Zoothamnium niveum harbors a monolayer of the gammaproteobacteria Cand. Thiobios zoothamnicoli on its outer surface. Cultivation experiments revealed maximal growth and survival under steady flow of high oxygen and low sulfide concentrations. We aimed at directly demonstrating the sulfur-oxidizing, chemoautotrophic nature of the symbionts and at investigating putative carbon transfer from the symbiont to the ciliate host. We performed pulse-chase incubations with 14C- and 13C-labeled bicarbonate under varying environmental conditions. A combination of tissue autoradiography and nanoscale secondary ion mass spectrometry coupled with transmission electron microscopy was used to follow the fate of the radioactive and stable isotopes of carbon, respectively. We show that symbiont cells fix substantial amounts of inorganic carbon in the presence of sulfide, but also (to a lesser degree) in the absence of sulfide by utilizing internally stored sulfur. Isotope labeling patterns point to translocation of organic carbon to the host through both release of these compounds and digestion of symbiont cells. The latter mechanism is also supported by ultracytochemical detection of acid phosphatase in lysosomes and in food vacuoles of ciliate cells. Fluorescence in situ hybridization of freshly collected ciliates revealed that the vast majority of ingested microbial cells were ectosymbionts.The giant colonial ciliate  Symbiodinium in corals [Rimicaris, for which transfer of fixed organic carbon from the ectosymbionts to the host was shown [In many symbiotic mutualisms, microbial symbionts provide benefits to their eukaryote host through nourishment , 2. Two n corals ; upside-n corals ; sulfur-n corals , 13; andn corals –16. Muchas shown .Kentrophoros ciliate [In mutualism, trait loss in the receiving partner may be the result of compensation of trait function by the providing partner , 19. Nou ciliate ). Other  ciliate .Protists frequently form associations with bacteria –28, but Zoothamnium niveum thrives in marine shallow waters on sulfide-emitting decaying plants and animal bones. The host forms a feather-shaped, sessile colony up to 1.5 cm in length. Ciliates are mostly unicellular, but the giant ciliate is a physiologically and functionally integrated, multicellular unit [The giant colonial ciliate lar unit . It is clar unit , 35). Colar unit , 37.Cand. Thiobios zoothamnicoli [−1 of sulfide (access to electron donor) [Except for the lower part of the stalk, all other host parts are covered exclusively by the gammaproteobacterial, thiotrophic ectosymbiont amnicoli , 38, 39.n donor) , 40. Symn donor) ). This sn donor) . Potentin donor) .Although this is the only reported thiotrophic symbiosis that can be cultivated over generations , direct (NanoSIMS) correlated with TEM to investigate the autotrophic behavior of the bacterial symbiont cells and the translocation of organic carbon to the ciliate host from populations collected from wood in the Adriatic Sea. In addition, we studied the in situ host diet using fluorescence in situ hybridization (FISH), and ultracytochemistry. We provide evidence for chemoautotrophy of the ectosymbiont using sulfide in the seawater, as well as internally stored sulfur as electron donor. We show that using bicarbonate in the seawater as a source of inorganic carbon, fixed organic carbon is rapidly released from the ectosymbiont cells to the host. Moreover, we demonstrate that the food vacuoles of the host are mainly filled by symbionts that are phagocytosed.We used a combination of tissue autoradiography (TA) and a newly developed cryo-preparation technique  coupled A detailed version of the materials and methods including the description of the sampling site is provided as −1 NaH14CO3 (DHI®). The following incubations were carried out: (1) 12.2 µmol L−1 ΣH2S (14C sulfidic pulse) and (2) no sulfide (14C oxic pulse) for 25 min each. Aiming at depleting internal sulfur storage compounds in the symbiont cells, five additional colonies were kept in oxic conditions for 24 h prior to incubation in 14C-bicarbonate at oxic conditions for 25 min (24-h oxic + 14C oxic pulse). Two negative controls were performed: (i) colonies were killed with absolute ethanol prior to the sulfidic pulse incubation for 25 min (dead control) and (ii) colonies were incubated in 12.2 µmol L−1 ΣH2S but without adding 14C-bicarbonate for 25 min . To follow the fate of labeled organic carbon, we performed the same sulfidic pulse incubation followed by a chase without 14C-bicarbonate in 12.4 µmol L−1 sulfide for 6 h (14C sulfidic pulse chase) (see Five colonies were incubated in normoxic seawater containing 2.5 µCi mL−1 of NaH13CO3 (Sigma-Aldrich®) and 27.1 µmol L−1 ΣH2S for 3 h and two colonies were analyzed in detail (13C sulfidic pulse). Another batch of five colonies was maintained in oxic seawater for 24 h prior to incubation in 13C-bicarbonate under oxic conditions for 3 h each (24-h oxic + 13C oxic pulse). As described above, a dead control and a natural carbon control were prepared  isotope fraction, given in at%). NanoSIMS and TEM images obtained from similar analysis areas were superimposed using the GIMP® software package.One colony per treatment was analyzed with NanoSIMS except for the sulfidic pulse incubation where two replicate colonies were analyzed separately  15.79–25.67; Fig. N = 291 symbiont cells, 13C isotope fraction 2.68 at%, IQR 2.45–3.04; Figs. N = 3 colonies, AGD 0.00, IQR 0.00–0.00; N = 50 symbiont cells, 13C isotope fraction 1.07 at%, IQR 1.06–1.07) and the same results were obtained for living colonies incubated without added bicarbonate , than after the sulfidic pulse  and 13C isotope fractions varied between 2.42 and 4.54 at% (N = 50) and 1.22 and 3.84 at% (N = 241), respectively  is sufficient to fuel an active thiotrophic metabolism maintaining sulfur storage. This is consistent with the experimental optimal conditions in oxic seawater supplemented with 3–33 μmol L−1 sulfide [−1 were recorded from the sunken wood surface colonized by Z. niveum [13C content and sulfur suggests that under sulfidic conditions the more carbon is fixed the more sulfur is also stored . Indeed, precise analyses of individual symbiont cells with NanoSIMS confirmed that 13C enrichment in symbiont cells covering microzooids  was significantly higher than that of cells on stalks  .To investigate whether symbionts located on different parts of the colony incorporate carbon to different degrees, we compared the RGDs of symbiont populations covering the microzooids and the stalk. The RGDs of the mixed symbiont populations on the microzooids  were siOverall, the symbiont cells on microzooids incorporated more labeled carbon than the rods on other colony parts. These results are in agreement with the ciliate behavior creating fine-scale differences in oxygen and sulfide concentrations along the microzooids . The oraRiftia pachyptila [14C-bicarbonate that is short enough to exclude digestion. Minimal time for digestion in ciliates is 30 min, with maxima of up to 5 h [Carbon fixation and release of organic carbon into the surrounding host tissue is virtually concomitant in endosymbionts of chyptila . We therchyptila –63. Somechyptila , 65. Ourp to 5 h , 67.14C-bicarbonate labeling for 25 min resulted in labeled carbon in host tissue after sulfidic  and oxic incubations  and in ubation; ). The RGSymbiodinum cells [N = 5 colonies; RGD 24.90%, IQR 9.99–33.75) and oxic  incubations. These results indicate a stable amount of released carbon correlated to the amount of fixed carbon governed by environmental conditions (which may be more or less favorable for chemosynthesis). Therefore, in contrast to corals, the ciliate host is apparently unable to enhance the release of fixed carbon from the symbiont under less favorable oxic conditions. Similarly, the symbiont apparently is also unable to actively reduce the release. About one order of magnitude lower AGDs in symbionts and host under less favorable oxic conditions compared with more favorable sulfidic conditions are consistent with this interpretation . We conclude that no further major leakage of labeled organic compounds occurred during the chase time. At the same time, however, the host RGD significantly increased 2.6-fold after the 6-h chase (Table The symbiont cells were highly labeled with Phagotrophy in microzooids was already hypothesized based on ultrastructural observations of a fully developed digestive system and symbiont-like bacteria in the cytopharynx and inside digestive vacuoles . In ciliTo unambiguously identify the ectosymbiont within the digestive vacuoles, we sequenced the 16S rRNA gene of the symbiont population from Slovenia. Interestingly, the obtained 16S rRNA gene had a single mismatch in the target region of the FISH probe ZNS196  that wasZoothamnium niveum—Cand. Thiobios zoothamnicoli association is a byproduct mutualism in which the symbiont cells benefit from the host behavior by gaining access to electron donors and acceptors for chemosynthesis and the host benefits from released organic carbon that the symbiont cells produce [Recently we proposed that the  produce . Here we produce  fueled bSupplemental Material


