Steroid hormones play essential roles in the reproductive biology of vertebrates. Estrogen exercises its effect through estrogen receptors and is not only a female reproductive hormone but acts virtually in all vertebrates, including fish, and is involved in the physiological and pathological states in all males and females. Estrogen has been implicated in mandible conservation and circulatory and central nervous systems as well as the reproductive system. This review intended to understand the structure, function, binding affinities, and activations of estrogens and estrogen receptors and to discuss the understanding of the role of sex steroid hormone estrogen receptors in mammals and fish. Estrogeβ2 genes , 5. Estrβ2 genes –10 In thγ, has been discovered for teleost fish [Estrogen is one of the essential ovarian hormones; it is a pleiotropic hormone and plays an important role in several organs such as the heart, the uterus, and the bone –16. Accoost fish .β-estradiol. An examination of the receptor's mechanism of exploit began ultimately after it was first cloned [β (ERβ) [α (ERα) was the first estrogen receptor that was characterized and used as an indicator for diagnosis and handling of breast cancer [β which is generally referred to as ERβ was discovered in the middle of the 1990s [Estrogen receptors are established in several reproductive target tissues, which include uterus and mammary glands of animals and oviduct and liver of oviparous species. Nevertheless, estrogen receptors may also be present in tissues outside the reproductive system , 20. Estt cloned , 22, 23.[β (ERβ) . The estt cancer , 20, 25.he 1990s . Estrogehe 1990s , 28. Thehe 1990s . Togetheα is mostly expressed in the uterus, liver, kidney, and heart, while ERβ is mainly in the ovaries, lung, gastrointestinal tract, bladder, and hematopoietic and essential nervous system [α, ERβ, and ERγ. The ERγ form in fish appears to be closely related to ERβ which suggests that a gene replication event has transpired within the teleosts. As a result, ERβ and ERγ have been named ERβ1 and ERβ2 [The ERs system , 30. Ests system . In fishand ERβ2 , 5.α and ERβ encrypting genes are situated on several chromosomes  [α and ERβ share 96 percent amino acid uniqueness in the DBD, nearly 53 percent “amino acid identity in the LBD and 30 percent or less in other domains, involved in transactivation and localization. Also as discussed by [α/β heterodimers plays a significant role in estrogen signaling, affecting patterns of gene directive divergent from those controlled by the ER homodimers.” Together, ER full-length mRNAs are encoded by eight exons and are expressed in a diversity of isoforms, owing to alternative splicing [ERctively) , 32, 33,ctively) –36. The ctively) –40. The ctively) , 41, ERαussed by , 28, it splicing , 38. “Esα and ERβ are located in the cell membrane [α-ERβ) or homodimers (ERα-ERα or ERβ-ERβ) [α identified a number of undesirable controllers of its expressions such as too many cell passages or too high cell density, lack of cell cycle synchronization within cell population, and serum starvation [There are several indications which show that both ERmembrane , 43. TheERβ-ERβ) , 43, 44.ERβ-ERβ) . Additioarvation , 47. Alsarvation , 47.α and mERβ, a small number of other proteins were categorized as mER within the past two decades. Among them, the most studied is the G-protein-coupled receptor 30, a rhodopsin-like protein unconnected to steroid nuclear receptors.” In recent times, it has been reported that G-protein-coupled receptor 30 is not only involved in speedy controlling mechanisms, for example, the mobilization of kinase or calcium activation but also play a substantial role in transcriptional activation of genes such as c-fos [α and ERβ, was identified by Almey et al. [α, ERβ, and Gpr30 was 67 kDa, 60 kDa, and 44 kDa, respectively [α than 17β estradiol [In addition to “mERas c-fos , 48. A dy et al.  and was ectively . Additiostradiol . There wstradiol  which arstradiol . The recstradiol .α and mERβ, the association between caveolin-rich light membranes complexes and mERs was also emphasized for ER-X as a way of rapid interaction with the mitogen-activated protein kinase (MAPK) cascade and other signaling pathways.” [α action in human endothelial cells [α and Gβ.” [α only. Therefore, comprehensive research of the mechanisms of estrogen exploit necessitates more broad research in view of the exact membrane and nuclear estrogen receptors and the nonspecific interactions [Although the details of the relationship of estrogen receptors with the membrane remain “unclear, two core interactions are understood to be critical: the palmitoylation of these ERs and their interaction with membrane/cytoplasmic scaffolding proteins.”  “Palmitothways.”  The meththways.” , 52, 53.thways.” . Li et aand Gβ.”  