine caused by mutations in the  A migraine attack is characterized by a severe, long lasting headache that often coincides with nausea and neurological signs and can be preceded by transient neurological symptoms; an aura2. Prevalence of migraine is higher in women than men and severity and frequency of headache episodes often decrease after menopause in women or with age in general3.Migraine is a highly prevalent, debilitating, episodic headache disorder affecting roughly 15% of the population4. In most cases, FHM2 is caused by a loss of function mutation in the ATP1A2 gene encoding the α2-isoform of the Na+/K+-ATPase, predominantly found in astrocytes6. The Na+/K+-ATPase is a pump that actively transports potassium into, and sodium out of, a cell to maintain stable transmembrane ionic gradients7. FHM2 related mutations in the ATP1A2 gene cause haploinsufficiency and result in diminished astrocyte function, rendering them insufficiently capable of clearing excess K+ from the extracellular space, and excess glutamate from the synaptic cleft9.A rare subtype of this disorder is familial hemiplegic migraine type 2 (FHM2), an autosomal dominant form of migraine characterized by the occurrence of an aura with hemiparesis (one-sided motor weakness) that shows a high degree of comorbidity with epilepsy11. This specific mutation causes a particularly severe phenotype with comorbidity of migraine with epilepsy, coma, motor symptoms and psychiatric disorders like depression and obsessive compulsive disorder (OCD)11. Based on this mutation, a new mouse model for FHM2 was generated and characterized12. Initial phenotypical investigation showed hypo-locomotion, OCD-like behavior and stress-induced depression. The phenotypes were generally more severe in females and could be rescued by blocking female sex hormones12.Two Italian families with several members suffering from FHM2 caused by the G301R mutation have been described15. Although the phenomenon has been described as early as 194414, mechanisms underlying spontaneous initiation and propagation of CSDs, and the relative contribution of glia and neurons, remain incompletely understood17. Several more recent studies have implicated CSD in causing the subsequent headache through activation of the trigeminal system22. Susceptibility to CSD, defined as a combination of CSD frequency and propagating speed, has been used to determine the validity of previously created FHM1 mouse models23. One mouse model harboring the human FHM2-related W887R mutation has been generated24 and α2+/W887R mice showed a reduced induction threshold for CSD using electrical stimulation in anesthetized animals25. Despite several studies, a deep understanding of the neurophysiology associated with CSD and migraine remains to be achieved in experiments in an awake setting to be able to verify and confirm observations from in vitro studies and anesthetized settings as well as to allow for assessment of epilepsy susceptibility.The neurobiological process underlying the migraine aura is thought to be cortical spreading depression (CSD); a wave of initial neuronal and glial depolarization followed by prolonged depression that slowly propagates over the cerebral cortex2+/G301R mice) shows an increased susceptibility to CSD and/or a decreased threshold for epileptiform activity. To address this, we performed experiments involving occipital application of KCl in awake, head-restrained heterozygous α2+/G301R mice and wild type littermates to determine differences in CSD susceptibility. Interestingly, in addition to generating CSDs, these experiments consistently resulted in epileptiform activity in α2+/G301R mice. About half of the α2+/G301R mice showed continuous epileptiform activity that was superimposed on CSDs.Here, we set to unravel whether a mouse model of FHM2 harboring the G301R mutation . Electrocorticographical (ECoG) recordings were made bilaterally from M1 and unilaterally from S1 (left side) while a small craniotomy over the left occipital lobe allowed for application of KCl  and their wild type littermates . Since migraine is more prevalent in women than men1, and often improves after menopause3 or with age in general26, we compared both genders in two different age groups (2–8 months and 14–20 months).We first investigated the susceptibility of αKCl Fig. . After s2+/G301R mice showed an increased CSD frequency as compared to their wild type littermates  = 2.08, p = 0.556; Kruskal-Wallis test), we did find significant differences between the groups of α2+/G301R mice (H(3) = 19.91, p < 0.001; Kruskal-Wallis test). More specifically, the post-menopausal females showed a significantly reduced CSD frequency as compared to males (t(16) = 13.22, p < 0.05), females (t(16) = 21.72, p < 0.001) and aged males (t(16) = 13.28, p < 0.05)  = 5.48, p = 0.140; Kruskal-Wallis test) or wild type groups (H(3) = 0.63, p = 0.890; Kruskal-Wallis test; Fig. 2+/G301R mice, indeed show a significantly increased susceptibility to cortical spreading depression and remarkably, this susceptibility decreases after menopause in females but not with age in males, suggesting that female hormones influence migraine pathophysiology.For propagating speed, calculated by dividing the distance between the two electrodes on the left hemisphere (2 mm) by the time difference in CSD onset, we found a similar pattern of differences between α2+/G301R mice and some wild type littermates showed various degrees of epileptiform activity during CSD experiments  = 20.16, p < 0.001; Kruskal-Wallis test; Fig. 2+/G301R males (t(17) = −19.94, p < 0.05), females (t(17) = −24.61, p ≤ 0.001) and aged males (t(17) = −20.17, p < 0.05) as compared to wild types, whereas the