Visual stimuli with emotional content appearing in close temporal proximity either before or after a target stimulus can hinder conscious perceptual processing of the target via an emotional attentional blink (EAB). This occurs for targets that appear after the emotional stimulus (forward EAB) and for those appearing before the emotional stimulus (retroactive EAB). Additionally, the traditional attentional blink (AB) occurs because detection of any target hinders detection of a subsequent target. The present study investigated the relations between these different attentional processes. Rapid sequences of landscape images were presented to thirty-one male participants with occasional landscape targets (rotated images). For the forward EAB, emotional or neutral distractor images of people were presented before the target; for the retroactive EAB, such images were also targets and presented after the landscape target. In the latter case, this design allowed investigation of the AB as well. Erotic and gory images caused more EABs than neutral images, but there were no differential effects on the AB. This pattern is striking because while using different target categories  appears to have eliminated the AB, the retroactive EAB still occurred, offering additional evidence for the power of emotional stimuli over conscious attention. Emotional visual stimuli gain access to conscious attention in a manner which is privileged over non-emotional stimuli, likely because of specialised neural mechanisms promoting detection of stimuli with ‘biological significance’137So great is the capacity of emotional stimuli to enter or hold awareness, that emotionally neutral stimuli that are being actively searched for can go completely undetected if they appear soon after a stimulus with affective contentThe EAB has similarities to the ‘standard’ attentional blink (AB)1091291214after a target stimulus. AndersonEmotional stimuli can also impair target detection if they appear immediately Thus there appear to be at least three situations in which emotional stimuli preferentially capture attention relative to neutral stimuli in a RSVP – forward EAB, retroactive EAB, and detection of a T2 emotional stimulus. A critical research question is whether we can disentangle the mechanisms underlying emotional influences on attention in the AB and EAB. Specifically, do these phenomena reflect a single mechanism, or are they caused by distinct mechanisms? Neurobiological models of emotion and attention have evolved from an initial focus on a thalamic path to the amygdala as the primary mediator of the processing benefits given to emotional visual stimuli17The present study investigated the relative strength of forward and retroactive EABs, and the ability of emotional stimuli to gain access to awareness during the refractory period of the standard AB. In order to try to measure a forward AB, a retroactive EAB, and the ability of emotional stimuli to break through an AB, we designed a novel dual-task RSVP paradigm in which subjects were asked to detect a T1 of a rotated landscape or architectural picture among a stream of upright landscape/architectural pictures, and then to detect a T2 target of persons who could be gory, erotic or neutral (clothed). This RSVP variant differs from a traditional AB paradigm in that the T1 and T2 are not drawn from the same class of stimuli. An immediate question arises as to whether an AB or a retroactive EAB will arise in such a design. If T1 and T2 detection rely on the same capacity-limited attentional resource, then both a traditional AB and a retroactive EAB should be observable. On the other hand, if detection and attentional resources can be committed in parallel, then neither phenomenon should be present. Thus an initial goal of this study was to determine whether AB and retroactive EABs were observable in a dual-task RSVP paradigm.A second question addressed in this study is whether the effects of emotional stimuli from different affective categories produce similar effects in the forward and retroactive EABs or in AB breakthroughs. In our own work, erotic stimuli have demonstrated substantial and robust forward EABs. Indeed, these stimuli have produced larger EABs than gory or threatening stimuliOur final goal was to determine the congruence of emotion effects across the three types of demonstrated emotion effects in AB and EAB paradigms. We reasoned that if these three effects rely on the same mechanisms, then the relative ability of emotional stimuli to produce a forward EAB, a retroactive EAB and to break through the traditional AB should be similar. By contrast, if they rely on independent mechanisms they may be triggered by different stimulus characteristics. Other studies1622To address these questions, we asked participants to complete two RSVP paradigms. The first paradigm was similar to the design of Most and colleaguesThe proportion of trials  in which participants both detected and accurately reported the direction of the rotated landscape target when it appeared at a lag of two and eight stimuli following distractor images of different emotional categories is displayed in 9p < 0.001, η2 = 0.69, in addition to a main effect of lag, F  = 186.53, p < 0.001, η2 = 0.88, and an interaction between distractor category and lag, F  = 75.41, p < 0.001, η2 = 0.74. Planned comparisons using Fisher’s least significant difference (LSD) method revealed significant differences between each stimulus class at lag 2 but not lag 8. At lag 2, attentional blinks following erotic stimuli occurred significantly more often than after gory pictures , and following gory ) and erotic ) stimuli more often than after neutral ones. In contrast, performance at lag 8 was consistently high. There were no significant differences in stimulus detection following the different distractor categories at this lag . At lag 8, across all categories, a mean of 79.12% of stimuli were detected (SE = 1.43).A significant main effect of distractor category was found, F  = 58.26, p = 0.011, η2 = 0.16. There was no main effect of lag, F  = 0.03, p = 0.6, η2 < 0.01 but a significant interaction between lag and T2 stimulus category was found, F  = 5.33, p = 0.008, η2 = 0.17. Planned comparisons using Fisher’s LSD revealed that retroactive blinks occurred more often for both gory ) and erotic ) T2 targets than for neutral T2 targets at lag minus-2. Additionally, at this lag there were more trials in which a T1 target appearing before an identified erotic stimulus was not identified than trials in which a T1 target appearing before an identified gory stimulus was not identified ). As expected, at lag minus-8, no effects of T2 category were found . At this lag, across all categories, a mean of 14.83% of rotated T1 targets were not detected when a T2 person stimulus was (SE = 1.07%). Thus, the dual-target paradigm demonstrates the presence of a retroactive EAB at lag minus-2 that is sensitive to the emotional content of stimuli, with erotic images producing the largest retroactive EAB, similar to erotica’s greater effect in the forward EAB paradigm. Results indicate no difference in the frequency of T2 detection without T1 across both lags.Trials in which the T2 target stimulus containing a person was identified correctly but the rotated landscape T1 target stimulus was not were classified as showing a retroactive EBA. This is represented in p = 0.06, η2 = 0.1; with no main effect of lag, F  = 0.49, p = 0.49, η2 = 0.02; and no significant interaction between lag and T2 stimulus category,  = 1.16, p = 0.32, η2 = 0.04. Across all conditions, 94.26% of person-containing stimuli were identified (SE = 0.48%).To examine the presence of an AB caused by detection of the T1 rotated target, we identified all trials in which a rotated landscape T1 was correctly detected , and then determined whether the T2 was correctly found. Strikingly, there was little evidence of a typical forward AB, in that T2 stimuli were detected at high levels despite appearing after a successfully detected T1. The percentage of T2 stimuli that were successfully detected for all stimulus categories at both lags ranged from 91.4% to 95.5% see . The higp < 0.05). Consequently Spearman’s rho was used in the correlation analyses. Alpha levels for this and the individual differences correlation below were set at α = 0.05 as it was hypothesised that a positive correlation would be found between proportion/number of blinks in all cases.In addition to looking at participant performance at detecting targets, we also investigated the performance of each stimulus in eliciting the different attentional processes. It was assumed that if the retroactive EAB relies on the same processes as the forward EAB, each stimulus would elicit these processes in similar proportions. As a means of determining this, the number of times emotional stimuli caused a forward (single target task/task 1) or retroactive  EAB or a breakthrough of the traditional AB across all subjects was calculated and submitted to correlation analysis.  