Worth meractions .Micropogonias undulates, the estrogen receptor-beta types have greater affinity which aimed at estradiol as compared to estrogen receptor-alpha [Homo sapien estrogen receptor-alpha exhibited no variation in affinity [Estrogen receptors have been identified conventionally as mammal-specific. The preliminary classification of estrogen receptors in mammals showed, fairly astonishingly, that even though mollusks have noticeable estradiol heights, their estrogen receptors could not affect estradiol, nor triggered by it. As an alternative, they exhibit constitutive action , 54. Nonor-alpha , 56, 60.affinity .Danio rerio and Ictalurus punctatus [Oncorhynchus mykiss estrogen receptor-alpha for estradiol is larger at 4°C than at 22°C [Oncorhynchus mykiss estrogen receptor-alpha has greater base action linked to the human estrogen receptor. According to [Oncorhynchus mykiss estrogen receptor alpha on a reporter per estrogen receptor estradiol only extended twenty-six percent of the human estrogen receptor. Nonetheless, on a reporter per three estrogen receptor estradiol Oncorhynchus mykiss ERα and human ER-alpha demonstrated comparable action. The focus on variances in estrogen receptors among vertebrates that may be flouted by depending exclusively on a single receptor reporter evaluate. Through generating fantasies among the Oncorhynchus mykiss ERα and human estrogen alpha, it is consequently established amino acids in the DNA-binding domain are accountable for a pronounced opinion that Oncorhynchus mykiss estrogen receptor alpha. This might only extend an extreme activating 74 percent lesser than human estrogen receptor alpha on reporter hypothesis per one estrogen receptor estradiol [Homo sapien estrogen receptor alpha spot 260. In the argument of the researchers, they define initial finding that incompletely incriminates the supplementary Arg in Homo sapien matched to fish estrogen receptor alpha. Furthermore, the different effects perceived among reporters of 1 estrogen receptor estradiol against three estrogen receptor estradiol indicates that the Oncorhynchus mykiss estrogen receptor needs the steadiness of DNA through protein-protein communications, of which these communications might be facilitated by the AF-1 domain [Oncorhynchus mykiss estrogen receptor, negligible section eleven amino acids situated from the beginning of B domain which is essential for AF-1 action [Oncorhynchus mykiss estrogen receptor alpha plays a hindering role, probably through the collaboration of the C-terminal section of the estrogen receptor [Homo sapien estrogen receptor alpha, the Oncorhynchus mykiss estrogen receptor alpha is triggered, in yeast functional unit, through choosy ER modulator, 4-hydroxytamoxifen, but seemingly not through pure antiestrogen, ICI 164 384, even though estrogen receptor action did seem to upsurge with dose of ICI 164 384 [The features of teleost species, estrogen receptor-beta types being capable of binding estradiol with greater affinity than estrogen receptor-alpha have now been defined in other fish types, explicitly, the unctatus , 26, 28. at 22°C . The pop at 22°C . The cel at 22°C . There a at 22°C , 62. Abo at 22°C , 64. Conrding to , the constradiol , 65. Owi1 domain . Inside 1 action . The desreceptor . A multi 164 384 , 30. It  164 384 .Estrogens are important in maintaining bone mass primarily by retarding bone resorption. They control the action of parathormone, a hormone which intakes calcium ions from bones and teeth, thus maintaining the calcium balance. They also cause salt and water retention that is edema. Additionally, blood coagulation is increased due to the formation of various clotting factors and also it promotes vasodilation by inducing secretion of nitric oxide synthase (NO) and PGI2. It also increases plasma “sex hormone binding globulin (SHBG), thyroxine binding globulin (TBG), and cortisol binding globulin (CBG).” , 69Estrogens are accountable for the advancement of pubertal changes and secondary sexual characteristics. They cause the growth and development of uterus, vagina, and fallopian tubes as well as also contribute to breast enlargement in females (humans). In addition, they add to the molding of the body contours and are responsible for the pubertal development spurt of the long bones and epiphyseal closure .Undoubtedly, estrogens play key roles in female hormone regulation and signaling and are responsible for metabolic, behavioral, and morphologic changes occurring during the stages of reproduction. They are also involved in growth, development, and homeostasis of a number of tissues. Again they also regulate reproduction, transport, and concentration of testicular liquid and anabolic activities of androgens in males , 72.Estrogens have beneficial effects in preventing heart diseases, osteoporosis, and Alzheimer's disease; however, there is also evidence that estrogens may promote cancer of the breasts, uterus, and other organs. Estrogen may cause adverse effects such as risk of thromboembolic events, postmenopausal uterine bleeding, and nausea, and breast tenderness .In vertebrates, the activities of estrogens are applied through ERs , and have shown that their expression profiles are tissues and cells specific. In mammalian immune cells, estrogen receptors (ERs) are receptive to estrogens. It must be noted that in recent times, ERs are also found in the teleost fish cells and immune organs, indicating estrogen immunomodulatory function conservation all through the evolution of vertebrates. In trout immune system, low mRNA numbers of ERs were reported with limited estrogen responsiveness. This is an indication that the estrogen