α2+/G301R post-menopausal females did not differ from either the other α2+/G301R groups or the wild types indicating that susceptibility to epileptiform activity too may decrease after menopause  = 3.18, p ≤ 0.001; linear regression; Fig. t(35) = −5.24, p < 0.001; linear regression; Fig. t(35) = −3.84, p ≤ 0.001; linear regression; Fig. 2+/G301R mice, likely related to increases in extracellular potassium concentration.Because both epileptiform activity and CSD can be caused by a high extracellular potassium concentration2+/G301R mice. Investigating raw data on a magnified time scale revealed differences in shape and timing between the channels  = 4.30, p ≤ 0.001 and t(32) = 3.74, p ≤ 0.001 respectively; paired t-tests; Fig. t(32) = 0.67, p = 0.507). This suggests, as expected, that whereas these correlations were generally quite high (~0.7 on average), epileptiform activity in either two different cortical areas of the same hemisphere or the same cortical area of different hemispheres was better correlated than the activity recorded from S1 and contralateral M1. Analyses of the timing of this peak in the cross-correlogram showed a somewhat different pattern. The epileptiform activity consistently occurs first in the primary motor cortex ipsilateral to the CSD induction site. The time difference between M1 and S1 on the ipsilateral hemisphere is very small and significantly shorter than the time difference between either of those channels and the contralateral M1 electrode (t(32) = −8.16, p ≤ 0.001 and t(32) = −11.09, p ≤ 0.001 respectively for M1 and S1; paired t-tests; Fig. t(32) = 1.58, p = 0.125). These results indicate that the epileptiform activity occurs first in the ipsilateral hemisphere and likely spreads to the contralateral side from there.Further investigation of the spatial and temporal relations between CSD and epileptiform activity, yielded interesting observations. First, whereas a CSD does not spread to the other hemisphere, the epileptiform activity is generalized Fig. . Furtherels Fig.  which waels Fig. . PairwisATP1A2 gene11. Results from in vivo recordings in awake, head-restrained mice, show a substantial increase in CSD susceptibility in α2+/G301R mice that was similar to data obtained from FHM1 mouse models23. Migraine prevalence in humans3, CSD susceptibility in FHM1 mice29, and prevalence of psychiatric and motor abnormalities in α2+/G301R mice12 have been shown to be higher in females and sensitive to blocking the female sex hormone cycle. We therefore assessed CSD susceptibility in adult and aged male and female mice. Whereas FHM1 models show an increased CSD susceptibility in females compared to males23, we do not find such an effect of gender. Interestingly, two of nine female α2+/G301R mice did show a higher CSD frequency than any of the other α2+/G301R mice, suggesting that a gender-based difference may be present in a limited period of the female menstrual cycle. This is in line with our finding that post-menopausal females show a significant decrease in CSD frequency comparable to aged and ovariectomized FHM1 models23 and with the rescue of OCD-like behavior, depressed mood and hypo-locomotion in female α2+/G301R mice upon suppression of the hormonal cycle12. In contrast, aged males did not show a decreased CSD susceptibility, suggesting that the partial rescue of the CSD phenotype in aged females is a consequence of menopause-like hormonal changes rather than age in general. Future pre-clinical studies should be carried out to investigate this phenomenon further, for example by examining whether pharmacological or surgical ablation of sex hormones would also decrease susceptibility to CSD and epileptiform activity in fertile females, and whether this can be reversed by hormone replacement therapy. This could provide a basis for potential clinical trials looking into hormone-based treatment options for women suffering from migraine.Migraine is a disabling disease which affects a large number of people at different levels. A key characteristic of FHM2 is the appearance of the aura phenomenon prior to the onset of the hemiplegic migraine, associated with CSD. Despite several lines of progress, the mechanisms underlying CSD remain poorly understood. In this study, we show that a mouse model for FHM2 harboring the G301R mutation shows an increased susceptibility to both CSD and generalized epileptiform activity in awake animals. These findings mimic symptoms described in FHM2 patients harboring the G301R mutation in the 2+/G301R mice, sometimes with lethal outcome. Interestingly, tonic-clonic seizures have also been described to co-occur with migraine attacks in a family with this mutation11. Additionally, the co-occurrence of epileptiform activity and CSD has been shown in rabbits30 and aneurismal subarachnoid hemorrhage patients31. High extracellular potassium concentrations have been known to result in epileptic discharges since the 1950s35. In addition, both epilepsy and CSD have also been shown to increase extracellular potassium concentrations38. Given that we are using a KCl solution, and that the resulting CSDs raise potassium levels even further, the occurrence of epileptiform discharges is not unexpected. The progression in the severity of the epileptiform activity suggests that extracellular potassium levels indeed increase over time. The dramatic increase in prevalence and severity of epileptiform activity in α2+/G301R mice can be attributed to the impaired functioning of astrocytes resulting in insufficient potassium clearance from the extracellular space40. In combination with a higher CSD frequency, this likely results in higher extracellular potassium levels in α2+/G301R mice compared to wild