The picture-based data were not normally distributed . The correlations between the forward EAB, the retroactive EBA and attentional breakthroughs broken down into stimulus category are listed in Descriptive data shows that neutral stimuli caused forward blinks of attention on 17.13% of the lag 2 trials in which they were present (SE = 2.45%). For gory stimuli, this was 35.03% (3.28%) and for erotic stimuli, it was 39.97% of trials (2.88%). The respective numbers for retroactive blinks per stimulus at lag 2 were neutral 12.19% (1.72%), gory 15.43% (2.43%); erotic 20.06% (3.15%) and for breaking through the attentional blink they were neutral 85.34% (2.02%); gory 80.4% (2.51%); and erotic 77.47% (3.79%). The number of times an image caused a forward EAB when it appeared at a lag of two stimuli prior to the target in the single target task was not correlated with the number of times it caused a retroactive EAB when it appeared as a T2 target at a lag of 2 stimuli following the landscape target in the dual target task, r = −0.092, p = 0.649) .In order to further investigate the relationship between the studied attentional processes, correlation analyses were conducted to determine if those participants who were particularly prone to forward EABs were also prone to retroactive EABs or to AB breakthrough by affective stimuli. The Pearson product moment correlation between the proportion of trials in which a participant experienced a forward EAB and the proportion of trials in which they experienced a retroactive EAB was not significant at lag 2  differs from the results of previous research by Anderson14In the dual-target task, it is noteworthy that no “traditional” attentional blink is seen, suggesting that T1 had little impact on T2 processing. The lack of AB effect prevents direct comparison of the processes involved in and manifestation of AB breakthrough versus the retroactive emotional blink in this study. There are many possible reasons for the asymmetry of T1 (AB) and T2 (retroactive EAB) effects in the dual-task RSVP task, reflecting the fact that T1 and T2 stimuli were of different types. In the dual-task RSVP stream, T1 stimuli were similar to the distractor images as both were comprised of landscape and architectural images, which only differed from each other in whether they were upright or rotated. In contrast, the T2 came from an entirely different class of images, and there was never more than one image of a person shown per trial. This may have allowed for some degree of parallel processing. Executive control of attention can allow more than one attentional focus to be maintained in this contextAnother reason for the asymmetry may have been that the questions about the T2 targets required high-level (clothing) details about these stimuli and thus participants were more vigilant for T2 than T1 targets. The emotional T2 stimuli shared low-level characteristics with the rotated T1 stimuli  whereas the neutral T2 stimuli were more vertical and thus more similar to the distractors. Thus, if a T1 identification lapse occurred, participant identification of an emotional (gory or erotic) T2 would be more likely than identification of a neutral T2 because the emotional T2s shared some T1 target criteria . A final possible explanation for the asymmetry of retroactive EABs and traditional ABs is that the T2 targets bore strong biological significanceThe differential effects of stimulus type accord with previous studies that have found that the particular emotional category of a stimulus affects its ability to capture or hold attention15The other research question addressed in this study – that of whether the forward and backward EABs rely on the same mechanism – was investigated by correlating the number of times each stimulus caused a forward blink (across subjects) with the number of times it caused a backward blink. There was no correlation between the two blinks, at either lag or for any of the stimulus categories. Because each stimulus is a static entity which presumably maintained the same arousal valueFurther support for the idea that the forward and retroactive EAB are not based on identical mechanisms comes from the lack of a correlation between susceptibility to the forward EAB and retroactive EAB. If identical mechanisms were employed for each of these processes, it would be predicted that those who are most susceptible to one form of EAB  and T2 targets in the dual-target (Task 2) came from multiple sources. Those with neutral affective content came from the International Affective Picture System (IAPS)Both the single target and dual target RSVP tasks followed a similar structure. In each trial, 17 images were presented for 100 ms each. Fifteen of these images were non-critical distractors . The other two were one each of a rotated landscape target and a stimulus that contained a person. Both tasks contained 6 blocks of 32 trials, making for 192 trials in total per task. In each task, there were 96 “lag 2” or “lag minus-2” and 96 “lag 8” or “lag minus-8” trials.th, 6th or 8th stimulus in the RSVP stream. The rotated landscape target appeared as the 2nd (lag 2) or 8th (lag 8) stimulus subsequent to the critical distractor. After each trial, participants were asked, “Did you see a rotated landscape?” and had to respond with a keypress indicating “yes” or “no”. If they responded with a “yes” keypress, they were then asked the second question, “Which way was it rotated?” and had to respond with a keypress indicating “left” or “right”. The next trial began immediately following their answer. Trials in which a person affirmed that they had seen a landscape but responded with the wrong orientation were classified as trials in which a blink had occurred.In each trial of the single target task , the stith, 6th or 8th stimulus in the RSVP stream and the other appeared 2 (lag minus-2) or 8 (lag minus-8) stimuli subsequent to it. The same stimuli as used in the first task were used in this second task but due to the short presentation time of stimuli, the large number of them, and a break given between the tasks, recognition or repeat exposure are unlikely to have caused significant problems with the data. In this dual target task, the rotated landscape stimulus appeared as the first target (T1) and the stimulus displaying a person appeared as the second target (T2). After each trial the following three questions were asked in sequence, with the third question only appearing if the answer to the second question was “yes”: 1) Which way was the rotated landscape turned?” (keypress “left” or “right”); 2) “Did you see a person?” (keypress “yes” or “no”); 3) “Was the person a) clothed b) naked c) injured?” . Again, trials in which the participant incorrectly identified the orientation of the landscape or the features of the person-containing stimulus were classified as trials in which a target was missed.In the second task, participants had to identify two targets . Like inHow to cite this article: Ní Choisdealbha, A. et al. Reaching back: the relative strength of the retroactive emotional attentional blink. Sci. Rep.7, 43645; doi: 10.1038/srep43645 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Remarkably, a centrosymmetric BiO6 octahedral-breathing distortion induces a sizable spontaneous ferroelectric polarization through Bi3+/Bi5+ charge disproportionation, which stabilizes simultaneously the insulating phase. The continuous attenuation of the Bi3+/Bi5+ disproportionation obtained by applying an external electric field reduces the band gap and RD spin splitting and drives the phase transition from a ferroelectric RD insulator to a paraelectric Dirac semimetal, through a topological Weyl semimetal intermediate state. These findings suggest that Ag2BiO3 is a promising material for spin-orbitonic applications.Controllable metal–insulator transitions (MIT), Rashba–Dresselhaus (RD) spin splitting, and Weyl semimetals are promising schemes for realizing processing devices. Complex oxides are a desirable materials platform for such devices, as they host delicate and tunable charge, spin, orbital, and lattice degrees of freedoms. Here, using first-principles calculations and symmetry analysis, we identify an electric-field tunable MIT, RD effect, and Weyl semimetal in a known, charge-ordered, and polar relativistic oxide Ag 2BiO3 can be tuned between ferroelectric and different topological semimetallic states using electric fields at room temperature.Many complex oxides combine multiple functionalities that can be manipulated by external fields, providing opportunities for creating devices. Here, He et al. predict that Ag For instance, metal-to-insulator transitions (MIT) driven by the application of an electric field represent a viable way for designing energy-efficient logic devices5. Ferroelectric compounds, whose spontaneous electric polarization can be reversed by the application of an external electric field, are among the most promising materials to achieve this type of switch6, owing to the many different driving forces enabling ferroelectric behavior, including cooperative interactions of lattice distortions, charge, spin, and orbital ordering10. Among the different types of MIT observed in nature11 , the Peierls MIT, associated with a crystal lattice distortion, frequently exhibits a higher transition temperature . Although Peierls-like MITs are ubiquitous in oxides, the breathing mode involving cation-oxygen bond length disproportionation usually does not couple with ferroelectric displacements21.Cross-controlling of order parameters by external stimuli in a single-phase material is of great interest in both fundamental research and technological applications24. The RD spin splitting is a consequence of lifting the spin degeneracy, typically occurring in materials with strong spin–orbit coupling (SOC) lacking inversion symmetry27. The most interesting outcome of the RD effect is that an electron moving under an electric field (E) or a gradient of the crystal potential (E = −∇V) behaves as it experiences an effective magnetic field Beff ∞ E × p/mc2, where p, m, and c are momentum, mass, and speed of light, respectively, that couples to the spin of the electron28. Owing to its promising applications in spin-orbitronics devices such as the Datta–Das spin field-effect transistor29, the RD effect has gained growing research attention32. The tunability of the RD splitting by applying an external field is primary possible due to the dependence of Beff on the crystal potential gradient, which is strongly affected by crystal structure distortions33.Recent studies have shown that an external electric field could also be employed to switch the Rashba–Dresselhaus (RD) spin splitting in ferroelectric materials34. Weyl semimetals are the realization of Weyl fermions in condensed matter systems and exhibit many exotic properties35. Since the Weyl semimetal typically only exists in crystals without either time-reversal or inversion symmetry, but not both, it is also possible to activate a Weyl semimetal phase in a topologically trivial material by applying an external field. Although the magnetic field induced Weyl semimetal has been discovered in GdPtBi recently36, the electric field