activities on trout immunity might not be principally facilitated through genomic activities, though it may comprise other mechanisms .The principal physiological estrogen function in vertebrates is regulation of sexual growth and propagation. Nonetheless, estrogens possess pleiotropic utilities away from the conventional utility in the propagative bloc, they influence many other systems of physiology which include the immune system , 75. Theβ2 was also found expressing with the same levels of ERα in the organs such as spleen and head kidney indicating a significant starring role of these ERs in teleost immune systems [Within the immune system of trout fish, estrogen receptor alpha was found to express conspicuously, whilst ER systems .Sparus aurata), which was reported to have no copies in the head kidney acidophilic granulocytes and testicular, however, “head kidney contained ERα mRNA, but absence of ERβ1 or ERβ2 mRNA, in channel catfish ,” [α and ERβ mRNA was found in head kidney and spleen, whereas only ERα was present in “peripheral blood leukocytes, in carp (Cyprinus carpio),” [α mRNA was highly expressed while there was no ERβ presence in peripheral blood leukocytes there was also a report of low-level ERβ in isolated leukocytes in head kidney. Accordingly, the tissue leukocytes exhibited high expression levels of ERα than of ERβ in all species mentioned above and were totally lacking in peripheral blood leukocytes [β forms in the rainbow trout's peripheral blood leukocytes and made some interesting revelation that the ERβ2 mRNA figures were the same as those of ERα in the head kidney and spleen. Therefore, we can conclude from numerous studies on nuclear ER that estrogen receptor alpha is the main estrogen receptor in the immune system of teleost of diverse species, “but not in the immune organs essentially while estrogen receptor beta expressions in the fish immune system seems to differ with the origin of the cells and across species.” [Many studies reported on ERs in immune system using quantification of relative mRNA method for several teleost fishes such as seabream (tatus),”  ERα and arpio),”  ERα mRNAukocytes –83. In cukocytes  reportedpecies.” β-estradiol (E2) modulates the functioning of immune cells, activation, differentiation, development, and life span, and can have both immunosuppressive and immunostimulating actions [α form has a more noticeable expression and distribution in the immune system of mammal's immune cells than estrogen receptor beta [It must be noted that in mammals, estrogens such as 17 actions , 84, 85.Prostate tissue manifested both estrogen receptor beta and estrogen receptor alpha and –88. Immuα behaves as an oncogene which facilitates the harmful influences of estrogen, such as prostate carcinogenesis, inflammation, and proliferation [β, which manifestation declines by promoter DNA methylation as prostate cancer advancements, appears to play “anti-oncogenic role.” Certainly, estrogen receptor beta agonist has been reported to have averted the propagation of prostatic epithelium [β knockout prompted dysplastic variations and premalignant revolution, ERα knockout mice persisted prostate cancer-free.” [β in a prostatic revolution. For example, the loss of estrogen beta manifestation shows a relationship, with amplified propagation in the “ERβ KO/transgenic adenocarcinoma of mouse prostate classic” [Several studies on a role “of estrogen receptor alpha or estrogen receptor beta in prostate cancer have been” carried out and linger on. Many of these discoveries recommend that ERferation . In dissithelium . Once mor-free.”  Some disclassic”  and humaclassic” , 93, 94.A. burtoni ERα and ERβ extensively expressed all through the telencephalon and diencephalon though the ERβ2 subtype seems to be distributed more than ERβ1, which are more than earlier available or reported in several teleost species, even though several of those literature studies define mRNA but not protein [α expressions or distributions have been studied in the midshipman [β1 and ERβ2 mRNA subtypes has been studied in the Atlantic croaker [Steroid hormone receptors are present in vital areas of brain identified to control social behavior in other vertebrates including “the mammalian amygdalar complex, hippocampus, striatum, preoptic area, anterior hypothalamus, ventromedial hypothalamus, and ventral tegmental area.”  For exam protein . The mRNdshipman , Atlantidshipman , rainbowdshipman , zebrafidshipman , and eeldshipman , while t croaker , sea bas croaker , and zeb croaker .Fundulus heteroclitus ablation of the preoptic area reduces the spawning reflex [α, ERβ1, and ERβ2, were present inside the three cell groups that are parvocellular, magnocellular, and gigantocellular composed of the preoptic area. These three cell sets could differentially control “social dominance, as dominant males have higher AVT expression in the gigantocellular subregion compared to subordinate males” according to [A. burtoni and Lepomis sunfish is the generation of body pigmentation [Several lesion and stimulus studies have point to the preoptic area (POA) as area of brain that controls antagonistic and reproductive activities in teleosts, in agreement with other studies in other vertebrates , 