types, resulting in more epileptiform activity. Another potential contributing factor concerns the dependency of the astrocytic glutamate clearance system on α2Na+/K+−ATPase43. Both astrocytic glutamate transporters, excitatory amino acid transporter 1 and 2 (EAAT1 and EAAT2) have been shown to be co-localized with α2Na+/K+-ATPase44. Glutamate clearance by these transporters depends on the Na+ gradient which relies on α2Na+/K+-ATPase45. Impairments in astrocytic glutamate uptake from the synaptic cleft have been shown in cultured hippocampal cells from α2G301R/G301R. embryonic mice12 and in another FHM2 model39. Increased extracellular glutamate concentration and EAAT2 deficiency have been shown to cause epilepsy47, suggesting a defective glutamate system may also contribute to the increased susceptibility in α2+/G301R mice. Susceptibility to epileptiform activity has also been shown in the S218L FHM1 mouse model; both spontaneous seizures and epileptic activity post CSD elicitation have been observed, yet such an occurrence has not been mentioned during KCl induced CSDs, likely due to the use of anesthesia29. Finally, epileptic seizures also occur during or immediately after migraine aura in patients with this mutation11. The exclusivity of epilepsy occurrence in relation to aura suggests that it is likely that CSD related changes may underlie the epilepsy phenotype in patients as well. Cross-correlation analyses revealed that this epileptiform activity occurs first in M1 ipsilateral to the CSD induction site. It then occurs in S1 and spreads to the contralateral side, suggesting that the epileptiform activity starts in ipsilateral M1and spreads from there. However, it cannot be excluded that the potassium diffuses into other brain areas, like the hippocampus, and that the epileptiform activity is initiated elsewhere. Yet, regardless of where it is initiated, our data clearly show the occurrence of epileptiform activity during CSD.The occurrence of generalized epileptiform activity and coinciding tonic-clonic behavioral manifestations was observed in some wild types and all αin vivo evidence for increased susceptibility to CSD and epileptiform activity in the α2+/G301R mice suggests that this mouse model closely resembles human symptoms and can be utilized to study the neuropathology underlying migraine disorders. Furthermore, the simultaneous occurrence of epileptiform activity and CSD in awake α2+/G301R mice provides a unique opportunity to study the role of neuronal activity in CSD. This could have important implications for the identification of novel therapeutic strategies.In summary, the neuro-electrophysiological 2+/G301R mice and their wild type littermates. The older group of female mice were well past the estimated age of 8 months at which murine menopause occurs48. Genotyping was done by Transnetyx . The presence of the α2+/G301R mutation was confirmed by polymerase chain reaction (PCR) using AGCATTTCATCCAGCTGATCACA (forward) and CCCAGGATGAGGGACAGAAC (reverse) primers. The α2+/G301R mice12 were maintained on a C57BL/6 J background . All animals were housed on a reversed light/dark cycle to prevent daytime experiments from interfering with their normal sleep cycle.All animal experiments were done in accordance with guidelines set, and approved, by IACUC at the Albert Einstein College of Medicine. Data were obtained from 2–8 and 14–20 month old male and female heterozygous α2) and kept under anesthesia on a lower dose (1.5–2% in 1.5 L/min O2). The head was shaved after which an incision was made to expose the skull. After thoroughly cleaning the exposed surface, a dental adhesive  was applied to the area. Five small burr holes (0.5 mm) were very carefully drilled to allow subdural implantation of three recording electrodes and a reference and ground electrode for electrocorticographical (ECoG) recordings. Recording electrodes were bilaterally placed over the primary motor cortex  and unilaterally over the primary sensory cortex  and −1 mm AP, relative to bregma). Reference and ground electrodes were placed over the superior colliculi (±1 mm ML and −1 mm AP relative to lambda). A larger craniotomy (~2 mm in diameter) was subsequently drilled over the occipital lobe to permit application of KCl. Dura was left intact during this procedure; if the dura was damaged, the mouse was excluded. Three PFA coated tungsten wires (50 μm) with a small loop at the end to prevent penetration of the dura, were used as recording electrodes and silver chloride balltip electrodes were used as reference and ground electrodes. The electrodes were cautiously positioned in the holes and secured with Charisma , a light cured composite. When all electrodes were implanted, the connector was lowered onto the skull and secured with Charisma. A small well was made around the craniotomy to hold the tip of a cotton-tipped applicator during experiments and non-toxic silicone  was subsequently used to close it off. Finally, two small 3D printed, low weight cylinders were secured to the pedestal using Charisma to allow for head-restraining using adjustable magnetic stands . Recordings were initiated at least 20 minutes after inserting them in the setup to allow for the effects of the anesthesia to wear off completely. After this waiting period, the silicon covering the craniotomy was removed and after visual inspection of the exposed brain  the experiment was initiated.23. In short: we placed the tip of a small cotton-tipped applicator drenched in KCl solution (300 mM) gently on the dura of the occipital craniotomy to evoke CSDs, and recorded DC ECoG for 1 hour. The cotton-tipped applicator was kept moist throughout the experiment to allow for continuous KCl