promoted Weyl semimetal has not been reported yet.Additionally, Weyl semimetals are a topological phase whose low energy excitations are the massless chiral fermions2BiO3. We identify an atypical polar structural distortion that arises from the octahedral-breathing mode associated with Bi3+/Bi5+ charge disproportion. This mechanism enables an unexpected route for tuning the MIT, RD spin splitting, and Weyl semimetallic state simultaneously by applying an external electric field. The modulation of the charge disproportion guides the transition from a polar insulating phase exhibiting RD spin splitting to a nonpolar spin-degenerate Dirac semimetallic state. Remarkably, we find that across the MIT transition there exists an intermediate topological Weyl semimetallic state, manifested by a non-degenerate band crossing around the Fermi level and non-trivial surface states connecting Weyl nodes with opposite chirality.Here, we explore the possibility of simultaneously controlling the MIT, RD spin splitting, and Weyl fermions in a single-phase ferroelectric oxide using an electric field by means of first-principles calculations and symmetry analysis. We demonstrate that this paradigm can be realized in the room temperature phase of the known Peierls-like semiconductor Ag2BiO3 crystallizes in a polar structure with Pnn2 (No. 34) space group37 . There is no further observed phase transition down to 2 K37. In the Pnn2 phase, each octahedra shares an edge  and a corner  characterized by different Bi3+/Bi5+–O bond lengths of 2.34 and 2.13 Å, respectively37. This charge-ordered pattern is responsible for opening a band gap (0.7 eV38), which has been observed in other Bi oxides40, and for the onset of ferroelectric behavior, which is observed for the first time. Our density functional theory (DFT) calculations find that the fully relaxed low-temperature Pn phase is nearly degenerate with the Pnn2 phase in energy, and correctly reproduce the bond length disproportionation, see Supplementary Tables 3+ and the unoccupied Bi5+ 6s orbitals hybridized with O-2p states at the R point k =   Agp37 Fig. . At 220 e  and a/2) Fig. . The slie  and aE↑ (k) = E↓ (−k)) are observed at the conduction band minimum and valence band maximum. Owing to the strong SOC of the Bi cation, both the lowest conduction band and the highest valence band split into two branches, forming inner and outer bands with opposite spin rotation patterns  is larger at the lowest conduction band and exhibits strong anisotropy, manifested by a larger ΔERD in the R–T kx direction (6.4 meV) compared to that in the R–U ky direction (0.7 meV). Therefore, a combined Rashba and Dresselhaus spin splitting is expected, similar to the case of the formamidinium tin iodide perovskite FASnI3 and consistent with a Cv2 symmetry, in which both Rashba and Dresselhaus spin splitting are symmetry allowed33. The dispersion relation of the resulting electronic states is given, to linear order, by the coupling Hamiltonian HRD = αR(kyσx−kxσy) + αD(−kxσx + kyσy), with αD > αR, is consistent with the calculated spin orientations showing also parallel components to the associated crystal momentum. The ground state band structure of the Pn phase is also insulating and exhibits very similar dispersion  to ferroelectric (Pnn2) transition, which produces a spontaneous polarization (Pz) of 8.87 μC cm−2  of Ag2BiO3 are comparable with known ferroelectric materials , about 0.08 eV above the Fermi level  surface electron structure by the Green’s function based tight-binding method based on the maximally localized Wannier functions2BiO2, indicating a potentially tunable MIT and spin splitting by applying an electric field. Since the energy difference between the semimetal paraelectric (Pnna) and insulating-ferroelectric (Pnn2) phases is only 5.3 meV per atom and the polarization is relatively large (8.87 μC cm−2), it is expected that the insulating phase could be suppressed (but not passed) by a relative small electric field (E) applied opposite to the direction of the spontaneous polarization (P) through the interaction P ⋅ E in the free energy. This is depicted in Fig. 56. If the frequency of the electric field pulse is carefully chosen, there should be an oscillation between the Pnn2 and Pnna phases. By increasing the strength of the electric field, the insulating phase of Ag2BiO3 becomes more conducting, as shown by the density of states provided in Fig. 57. If the applied pulse electric field is strong enough, however, the Bi3+/Bi5+ charge-order can be melted and converted into a metallic phase, a phenomena called colossal ER, which has been observed in charge-ordered insulators with strongly competing metallic phases, Pr0.7Ca0.3MnO3, R0.5Ca0.5MnO3 2, and LuFe2O46.We have established that two pairs of distinct properties, insulator/metal and RD/non-RD, through a Weyl semimetal state, are closely connected with the ferroelectric structure distortion in Ag2BiO3 by means of first-principles calculations. The functionalized electric-field tunable MIT could make Ag2BiO3 a suitable material for lower power memory applications compared to magnetic field controlled MITs. Moreover, the semiconducting nature of Ag2BiO3 could allow for the injection of a reasonable number of carriers (if the Fermi level is properly tuned) without suppressing the ferroelectric instability, eventually resulting in electrically controllable spin polarized currents as a consequence of the Rashba–Dresselhaus effect58. The higher stability of oxides in atmospheric conditions and the higher ferroelectric Curie temperature of Ag2BiO3 over organic–inorganic metal halide perovskites and GeTe, the prototypical ferroelectric Rashba semiconductors33, make this bismuthate more practical for realistic applications59. Finally, the electric-field tunable Weyl semimetallic state proposed here will enable future studies on intertwined ferroelectric and topological phenomena.We have demonstrated a controllable metal–insulator transition, Rashba–Dresselhaus spin splitting effect, and Weyl semimetallic state by applying an electric field in the experimentally available charge-ordered oxide Ag61 as implemented in the Vienna ab intio Simulation Package (VASP)63. PBEsol exchange-correlation functional64 and the plane-wave basis set with energy cutoff of 520 eV were used. The Monkhorst-Pack k-points grids of 10×10×6, was used to sample the Brillouin zones. All the crystal structures were fully relaxed until the Hellmann–Feynman foreces acting on each atom were less than 0.01 eV Å−1. The phonon dispersion was calculated by using finite displacement method as implemented in the Phonopy code65. Spin orbital coupling is included in all the electronic structures calculations. The symmetry analysis was conducted using the PSEUDO program provided by the Bilbao crystallographic server66. The spontaneous ferroelectric polarization was calculated by using Born effective charges (Z*) of the ferroelectric phase (Pnn2) and the structure distortion (u) of ferroelectric phase with respect to reference paraelectric phase (Pnna) as Ω and e are the volume of unit cell and elementary charge, respectively. The spin texture of the lowest conduction band have been computed by plotting, for each momentum vector on the kx–ky plane, the expectation values of the Pauli σ-matrices onto the Kohn–Sham wavefunctions, i.e., the vectorial quantity Si = i = x,y,z and n referring to either the inner or the outer branch of the conduction bands. Represented as a vector, the k-space distribution of the Si resulted in a combined Rashba and Dresselhaus spin-pattern.All the first-principles calculations were conducted by using the projector-augmented wave pseudopotential methed51. The TB model Hamiltonian was constructed by means of maximally localized Wannier functions (MLWFs53) obtained by the the Wannier90 code54 and constructed from Bi 5s and O 6p orbitals by employing VASP2WANNIER9055. The TB parameters were obtained from the MLWFs overlap matrix. Finally, the berry curvatures based on the first-principles Bloch functions provided by VASP following the recipe described in ref. 67. The topological charge of each Weyl point (WP) is defined by the integration of the Berry curvature over a closed surface enclosing that WP, and was computed by employing the Wilson-loop method68.The surface state calculations have been performed using a Green’s function based tight-binding (TB) approachAll data are available from the corresponding authors upon reasonable request. All codes used in this work are either publicly available or available from the authors upon reasonable request.Supplementary InformationPeer Review File


The structure–property relationship between biomineralized calcium phosphate compounds upon a fluorescent quenching–recovery platform and their distinct crystalline structure and surficial functional groups are investigated. A fluorescence-based sensing platform is shown to be viable for the sensing of 8-hydroxy-2-deoxy-guanosine in simulated systems. The structure−property relationships compared to their non-biogenic counterparts of original biomass are explored upon their florescence properties. A fluorescence quenching–recovery platform determination platform was constructed using a classical DNA beacon strategy and used for the sensing of 8-OH-dG in simulated system to achieve a convenient detection strategy via a simple colour differentiation . The ins2.4H2PO4 solution (0.12 g ml−1) and heated at 160°C for 3 to 9 days. For visualization of these surface/substrates, the surface closest to the membrane was marked as ‘inner surface' while the outside was defined as ‘outer surface'. The morphology of all the samples was observed to be within the range of micrometres with both the outer and inner surfaces of the original shells experiencing a series of morphology evolutions during this hydrothermal treatment process, as clearly illustrated within The shells of the biomass were treated as described in the experimental section  to produce the fluorescence sensing platforms. In brief, the shells of the four natural calcium carbonate biomasses: seashell, conch, eggshell and coral were cut into approximately 1 × 1 cm pieces and ultrasonically cleaned. These pieces were then placed into autoclaves containing 80 ml of NHIn the case of calcium phosphate compounds, the Ca : P ratio roughly corresponds to the crystalline phase composite. EDAX characterization was performed in order to investigate the evolution of elemental composition  and the corresponding Ca : P values were calculated and summarized within electronic supplementary material, table S1, which also reflects the distinguished transformation between the outer and inner surfaces. It can be inferred that these evolution morphological differences among these biomass shells were closely related to their distinct organic composition even for the same piece of material.−1 represent the phosphate group (ν3 triply asymmetric stretching mode of the P–O bond) and approximately 963 cm−1 (ν1 symmetric stretching mode of the P–O bond) for both outer and inner surface. Bands at approximately 3300 and 1631 cm−1 are due to O–H stretching and ν2 (H–O–H) bending modes of lattice water molecule. The band at 896–900 cm−1 can be attributed to the P-OH deformation indicating the protonation of the phosphate groups [−1 region indicate the existence of carbonate groups, suggesting their incorporation into the crystal structure, which is possibly due to the absorption of carbon dioxide from the air during the treatment time.In order to characterize the superficial functional groups and crystalline phase composition of all the biomass shells, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray diffraction (XRD) measurements were performed . This ane groups . Additio3) → brushite/monetite(CaHPO4) → hydroxyapatite (Ca5(PO4)3OH) → monetite/whitlockite (CaHPO4), which experiences a nucleation dissolution–recrystallization–self-assembly process, consistent with our previous report [XRD was also used in order to help characterize the material where the difference among these spectroscopy curves could be attributed to the evolution of crystalline composition during a holding/treatment time from 3 to 9 days. Detailed identification of crystalline structural parameters are listed in electronic supplementary material, table S2. It can be observed that the main transform process for all the substrates was calcite/aragonite  have more considerable quenching–recovery capabilities than Ca5(PO4)3OH or other calcium phosphate compounds . The negative response of sea snails is attributed to their different surficial functional groups than the other three samples. From −1 in ATR-FTIR spectra of sea snails are speculated to be due to their fluorescence reflection property in this area. Another likely reason speculated to be associated with the surficial functional groups 3OH or other calcium phosphate compounds. The outer/inner surface of seashells and outer surface of eggshells after 5 days' treatment exhibit distinct expected properties and can be used for fluorescent discrimination sensing of 8-OH-dG. This work forms the basis of extending further HAps application into clinical detection as a biomarker of real DNA damage samples.We have reported a hydrothermal biomineralization strategy for the fluorescent discrimination sensing of 8-OH-dG by quenching–recovery conversion. We have demonstrated that the appropriate crystalline structure and surficial functional groups are key factors for constructing a platform with fluorescent quenching–recovery capabilities by investigating the duration time and composition of biomass shells. It is revealed that the as-prepared substrates mainly consisting of CaHPO


Surface functional groups constitute major electroactive components in pyrogenic carbon. However, the electrochemical properties of pyrogenic carbon matrices and the kinetic preference of functional groups or carbon matrices for electron transfer remain unknown. Here we show that environmentally relevant pyrogenic carbon with average H/C and O/C ratios of less than 0.35 and 0.09 can directly transfer electrons more than three times faster than the charging and discharging cycles of surface functional groups and have a 1.5 V potential range for biogeochemical reactions that invoke electron transfer processes. Surface functional groups contribute to the overall electron flux of pyrogenic carbon to a lesser extent with greater pyrolysis temperature due to lower charging and discharging capacities, although the charging and discharging kinetics remain unchanged. This study could spur the development of a new generation of biogeochemical electron flux models that focus on the bacteria–carbon–mineral conductive network. Electron transfer reactions govern most biogeochemical processes, yet we have a limited knowledge of the electrochemistry of pyrogenic carbon, a major component of organic matter. Here, the authors quantify electron transfers between pyrogenic carbon and mineral phases under different pyrolysis temperatures. Electron transfer reactions are the basis of biogeochemical cycles in water, soil, and sediment and govern most geochemical and biochemical transformations132641316−1 was obtained (−1 with molar H/C and O/C ratios of 0.11 and 0.05). Therefore, further increases in electron transfer kinetics will be marginal with lower H/C and O/C ratios at pyrolysis temperatures exceeding 800 °C.Electrochemical tests demonstrated that pyrogenic carbons can transfer electrons by means of direct electron transfer of carbon matrices  and the obtained , which wDue to the inherent inner resistance caused by minerals and non-aromatic carbon structures, it was not possible to precisely calculate the rate constant of pyrogenic carbon between 650 and 725 °C, and the actual values lay in the grey area between estimated upper and lower limits . For pyr−1. The increased absorption ratio of aromatic C–H to aromatic C=C bonds suggested the formation of larger and increasingly condensed aromatic sheets. Nearly all featured vibrations disappeared above a temperature of 700 °C indicating attenuated functionalities. Further carbon k-edge EELS examination of the pyrogenic carbons at fine spatial scale revealed similar trends in which diminished surface functionality occurred with increased pyrolysis temperature, as shown by the lower intensity of transitions between 285 and 292 eV. The 1s-π* transition at around 284.1 eV (expressed as shoulders at a lower energy of the C=C transition peak at 284.8 eV) along with the transition peak at 286.1 eV suggest the existence of olefins2411−1 carbon made at pyrolysis temperatures of 400–700 °C.Reversible charging and discharging current peaks revealed that surface functional groups of pyrogenic carbon can act as both an electron acceptor and donator . Quinone1119−1 for pyrogenic carbon produced at 450 and 550 °C, respectively (−1 (for a two-electron and two-proton transfer process). We attributed the discrepancy  to the impaired pH buffering on pyrogenic carbon in the microenvironment protected by Nafion than in a theoretical Nafion-free environment.The charging and discharging capacities increased from 400 to 550 °C  and fully buffered  solutions  was reported34353The estimated rate constants of benzoquinone were in the range of direct electron transfer by carbon matrices produced at 600–700 °C . A widerin situ examination of the carbon surfaces through which the electron transfer occurs. G peak in the Raman spectra  due to a combination of slow electron transfer kinetics and a distribution of reduction potentials of redox-active moieties2627We argue for the benzo/hydroquinone couple as the most likely model compound because of the correspondence of the peak potentials, identical electron transfer kinetics and spectroscopic consistency with the surface quinone groups of pyrogenic carbon. The benzo/hydroquinone couple was immobilized on a working electrode in a crystalized solid phase  as used 325253It is expected that increasing amounts of pyrogenic carbon will accumulate in the environment due to predicted increases in fire frequency as a result of climate change6]3−, min. 99%), hexa-ammineruthenium(III) chloride ([Ru(NH3)6]3+, 98%), (dimethylaminomethyl)ferrocene , iron(III) chloride (97%), hydroquinone , anthraquinone-2-sulfonic acid , Nafion perfluorinated resin solution (5 wt. %), graphite rod , graphite powder  and Manganese(IV) oxide  were obtained from Sigma-Aldrich . 