diffusion. After the hour, the exposed brain was thoroughly flushed with NaCl and the craniotomy was sealed off with silicone again. All mice that showed epileptiform activity during this experiment were anesthetized before being put back in their home cage to stop the pathological activity. Still 4 mice died shortly after a tonic-clonic seizure-like episode. If mice showed extremely epileptiform activity for prolonged periods of time (>20 min), the experiment was aborted prematurely to attempt to prevent death.In order to assess susceptibility to cortical spreading depression, we performed experiments as described previously49.All ECoG data was recorded using a custom made amplifier , digitized  and stored for further analysis. The extremely high input impedance minimized all potential temporal errors with regard to CSD waveformCustom written algorithms  were used for all analyses. All ECoG data was down sampled to 300 Hz and filtered offline . A CSD was identified by the characteristic shifts in DC potential; an initial wave of depolarization followed by pronounced depression. The CSD frequency was determined by counting the number of CSDs per hour and propagating speed was assessed by dividing the distance between the two electrodes (2 mm) by the difference in onset of the wave of depolarization of the first CSD.2+/G301R mice using a custom written LabView program. We used a Matlab node in LabView and applied the standard xcorr (coeff) function to assess the similarity between two vectors as a function of lag. Three out of the 36 mice were excluded because one of the channels was not working properly.Epileptiform activity was identified by the synchronous occurrence of low frequency spikes in the ECoG signal and the coinciding tonic-clonic behavioral correlates. Activity in the contralateral hemisphere was checked in each mouse to assess whether the epileptiform activity was generalized. In each mouse that showed epileptiform activity, it would start in bouts. In a subset of mice, the bouts progressed into continuous epileptiform activity without interruptions. Onset of epileptiform activity was defined as the first moment at which clearly distinguishable spikes occurred in the ECoG. In order to objectively measure the severity of the epileptiform activity, we performed Fast Fourier transforms and calculated the average power at spike frequency (0.5–4 Hz) in the last 10 minutes of a trace and normalized this to the average power at this frequency range in the first 10 minutes. To assess the relation in shape and timing of spikes between channels, cross correlation analyses were performed in α2+/G301R mice and their wild type littermates) were determined using non-parametric Mann Whitney U tests and Kruskal-Wallis tests followed by Dunn’s Multiple Comparison tests with Bonferroni correction in case of a significant result. Predictability of the degree of severity of the epileptiform activity and onset by CSD frequency was tested using linear regression analyses. Differences in maximum cross correlation and timing thereof between the different ECoG channels during epileptiform activity were assessed using paired t-tests. Two tailed testing was used in all cases in which a p-value was considered significant if below 0.05 (α).Due to inequality of variances and because not all variables were normally distributed, statistical differences in CSD frequency, propagating speed and onset of epileptiform activity between the independent groups of mice .


Patients with Borderline Personality Disorder (BPD) feel ostracized even when they are included. This might be due to a biased processing of social participation in BPD. We examined whether patients with BPD also process social overinclusion in a biased manner, i.e., whether they feel ostracized even when the degree of social participation is increased.An EEG-compatible version of Cyberball was used to investigate the effects of inclusion and overinclusion  on perceived ostracism, need threat and P3 amplitude, an EEG indicator for expectancy violation. Twenty-nine patients with BPD, 28 patients with Social Anxiety Disorder (SAD) and 28 healthy controls (HC) participated.The P3 amplitude was enhanced for patients with BPD and SAD compared to HCs independent of condition. Both patient groups reported more perceived ostracism relative to HCs in the inclusion but not in the overinclusion condition. Only patients with BPD reported stronger need threat in both conditions.The EEG results imply that being socially included violates the expectations of patients with BPD, irrespective of the actual degree of social participation. However, when overincluded, patients with BPD no longer feel ostracized. Except for need threat, patients with SAD might show a comparable bias in the processing of social participation as patients with BPD. •In social situations, patients with Borderline Personality Disorder generally expect to be excluded.•This is indicated by their neurophysiological response .