1,4-benzoquinone  and 1,4-naphthoquinone  were purchased from TCI America and Alfa Aesar, respectively. Strem Chemicals  was the supplier of iron oxide  and iron(III) oxide . The measured Brunauer–Emmett–Teller areas (N2) of manganese(IV) oxide, hematite and magnetite are 108±0.5, 10±0.2 and 7.7±0.1 m2 g−1, respectively. Glassy carbon electrodes (3 mm diameter) and an electrode polishing kit was purchased from CH Instruments . Millipore water (18 MΩ cm) was used to clean all glassware and prepare all aqueous solution.All chemicals were used as received unless otherwise noted. Potassium hexacyanoferrate(III) (3−/4−), hexacyanoferrate ([Fe(CN)6]3−/4−) and octacyanomolybdate ([Mo(CN)8]3−/4−), the second category includes mainly ferrocene and its derivatives+/0, and the third category includes compounds such as trisruthenium ([Ru(bpy)3]3+/2+), hexaammineruthenium ([Ru(NH3)6]3+/2+) and methyl viologen2+/1+ with an electron transfer rate constant higher than ferrocene compounds but in the same order of magnitude. During preliminary testing of three redox couples with relatively neutral formal potential from each category using the pyrogenic carbon electrode (6]3− had the lowest electron transfer rate constant (0.004 cm s−1 compared to 0.017 and 0.018 cm s−1 for [Ru(NH3)6]3+ and FcDMAM, respectively). The formal potential of [Ru(NH3)6]3+ is too negative (−0.134 V versus Ag/AgCl compared to 0.388 V for FcDMAM and 0.256 V for [Fe(CN)6]3−) for the characterization of pyrogenic carbon matrices and the electron transfer rate constant is slightly lower than that estimated by FcDMAM.The redox species used to describe the electron transfer kinetics of materials can be generally classified into three categories from low to high kineticslectrode , we founk0) were calculated. The electron transfer kinetics of pyrogenic carbon matrices were divided into two classes based on the potential difference between the cathode and anode peaks (ΔEP): quasi-reversible kinetics for pyrogenic carbon pyrolysed at 725–800 °C and irreversible kinetics for pyrogenic carbon pyrolysed at 650–700 °C, which leads to two methods of calculation.EP can be converted into a dimensionless kinetic parameter ψ that is directly proportional to the reciprocal of the square root of scan rate v−1/2. The rate constant can thus be calculated by For pyrogenic carbon with quasi-reversible kinetics, the Nicholson method was used to calculate the rate constantsk0 is given in cm s−1, the slope is obtained from a linear fit of the ψ∼v−1/2 relationship (n is the number of electrons transferred (n=1), F is the Faraday constant and D is the diffusion coefficient of FcDMAM in aqueous media (D=5.1 × 10−6 cm2 s−1).where tionship , n is th|iPC|) and overpotential  and formal potential (E0′)) at different potential scan rates in the Tafel plotsFor pyrogenic carbon with irreversible kinetics, the rate constant was calculated using |iPC|∼(EPC−E0′) (A is the surface area of electrode (0.196 cm2) and C* is the bulk concentration of FcDMAM in solution (3 mM).Here, the intercept is obtained from a linear fit of the relationship lnEPC−E0′) , A is thSlower scan rates were applied with low-temperature pyrogenic carbon electrodes than with high-temperature pyrogenic carbon electrodes to properly capture the redox peaks for the estimation of kinetics. As can be seen from RInner, The inner resistance of pyrogenic carbon matrices RInner,  have beeEPC) was only corrected by the measured current using The interference of inner resistance in pyrogenic carbon with quasi-reversible kinetics was minor, as most of the nonconductive impurities had been removed during the high-temperature pyrolysis processes, and only <10% of the kinetics loss was found without any correction of inner resistance. However, for the pyrogenic carbon matrices with irreversible kinetics, the interference of inner resistance could induce a significant change to the calculation of the rate constants. As given in 6]3− . Back contact was ensured by using a spring-pressed copper rod.The influence of the inner resistance on cyclic voltammograms of pyrogenic carbon with irreversible kinetics can be clearly seen in k0′) of surface quinone groups and benzoquinone were calculated using the Laviron methodα) of 0.5 , the peak potential difference can be converted into a dimensionless kinetic parameter m−1, which is directly proportional to the scan rate v, using From the cyclic voltammograms with immobilized pyrogenic carbon as redox-active species , the chak0′ is in s−1 and the slope is obtained from a linear fit of the m−1∼v relationship −1) of surface quinone groups was calculated by integrating the cathodic peak current  and the reaction time (t) based on the potential scan rate, that is, the Faradaic area, divided by the mass of pyrogenic carbon (m), using The charging and discharging capacity (Ccapacitance) was calculated by The electrochemical capacitance of pyrogenic carbon was determined from the non-Faradaic current response to the potential scan in a supporting electrolyte containing 0.1 M KCl and using pyrogenic carbon as the working electrode . The capCcapacitance is in mF cm−2, icapacitance (A) is the capacitive current determined by re-plotting the cyclic voltammogram to the current–time form as shown in A is the geometric surface area of the pyrogenic carbon working electrode (0.196 cm2), and v is the scan rate (V s−1). Due to the inner resistance and linear current–potential relationship shown in the cyclic voltammogram, the capacitive current at steady state could not be identified on 600 °C pyrogenic carbon, and its capacitance was approximately estimated using the current separation at mid-point potential  spectroscopy was performed using a Vertex 70 FTIR spectrometer  equipped with a deuterated L-alanine-doped triglycine sulfate (DLaTGS) detector. A Pike GladiATR accessory with a single-bounce diamond internal reflection element (IRE) and flow cell  was used for sampling. Complete temperature series of pyrogenic carbon samples were scanned 128 times in the mid-infrared region from 4,000 to 600 cmPyrogenic carbon particles were dispersed onto a Copper TEM grid with a SiO support film. EELS was conducted using a FEI Tecnai F20 STEM/TEM  with a Gatan 865 HR-GIF spectrometer. The microscope was operated at 200 kV in scanning TEM mode with a ∼10 pA probe current. The resolution of the EELS spectra was 1.1 eV. Detailed peak assignments of FTIR and EELS spectra can be found in D peak and G peak over the area mapped using ImageJ CSIRaman spectral mapping of pyrogenic carbon matrices was carried out using a Renishaw InVia confocal Raman microscope with a 532 nm wavelength laser source. Samples of pyrogenic carbon at different pyrolysis temperatures were finely polished with diamond polishing paper for Raman spatial mapping . The ageing simulated here generated molar O:C ratios2O2 for oxidation because except for H2O all other chemical species may potentially interfere with further electrochemical tests performed on pyrogenic carbon after the oxidation treatment. It can be seen from The ageing process was simulated by oxidation of pyrogenic carbon using hydrogen peroxide .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, and Supplementary References


Among MCI due to AD subjects, the mean worsening in cognitive score was significantly greater in females (11.58 ± 14) than in males . Our findings highlight the need to further investigate these findings in other populations and develop sex specific timelines for Alzheimer’s disease progression.Sex differences in Alzheimer’s disease (AD) biology and progression are not yet fully characterized. The goal of this study is to examine the effect of sex on cognitive progression in subjects with high likelihood of mild cognitive impairment (MCI) due to Alzheimer’s and followed up to 10 years in the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Cerebrospinal fluid total-tau and amyloid-beta (Aβ42) ratio values were used to sub-classify 559 MCI subjects  as having “high” or “low” likelihood for MCI due to Alzheimer’s. Data were analyzed using mixed-effects models incorporating all follow-ups. The worsening from baseline in Alzheimer’s Disease Assessment Scale-Cognitive score  (9 ± 12) in subjects with high likelihood of MCI due to Alzheimer’s was markedly greater than that in subjects with low likelihood (1 ± 6,  The high failure rate of clinical drug trials in Alzheimer’s disease (AD) over the past decade3 has increased the urgency to better dissect the heterogeneity of AD4 in order to facilitate more personalized therapies. Females have been noted to be at the epicenter of the AD epidemic due to the fact that they account for roughly two-thirds of AD patients in the US and also the majority of caregivers6. However, despite substantial research investment in AD over decades, the biological role of sex in the neurodegenerative process has been relatively understudied. Laboratory research into AD mechanisms is largely done on male rodents - mirroring the sex bias that exists in many areas of biomedical research where findings from male animals are viewed as generalizable to humans of both sexes6.Understanding the role of sex in health and disease is a cornerstone of personalized medicine23. In addition to lifespan differences, there are also well known sex differences in other possible AD risk factors such as in genetics, sex hormone changes in midlife, cognitive reserve, and age of onset of comorbid cardio metabolic diseases (reviewed in6) whose interactive effects remain poorly studied. Emerging evidence suggests that female sex may be linked to a greater effect of apolipoprotein ε4 allele (APOE ε4) on amyloid pathology and dementia risk as well as a faster rate of cognitive decline after onset of mild cognitive impairment (MCI) or AD17. In contrast, other studies note that men may have faster verbal memory decline in normal aging17, an earlier onset of cardiovascular disease, greater risk for cerebral micro-hemorrhage10 and a higher risk for incident MCI18. Initial studies of pathological (e.g. beta-amyloid and tau measurements) and neuronal loss  biomarkers has also suggested there may be sex differences in the evolution of AD pathophysiology23, reviewed in1 and6. These findings, while preliminary, raise the possibility of multiple points of interaction between sex and AD progression.The higher prevalence of AD in females was largely assumed to be due to their longer life spans compared to men but recent studies are beginning to paint a more complex picturewww.adni-info.org), conducted at sites in the US and Canada, has provided new insights into the timeline of evolution of AD biomarkers26. New NIA-AA recommendations for defining “MCI due to AD – high likelihood”27, which require positive pathological  and/or neuronal loss  biomarkers in addition to clinical criteria, were, in part, based on MCI data from ADNI. However, these data have not yet been fully examined to study potential sex differences in the progression of subjects with MCI due to AD.The Alzheimer’s Disease Neuroimaging Initiative (ADNI), a multicenter, prospective, naturalistic study  (adni.loni.ucla.edu). ADNI was launched in 2003 as a large-scale public-private partnership with a primary goal to investigate whether the integration of clinical assessments, serial imaging studies, and other biological markers can be used to discover early signs of Alzheimer’s Disease (AD) progression. ADNI  involved over 60 sites across the United States and Canada. ADNI-1 recruited approximately 400 MCI subjects and followed them up to 5 years. These subjects could then choose to continue in ADNI-2 and hence had total follow up of up to 10 years. ADNI-2 recruited approximately 150 new MCI subjects and followed them up to 5 years. Details of protocols and methods can be found in the procedures manual .MCI subjects were monitored in both ADNI-1 and ADNI-2 at 12 month intervals for up to 5 years. In addition, ADNI-1 MCI subjects could be followed for an additional 5 years if they chose to continue into ADNI-2 thus had a maximum possible follow up of 10 years. At each annual follow visit, subjects underwent cognitive assessments [We pooled MCI subjects from ADNI-1 and ADNI-2 studies. Sex-differences in baseline demographic and cognitive variables were tested using either analysis of variance (ANOVA) or analysis of covariance (ANCOVA).APOE ε4 allele status as follows:Next we ran three mixed-effect models to examine the effect of sex on change from baseline in ADAS-Cog11. The first model adjusted for age, education, baseline ADAS-Cog11, and APOE ε4- male with 16 years of education and an ADAS-Cog11 score of 11. We included both random slope and random intercept of each subject in this mixed effect model to account for subject-specific variability in each baseline dependent variable and rate of change, respectively, as reported previously14. Square root transformations were used for all dependent variables in all models to obtain approximate normality of estimated error distribution and homoscedasticity (constant variance) of the errors across fitted values of each dependent variable. In the models, APOE ε4 status was treated as a categorical variable while age, education, cognitive scores were treated as continuous variables. In the equation, APOE ε4 ++ indicates carriers of two APOE ε4 alleles (homozygous) while APOE ε4 + indicates carriers of one APOE ε4 allele (heterozygous). We also ran a second mixed effect model replacing APOE ε4 with biomarkers  as covariates. Next, we ran a mixed effects model in subjects with high likelihood of MCI due to AD selected two ways. The first was based on high t-tau/Aβ42 ratio. In this model, APOE ε4 status was not included as a term in this last model due to its collinearity with Aβ42. We also ran a mixed effects model in MCI subjects who are APOE ε4 positive. The model terms and covariates are all described in the Tables. Not all analyses had the same sample sizes due to missing values or drop outs. All statistical analyses were conducted in the R (www.r-project.org); mixed-effect models for longitudinal analyses were conducted using the nlme package in the R. All methods were performed in accordance with the relevant guidelines and regulations.In this model, the follow-up time (month) was centered with the median follow-up time and covariates were centered i.e. a 75 years old p = 0.002). There was no statistically significant difference in APOE ε4 carrier status between males and females.Table p = 0.001). The mean change from baseline in ADAS-Cog11 in female APOE ε4 carriers (10 ± 14) and non-carriers (7 ± 10) was greater than in male APOE ε4 carriers (8 ± 11) (p = 0.04) and non-carriers (3 ± 9) (p = 0.003), respectively.Mean follow up duration (months) for males (44.8 ± 30.9) did not significantly differ from that of females (42.4 ± 27.3). The mean (±SD) change from baseline in ADAS-Cog11 in females (8.7 ± 12.6) was greater than in males  with the cognitive decline being greater in females than males. Baseline cognition, education and APOE ε4 status also had a significant effect. Subjects with worse baseline cognition and higher education declined faster. APOE ε4 heterozygotes and homozygotes declined faster, and APOE ε4 had a significant effect on both slope and curvature of ADAS-Cog11 change (compared to non-carriers). The effect of interaction between sex and APOE ε4 on ADAS-Cog11 change was not significant (Supplementary Table Table p = 0.027), baseline CSF Aβ42 (p < 0.001) had a significant effect on ADAS-Cog11 change. Females and subjects with lower CSF Aβ42 declined faster. Aβ42 also had a significant effect on curvature of ADAS-Cog11 change (p < 0.001). The effect of age, education, baseline cognition was not significant. Supplementary Table p < 0.001). Supplementary Table p < 0.0001).Table p < 0.0001). Among subjects with “MCI due to AD – high likelihood”, the mean worsening in ADAS-Cog11 was significantly greater in females (12 ± 14) than in males  . In this model, age, education and baseline cognition did not have a significant effect.As described in Methods, we used t-tau/Aβ42 ratio cut-off to identify subjects with “MCI due to AD – high likelihood”. 70% of all MCI subjects, 73% of females and 67% of males met this criterion for MCI due to AD Table . The meaAPOE ε4 positive MCI subjects (including both heterozygotes and homozygotes). In this model, sex had a near significant effect on ADAS-Cog11 slope (p = 0.05) with the cognitive decline being greater in females than males.Table Supplementary Figures 7. Our study systematically examined sex differences in longitudinal cognitive outcomes using pooled MCI data from two multicenter studies, ADNI-1 and ADNI-2. To our knowledge, this is also the first report to examine sex differences in outcomes of MCI subjects defined using pathological CSF biomarkers to have a high likelihood for MCI due to Alzheimer’s.Understanding the potential underpinnings of sex-related differences in the risk for dementia is an important research priority for the fieldAPOE ε4 has an effect on both slope and curvature of ADAS-Cog11 decline and with both heterozygotes and homozygotes declining faster than non-carriers. Further among MCI APOE ε4 carriers, we found that females declined faster than males. We found no interaction effect between sex and APOE ε4 suggesting these variables may potentially have additive but not multiplicative effects. Lastly, we used CSF biomarkers to identify subjects with “high” or “low” probability of having MCI due to AD. Although not a perfect classifier, the tau/Aβ42 ratio cut-off we used has been validated in a clinic-pathological study25, used in published studies e.g.31, and cited in the NIA-AA guideline report on diagnosing MCI due to AD27. Approximately 70% of the ADNI MCI sample met these surrogate criteria for “MCI due to AD – high likelihood”. MCI due to AD subjects showed a markedly greater cognitive decline  than MCI subjects not meeting these criteria. This further supports the utility of the CSF ratio as a potential prognostic marker for selecting subjects at high risk for decline in clinical trials. Further, among the “MCI due to AD – high likelihood” group, females showed greater cognitive decline than males. This finding extends prior reports of sex differences in MCI23 and to our knowledge is the first study to examine sex differences in subjects with “MCI due to AD – high likelihood”.Several interesting findings emerged from this study. Our longitudinal cognitive analyses of the pooled dataset found that MCI females showed greater cognitive decline than males. Our study also found that 24. ADNI results have, in part, formed the basis for entry criteria in many prevention trials and hence ADNI is a highly relevant dataset. Our analyses tried to mimic the emerging new criteria for MCI due to AD – high likelihood using pathological CSF biomarkers. There is as such no definitive binary marker for neuronal loss and hence we relied on pathological markers. The relatively large sample size and long duration of follow up are other strengths of the analyses. There are also some limitations. ADNI subjects were recruited largely at clinic-based research sites for a biomarker study and as such may not reflect milder subjects seen in general practice, especially in primary care settings. While we relied on a pathologically validated25 tau/Aβ42 ratio cut-off as a surrogate to identify biomarker positive MCI subjects, there is as yet no perfect in-vivo method to identify MCI due to AD. While the diagnostic utility of CSF tau/Aβ42 ratio may differ by setting and laboratory32, it still remains useful for identifying a subset of rapid decliners. While baseline sex differences were seen in both studies, longitudinal sex differences were driven primarily by ADNI-1 data and were not significant in ADNI-2. We do not know why, but one possibility may be that ADNI-1 recruited twice as many MCI subjects as ADNI-2 and ADNI-1 subjects could have a 120 month maximum follow up (versus 60-month maximum follow up in ADNI-2). Although entry criteria were the same for late MCI between ADNI-1 and ADNI-2, we cannot rule out the possibility of selection bias since the two studies were done 5 years apart. Differences in comorbid conditions and concomitant medications between ADNI studies may also have contributed. Since the follow up period was roughly the same between males and females, the observed differences are less likely to be due to attrition or survival biases but we cannot rule them out. We studied sex effects on the ADAS-Cog11 as it is frequently used in clinical trials. However, it is not necessarily perfectly balanced across all cognitive domains and there is some evidence that females may show a slightly different profile (as a group) than