•Our data provide preliminary evidence for a similar bias in Social Anxiety Disorder. The CogPrevious research showed a characteristic bias of social information processing in BPD e.g.,  and suppambiguous social situations. Being included is an ambiguous situation and leaves space for biased interpretations. Being excluded and being overincluded, by contrast, mean getting the ball almost never or almost all of the time during the Cyberball game, and cannot be seen as ambiguous. Following this line of thought, we argue that individuals with BPD should process social inclusion in a biased manner, but should show no bias in the processing of social exclusion and overinclusion.According to the Cognitive Theory of Personality Disorders , the afo1.1Results of previous Cyberball studies indicated that patients with BPD process social inclusion in a biased manner: Compared to healthy controls (HCs), they estimated that the co-players tossed the ball less often to them than to the other player , and theThe study by 1.2To our knowledge, only one study has looked at overinclusion in patients with BPD . De PanfTo summarize, there is sound evidence that patients with BPD process social participation in a biased manner. However, most previous studies focused on social inclusion and exclusion . To our The primary aim was to examine whether the biased processing in BPD is specific to the ambiguous situation of social inclusion or whether patients with BPD also process overinclusion in a biased manner. Relying on the Cognitive Theory of Personality Disorders , we hypo22.1Overall, 85 participants took part in the study: 28 HCs, 28 SAD patients and 29 BPD patients. All HCs, 11 BPD patients and 12 SAD patients were recruited via media advertisements. The remaining 18 BPD patients were recruited at the Department of Psychiatry (Charité Berlin) and the remaining 16 SAD patients were recruited at two university outpatient departments in Berlin. Outpatients and HCs were reimbursed for their participation (30 €). The 18 BPD inpatients from the Department of Psychiatry did not receive financial compensation for their participation. The study was approved by the ethics committee of the Freie Universität Berlin (ID 97 II/2016). Participation was voluntary.p > 0.6). Inclusion criteria for all participants were age between 18 and 40 years and the absence of mental retardation, epilepsy or organic brain disease. Exclusion criteria for the patients were any psychotic disorder, current substance abuse/dependency and intake of psychotropic medication within the last 4 weeks .We confirmed DSM-IV diagnoses using the German versions  of the S2.22.2.1not at all to 5 = very much). Results indicated that participants tended to believe the cover story .To manipulate social inclusion and overinclusion, we used the EEG-compatible version  of CyberTo induce inclusion, each player received the ball equally often . To induce overinclusion, the participant received the ball in 45% of all throws . The participants were told a cover story that the study was aiming to examine visual mentalization capabilities, and were thus instructed to mentally visualize the ball-tossing procedure throughout the Cyberball game. After completing the experiment, all participants were debriefed.The participant sat in front of a computer screen (7° × 7° at a viewing distance of 140 cm) on which the participant and the putatively connected co-players were displayed. 2.2.2not at all to 5 = very much). Like We assessed need threat via the German version of the NTQ . The NTQMoreover, the NTQ also assesses ostracism intensity, negative mood  and the estimated percentage of ball tosses received , what percentage of the throws was directed to you?”). Ostracism intensity was measured by creating the sum score of two items (“I was ignored” and “I was excluded”), which were answered on the 5-point Likert scale described above (range 2–10).2.3Prior to the lab session, participants completed a web-based battery of questionnaires. At the lab, electrodes were attached and participants completed further questionnaires, specifically the LPS-4 to measure IQ  and the This study was part of a larger project. Here, we only report the part relevant to a bias in the processing of social participation. Results referring to the hypothesis of impaired positivity in SAD will be reported elsewhere.2.4We recorded EEG data at frontal, central and parietal positions with Ag/AgCl electrodes. We embedded the electrodes in an electrode cap  and filled them with electrode cream . Electrodes attached at the earlobes served as the reference . In addition, vertical and horizontal electrooculogram were recorded to control for ocular artifacts (impedance <20 kΩ). EEG data were band-pass filtered online (0.1–200 Hz) and sampled at 500 Hz.“Brain Vision Analyzer” was used to analyze EEG data offline . Two discrete events on the screen were of interest: (1) participant receives the ball (“self”), (2) co-player receives the ball from the other co-player (“others”). For each ball reception event, a trigger was provided and EEG was segmented accordingly (−200 to 600 ms epoch length). These EEG segments were baseline corrected (−150 to 50 ms) and filtered (0.3–30 Hz and 50 Hz). Subsequently, EEG segments with muscular or ocular artifacts as well as high Alpha activity were removed manually. A minimum number of 15 segments per event “self inclusion” and per event “self overinclusion” was defined to ensure the stable averaging of noise. Ten participants  had to be excluded due to an insufficient number of segments, leading to the sample of 85 participants described above. We were not able to consider the events “others” in the analysis because of the reduced number of EEG segments in the overinclusion condition . We matched the number of segments for the event “self overinclusion” to the number of segments for the event “self inclusion” to obtain comparable signal-to-noise ratios.Averages for each participant were calculated, separately for condition , ball possession  and electrode position . Subsequently, grand averages were calculated for the three groups  and the P3 time window (310–390 ms). The time window was determined based on the grand averages of the event-related potentials.2.5group  and condition . Significant effects of the ANOVA were further examined in Bonferroni-corrected post-hoc analyses. Pearson's r was used as a measure of effect size .We used