males on this test. To rule out potential testing bias, it is important to also examine sex differences on other cognitive and functional domains. Hence, these issues must be kept in mind while interpreting the data and our findings must be viewed as initial pending replication in population samples.There are some strengths and limitations to our study. A major strength of the ADNI study data is that it represents a multicenter biomarker study that recruited subjects from over 60 sites in the US and Canada and performed longitudinal clinical and biomarker assessments using a highly standardized protocolAPOE ε4 allele and the BDNF Met66 allele in females, differences in sex hormones, smaller head size, lower cognitive reserve, as well as the possibility of differential expression of a variety of genes (reviewed in23). The disappearance of sex effects after co-varying for total hippocampal volumes and total tau levels suggest these may be somehow related. However, because AD involves multiple biochemical alterations, systems biology approaches to examine sex differences at a network and pathway level are warranted and may yield deeper insights34. In summary, our findings of sex differences in both baseline biomarkers and cognitive progression in biomarker defined MCI subjects highlight the need to further investigate sex specific biomarker evolution and disease progression in AD.Our study does not directly address mechanisms that may underlie potential sex differences in MCI progression. Theories proposed have included a greater potency of the AD risk associated with the Supplementary Information


Magnetic resonance imaging (MRI) and in particular diffusion-weighted imaging (DWI) have been broadly proven to be the reference imaging method to discriminate between cholesteatoma and noncholesteatomatous middle ear lesions, especially when high tissue specificity is required. The aim of this study is to define a range of apparent diffusion coefficient (ADC) values within which the diagnosis of cholesteatoma is almost certain. The study was retrospectively conducted on a cohort of 124 patients. All patients underwent first- or second-look surgery because primary or secondary acquired cholesteatoma was clinically suspected; they all had preoperative MRI examination 15 days before surgery, including DWI from which the ADC maps were calculated.−6 mm2/s , while for noncholesteatomatous inflammatory lesions, it was 2216,3 × 10−6 mm2/s . Interobserver agreement with Fleiss' Kappa statistics was 0,96. No overlap between two groups' range of values was found and the difference was statistically significant for p < 0.0001. Average ADC value for cholesteatomas was 859,4 × 10 We propose an interval of ADC values that should represent an appropriate benchmark range for a correct differentiation between cholesteatoma and granulation tissue or fibrosis of noncholesteatomatous inflammatory lesions. Middle ear cholesteatoma is an enlarging cystic keratin-filled mass surrounded by stratified squamous epithelium often originating from Prussak space or more generally in epitympanum. The most common classification of cholesteatomas is based on pathogenesis, and it differentiates three types of lesion: congenital 2%), primary acquired (80%), and secondary acquired 18%) [8% [1]. A%, primarHigh resolution computed tomography (HRCT) frequently is the first step in the diagnostic imaging assessment of suspected cholesteatoma because of optimal spatial resolution and high sensitivity for bony erosions; at present, CT is considered a key element in presurgical planning, helpful in depicting precise disease extension , 6. NeveFrom April 2011 to March 2016, we recruited 124 consecutive unrelated patients  with clinical suspicion of unilateral or bilateral middle ear cholesteatoma, candidate to surgical procedure. We included both primary and secondary acquired suspected lesions. In our sample no congenital cholesteatoma was present. All patients underwent preoperative MRI 15 days before first- or second-look surgery. The diagnosis of cholesteatoma was always confirmed histologically. Overall, 15 patients were unable to complete MRI examination, while 9 additional patients were excluded because of their refusal to undergo surgery. Finally, from the original sample, 100 patients  were recruited. Demographic and clinical information of all patients included in the study is shown in b = 0 and b = 800 s/mm2; 5 averages); during Msh-TSE DWI, cardiac gating was performed in order to limit patient-related artefacts due to heart pulse and blood flow. ADC maps were obtained by using the free Osirix plugin “ADC Map Calculation.” For each patient, three different neuroradiologists measured ADC values separately. A circular region of interest (ROI) of 1 mm in diameter was placed in the area where abnormal restricted diffusion was more evident on DWI sequence with the highest b-values (b = 800) by every single observer and then automatically transferred to the coregistered ADC map, as shown in MRI was performed at 1.5 T MR unit  with an 8-channel head coil. The imaging protocol consisted in TSE T2w on the axial plane ; T1w SE on three orthogonal axes before and after intravenous administration of contrast media ; T2 3D DRIVE on the axial plane for the study of inner ear and cerebellopontine angle ; coronal multi-shot non-echo-planar diffusion-weighted imaging (MSH non-EPI DWI). DWI acquisition was performed on the coronal plane . We compared the average ADC values of histologically confirmed cases of cholesteatoma with the ones of histologically identified noncholesteatomatous inflammatory lesions.For final analysis, we considered the arithmetic mean of measured ADC values obtained by the three neuroradiologists. Considering the normal distribution of the collected data assessed by using the Shapiro-Wilk Test for normality, parametric statistical analysis was performed with Student's −6 mm2/s . Average ADC value for noncholesteatomatous inflammatory lesions was 2216,3 × 10−6 mm2/s . No overlapping value between the two groups was found; two outlier values (below Q1 − 1.5 × IQR or above Q3 + 1.5 × IQR) were identified, both in the group of patient with histologically confirmed cholesteatoma and candidate to second-look surgery  primary or secondary acquired cholesteatoma was confirmed histologically, while in 28 cases  postoperative findings indicated the presence of noncholesteatomatous inflammatory tissue. Overall agreement among raters calculated using Fleiss' Kappa statistics was 0,96 . Average ADC value for cholesteatomas was 859,4 × 10 surgery . The difTo our knowledge, this report is the first quantitative assessment of a specific range of ADC values for differentiating between middle ear cholesteatoma and noncholesteatomatous tissue on a relatively large number of patients.Reproducibility and reliability of ADC values in the head and neck region of healthy subjects have been recently assessed, and the importance of MR imaging systems and sequences performed has been pointed out . With thPrevious consensus conferences proposed standardized criteria for ROI placement in diagnosis and response assessment of expansive and tumoral lesions , suggestAs the hyperintensity of cholesteatoma on DWI is a combination of T2 shine-through effect and restricted water diffusion, to confirm the diagnostic suspicion it is always desirable to compare the DWI image to the ADC maps . In case−6 mm2/s) compared to patients without cholesteatoma  (Q1 − 1.5 × IQR or above Q3 + 1.5 × IQR) were identified in the group of patient candidate to second-look surgery and with histologically confirmed cholesteatoma. In both cases, these findings are probably attributable to a small residual/recurrent cholesteatoma surrounded by a large amount of granulation tissue. In fact, DWI and ADC ability to detect small cholesteatomas is limited to lesions bigger than 2-3 mm [In the light of previously described results on smaller samples of patients , 32, 34,6 mm2/s) . The difn 2-3 mm ; therefoσ . For cholesteatoma, after excluding the two above-mentioned outliers, we suggest a reference range between 318 × 10−6 mm2/s and 1265 × 10−6 mm2/s, while for noncholesteatomatous inflammatory lesions we suggest a reference range between 1774 × 10−6 mm2/s and 2658 × 10−6 mm2/s (Given the sufficiently large sample of this study and the distribution of the collected data, we propose a benchmark interval of ADC for both cholesteatoma and noncholesteatomatous inflammatory lesions ranging from ADC ±1,96 standard deviation −6 mm2/s .b-values DW images can be prone to error because of T2-shine-through effect, occasionally affecting the goodness of final result.No significant difference was found stratifying patients by primary and secondary acquired cholesteatoma or by age and sex. For second-look surgery, neither autologous nor heterologous materials have been used in middle ear surgical repair in our sample, in order to prevent scan artefacts that could potentially affect the interpretation of the ADC map in secondary lesions. A further core strength of this work is the large number of highly selected patients recruited over the years compared to the small sample size of previous studies , 32, 34,−6 mm2/s and 1265 × 10−6 mm2/s for cholesteatoma and between 1774 × 10−6 mm2/s and 2658 × 10−6 mm2/s for noncholesteatomatous lesions. The proposed interval of ADC values should represent an appropriate benchmark range for a correct and unambiguous interpretation of what is observed in non-EPI DWI sequences.In conclusion, ADC maps could be helpful in differentiating cholesteatoma from noncholesteatomatous inflammatory lesions, in particular when postoperative residual or recurrent disease is suspected and differential diagnosis is difficult to perform. We suggest a reference range between 318 × 10