mixed ANOVAs to examine group differences in the processing of social inclusion and overinclusion. Dependent variables were a) need threat, b) ostracism intensity, c) estimated percentage of ball tosses received and d) P3 amplitudes. Independent variables were Analyses were conducted using R version 3.4.0  and an a33.1χ2(2) = 199.58, p < 0.001). A contrast analysis revealed that, as expected, the P3 amplitude was more pronounced at the parietal compared to the frontal/central position (t(338) = 13.82 p < 0.001, r = 0.60). Means and SDs of the P3 amplitude at all electrode positions can be found in In a pre-analysis, we checked the effect of the electrode position on the P3 amplitude . The clinical groups did not differ in their P3 amplitude .Mean amplitudes of the parietal position indicated that the P3 was more pronounced in the inclusion compared to the overinclusion condition and that SAD patients and BPD patients showed a more pronounced P3 amplitude than HCs see . Both ma3.2p = 0.001, r = 0.38; HC vs. BPD: p = 0.006, r = 0.33). SAD patients and BPD patients did not differ in ostracism intensity in the inclusion condition . In the overinclusion condition, the three groups did not differ in their reported ostracism intensity .The interaction effect of condition and group on ostracism intensity was significant see . Bonferrp = 0.01, r = 0.32; HC vs. BPD: p = 0.04, r = 0.27; SAD vs. BPD: p = 1, r = 0.06). In the overinclusion condition, the groups did not differ in terms of negative mood .The interaction effect of condition and group on negative mood was significant see . The Bonp < 0.001, r = 0.47; HC vs. BPD: p < 0.001, r = 0.45; SAD vs. BPD: p = 1, r = 0.03). In the overinclusion condition, only BPD patients reported higher need threat than HCs .The interaction effect of condition and group on need threat was significant see . Post-hop > 0.17).Participants estimated having received the ball more often in the overinclusion than in the inclusion condition see . Thus, o3.3group .Finally, we examined whether we can replicate the results of χ2(2) = 116.65, p < 0.001). As expected, the contrast analysis revealed that the P3 was more pronounced at the parietal compared to the frontal/central position (t(168) = 10.48, p < 0.001, r = 0.63).First, we checked the effect of the electrode position on the P3 amplitude in the inclusion condition (SD). The one-way ANOVA showed that group had a significant effect on the P3 amplitude in the inclusion condition (χ2(2) = 7.25, p = 0.03). Bonferroni-corrected post-hoc analyses revealed that the P3 amplitude was only significantly more pronounced in BPD patients compared to HCs . Differences in the P3 amplitude between SAD patients and HC and differences between SAD patients and BPD patients were not significant .In the inclusion condition, the P3 amplitude was more pronounced in BPD patients compared to HCs  social inclusion in a biased manner. When included, patients with BPD reported more negative mood as well as ostracism and experienced more need threat compared to healthy participants.As part of the study, we replicated the finding of and when excluded. Moreover, this finding corresponds with the negative thinking patterns in BPD  to the current situation . Hence, the P3 amplitude might be a possibility to measure the persistence of expectation, which seems to be a core feature of mental disorders . It shouIn contrast to the results of Patients with SAD reported more need threat, ostracism intensity and negative mood compared to HCs in the inclusion but not in the overinclusion condition. Moreover, patients with SAD showed a generally enhanced P3 amplitude compared to HCs. Thus, our results further imply that individuals with SAD show deviations in the processing of social participation as well. This extends previous findings that individuals high in social anxiety need longer to recover from social exclusion than individuals low in social anxiety ; and tha4.1Several limitations of the study design need to be mentioned: First, we had no exclusion condition and were thus only able to confirm the results of 4.2This is the first study to examine the processing of social inclusion and overinclusion in BPD and in SAD relying on EEG data. Our study replicated previous findings that individuals with BPD process and experience social inclusion in a biased manner. Moreover, we provided evidence that individuals with BPD are well able to recognize when they are extremely included, and consequently no longer feel ostracized. However, they seem to expect to be excluded and feel a threat to their social needs irrespective of their current degree of social participation.In BPD, these deviations in the processing of social participation may decrease the probability of positive social interactions and may explain interpersonal problems of individuals with this disorder. These results have implications for clinical practice. Psychoeducation is needed to inform BPD patients about the possibility that they may feel excluded even though they were part of a group. BPD patients could be advised to behave in social situations as if they are included, even though they feel rejected . This might enable them to interrupt the vicious cycle of interpersonal problems related to perceived ostracism. Future research should target adaptive processes in social interactive situations that influence the modification of social expectation in individuals with BPD.No external funding was received for this study. We will apply for the Open Access Publication Fund of the Freie Universität Berlin.


Hexagonal boron nitride (hBN) ceramics are expected to have wide applications at high temperatures as both a structural and functional material. However, because of its flake structure and general inertness, it is currently impossible to sinter hBN powder to a dense bulk (with a relative density of above 96%) even at 2000 °C. Here, we report dense bulk hBN with 97.6% theoretical density achieved at a lower preparation temperature (1700 °C) via a self-densifying mechanism without sintering additives. During the sintering process, cubic boron nitride particles incorporated into the hBN flake powders transform into BN onions with a significant volume increase, thus filling in voids among the hBN flakes and highly densifying the hBN bulks. The resulting dense hBN ceramics possess 2–3 times the strength of traditional hBN ceramics. This phase-transition-induced volume expansion strategy could lead to dense sintered compacts with high performance in other ceramic systems. Sintering hexagonal boron nitride until it is more than 96% dense remains a challenge. Here, the authors mix cubic boron nitride particles into hexagonal boron nitride flakes and sinter the combined powders to obtain dense hexagonal boron nitride ceramics with significantly increased strength. However, the generally low density and weak mechanical strengths attainable by current consolidation techniques reduce the applicability of the material. As such, the main challenge with hBN ceramics is to improve the density of hBN ceramics, which seems a challenging task to overcome using conventional consolidation practices.Hexagonal boron nitride (hBN) ceramics are widely-used materials with excellent properties, characterized by its excellent refractory behaviour at high-temperatures, chemical inertness, high lubricity, excellent thermal shock resistance, and favourable dielectric properties2 hybridization within a two-dimensional layer. However, the material also exhibits remarkable ionic character6, resulting in electrons in the π bond that cannot be delocalized. As such, hBN is not a conductor like graphite, where the 2D layers are held together by weak van der Waals forces7, which also allows the layers to easily slide over each other. The special layered structure of hBN makes it difficult for the hBN flakes to stack densely and the strong in-layer covalent bonding results in the sluggish self-diffusion during sintering. All these hinder the densification of the hBN ceramics. Conventional pressureless sintering of hBN powder without any additives results in ceramics of less than 70% relative density10. Therefore, hot-pressing (HP) is typically adopted to prepare dense hBN ceramics, although sintering additives are still required to achieve suitable densities.hBN has a typical layered structure similar to graphite, with an equal number of alternating B and N atoms that are covalently bonded by sp2O3 and a number of other oxides  under an applied pressure of 100 MPa and achieved a relative density of 95.1% at a temperature of 2000 °C. While Zhang’s results are encouraging, the sintering conditions are too harsh to be feasible for the mass production of dense, high-purity dense hBN ceramics. Additionally, while the achieved density is an improvement on past results, significant room for improvement still exists.Recently, Zhang et al.12, we sintered diamond powders at 1600 °C by SPS and obtained graphite onion bulks with superior strength. These graphite onions were derived from nano-diamond particles. This achievement has made us draw upon an alternative route to synthesize high strength hBN (HSHBN) bulk materials using BN onions produced in situ from cubic boron nitride (cBN) as sintering aids and reinforcements.All previous studies seemingly indicated that the upper limit of the relative density of hBN ceramics is approximately 95% and that the preparation of dense, additive-free hBN ceramics is all but an insoluble problem, still, efforts have been made. In our recent research15. When heated to a high temperature above 1400 °C, cBN particles gradually transform into a structure of the BN concentric-shell onion morphology16.cBN, a polymorph of hBN, has very similar crystal structure and lattice constant as diamond and transforms to hBN at approximately 1400 °C under ambient pressure conditions (more information in Supplementary Note −3) than hBN (2.27 g cm−3), the transformation of cBN particles to BN onions during sintering is accompanied by a significant volumetric expansion. The expanding material serves to both fill the voids between larger hBN particles, and subsequently reduce diffusion distances and allows it to assist in the densification process. With this method, we achieve low-temperature sintering of high density and high strength BN bulk materials without any additives. The strategy is efficient and opens up new possibilities for applications in many other material systems.Herein, we report the preparation of pure, dense and high strength hBN ceramics through the sintering of cBN/hBN powder mixtures via SPS Fig. , Table 2θ, correspond to the (002), (100), (101), (102), (004) and (110) reflections of hBN, respectively17. No diffraction peaks corresponding to cBN, located at 43°, 50° and 74° 2θ, can be detected, indicating that all of the starting cBN has transformed to hBN during sintering.The X-ray diffraction (XRD) patterns of the raw materials used in this study and the sintered HSHBN bulks with 0–100 wt% cBN sintered at 1700 °C for 5 min are shown in Fig. 5. This preferred orientation can be substantiated by XRD analysis. The anisotropy of hBN ceramics is apparent in its strong (002) reflection  micrographs of different regions of HSHBN-30 are shown in Fig. The inset of Fig. 21 addresses the relationship between the particle size distribution, volume fraction and porosity of mixed powder compacts. Based on this rule, two different cBN powder particle sizes (200 nm and 400 nm) were added a ratio of 1:3 to fill the voids between hBN flakes to achieve a high packing density.The sintering of powders is usually associated with volumetric shrinkage of the compact due to the elimination of voids between particles. To achieve a high packing density and reduce pores in the powder compact, particle size gradation is very important. Smaller particles are added to fill the voids between larger particles to obtain a high packing density. Horsfield filling theory11. When the sintering temperature is increased to 1700 °C, the relative density of the HSHBN-30 correspondingly increases to 97.6%. Supplementary Figure The relative densities of the HSHBN samples are shown in Fig. 22. The initial stage consists of particle rearrangement, followed by volumetric shrinkage. Then, particle neck growth occurs, and densification proceeds via mass transport and atom diffusion. During the third stage, pore elimination and rapid grain growth occur. During SPS, the grain growth typically taking place during the third stage is reduced by the short time interval over which the process is conducted; however, coarsening is not completely inhibited.For traditional powder consolidation, the sintering process is composed of three stages223, two major driving forces contribute to the sintering. One is the enthalpy change of the anatase to rutile transformation, and the enthalpy of anatase-rutile transformation is 6.51 kJ mol−1. The other is the release of surface energy due to grain growth. However, in our present work, no significant grain growth is observed that reduces the surface energy. The enthalpy change (∆trHc-h) of the cBN-hBN phase transition was calculated according to the Kirchhoff’s law. In the case of little change in pressure, ∆trHc-h (1673.15 K) = 15.4867 kJ mol−1, i.e., 15.4867 kJ mol−1 of heat is absorbed at 1400 °C  crystal planes, due to the weak van der Waals forces between (002) planes, while much more energy is required to initiate fracture in the (002) layers of the BN onions in the HSHBN bulks.The nanoindentation test results are shown in Fig. 26, respectively. Uniaxial pressure of 100 MPa was applied perpendicular to force surfaces with a radius of 15 mm on the models to analyse the strain and stress distribution, and the support surfaces are the entire bottom surfaces of the both models.To determine the relative strengths of the BN onions and hBN flakes, modelling and analysis of both morphologies were performed with ANSYS software Fig. . The YouAs shown in Fig. 27. The mechanical properties of HSHBN-30 are nearly double those of the pure hBN sample. The flexural strength of HSHBN-100 is 94.51 MPa, approximately 3 times that of pure hBN ceramics. The onion morphology ensures an intrinsically higher strength relative to layered hBN because it isn’t prone to the easy cleavage that occurs in the latter material. Furthermore, some of the outer layers of the BN onions are observed to be entwined with the layers of the hBN flakes, forming an interlocking structure where the onions serve as a sort of binder.Due to the morphology of the BN onion being composed of multiple curved BN layers, the compressive strength in the direction parallel to the plane and flexural strength in the perpendicular direction increase significantly with the cBN addition, as shown in Fig. σc) with the elastic modulus (E), fracture surface energy (γ), and crack size (c) is described by the relation28:Griffith’s theory of fracture for brittle materials states that the relationship of the fracture strength  and two types of cBN powders  were used as raw materials. Prior to use, the powders were treated for 10 h in a 5 wt% HCl solution to remove B−1.The obtained mixtures were subsequently hot pressed at 1700 °C under an applied pressure of 80 MPa on an SPS apparatus , while HSHBN-30 and pure hBN ceramics were also consolidated at temperatures of 1400 °C, 1500 °C and 1600 °C for 5 min, at a heating rate of 150 °C min31.The sintering shrinkage curves were derived from the curves of the pressure head displacement vs time/temperature from 1000 °C upwards, where the thermal expansion of graphite dies and samples were taken into considerationθ over a scanning range of 15–85° 2θ. Microstructural characterization was performed using FESEM (Hitachi S-4800) and HRTEM (JEOL 200CX) at an accelerating voltage of 200 kV.The Archimedes’ principle density measurements were employed to determine the relative densities of all samples. The phase composition of all samples was determined using XRD (Bruker D8 Advance) with Cu-Kα radiation with a step size of 0.02° 2−1.FTIR spectra were recorded on a Bruker TENSOR27 spectrometer with sample material embedded in KBr disks. Grind the samples thoroughly with an agate mortar. Transfer weighed amounts of sample powder (particle size <10 μm) approximate 1 mg, and KBr (GR) dry powder approximate 200 mg. The two powders were mixed evenly and ground thoroughly while baked with an infrared heat lamp. Transfer the ground mixture into a mold with 10 mm inner diameter and pressurize to 10 MPa, then remove the pressed translucent disk. The spectrometer runs the background channel without a sample, then puts the disk and runs the sample channel. The scanning range is 3000–400 cm−1.Nanoindentation testing was carried out on a Bruker UMT TriboLab nanoindentation device equipped with a Berkovich pyramidal indenter. The three-point bending strength was measured on a universal testing machine , and a crosshead speed of 0.1 mm minThe models of the hBN flake and BN onion were built by SolidWorks software (Supplementary Note Supplementary InformationPeer Review File