102. Acg reflex . Accordig reflex , all sexrding to . Similarentation , 107, anentation , 108. ThVertebrates and other mammals including species of teleosts have no doubt become an appropriate and widely held suitable species to study the activities and function of estrogens and estrogen receptors. Estrogen receptor subforms expressed broadly, and hepaticvitellogeneisis offers a physiologically significant endpoint of estrogen receptor activation. Certainly, hepatic vitellogenesis and estrogen receptor alpha gene's distribution are frequently utilized as bioassays aimed at endocrine disruptors that have estrogen-like action , 61. It There is also a need to investigate whether the exposure to exogenous estrogens from various sources could trigger cancer development in experimental animals including fish. This has become necessary due to studies suggesting that the cancer-causing effect of estrogens and metabolites of some estrogens is linked with the stimulation of estrogen receptors alpha and beta that activate transcription of genetic material stimulating cell propagation. Additionally, numerous cancers such as ovarian cancer, uterine cancer, and breast cancer which are mostly associated with a female are mostly suggested to be causative of estrogens. We believe that the increased understanding of the molecular signaling nature of ERs will also flourish our capacity to precisely target the receptor and its signaling pathways for varied arrays of research.


Crowding, the failure to identify a peripheral item in clutter, is an essential bottleneck in visual information processing. A hallmark characteristic of crowding is the inner–outer asymmetry in which the outer flanker (more eccentric) produces stronger interference than the inner one (closer to the fovea). We tested the contribution of the inner-outer asymmetry to the pattern of crowding errors in a typical radial crowding display in which both flankers are presented simultaneously on the horizontal meridian. In two experiments, observers were asked to estimate the orientation of a Gabor target. Instead of the target, observers reported the outer flanker much more frequently than the inner one. When the target was the outer Gabor, crowding was reduced. Furthermore, when there were four flankers, two on each side of the target, observers misreported the outer flanker adjacent to the target, not the outermost flanker. Model comparisons suggested that orientation crowding reflects sampling over a weighted sum of the represented features, in which the outer flanker is more heavily weighted compared to the inner one. Our findings reveal a counterintuitive phenomenon: in a radial arrangement of orientation crowding, within a region of selection, the outer item dominates appearance more than the inner one. Crowding is considered to be an impediment to reading7, face recognition8, eye movements10, visual search3 as well as deficits like macular degeneration11, amblyopia12 and dyslexia60. Research has characterized crowding and distinguished it from other spatial interferences, such as masking13, lateral interaction14 and surround suppression15. Yet, the underlying processes of crowding are still unclear17. Researchers have associated crowding with a variety of factors: the reduction in spatial resolution in the periphery due to increased receptive field size of cells at early visual areas20, decreased cortical magnification21 and reduced attentional resolution25.Crowding refers to our inability to identify an object  because it is presented with nearby objects34 (but see57). The asymmetry effect has been demonstrated with different stimulus types and tasks, such as letter recognition34, face recognition27 and Gabor orientation discrimination15. Nevertheless, the mechanisms underlying this phenomenon remain a matter of debate.A hallmark characteristic of crowding is the inner-outer asymmetry: an outer flanker  produces stronger interference than an inner one (closer to the fovea)35. Other accounts postulate that increased receptive field size in the periphery biases sampling rate toward the outer flanker26 or leads to sparse selection in the visual periphery30. Finally, the attentional account attributes display layout and experimental task in biasing spatial attention outwards, leading to the selection of the outer flanker23. Yet, none of the proposed accounts directly link the inner-outer asymmetry with recent models of crowding.Researchers have proposed various causes of the inner-outer asymmetry. According to the cortical magnification account, the smaller cortical distance between the outer flanker and the target leads to stronger interference between these two items41 or substitution models44. According to pooling models, under crowding, due to inappropriate integration field size in the periphery, observers simultaneously detect and pool excessive information of low-level features, including those that belong to the flankers39. In the more simple form of pooling models, crowding reflects an averaging of target and flanker features. For example, simple averaging errors were observed in judging target orientation39 and target position36. However, simple pooling models cannot explain the well-documented finding showing that under crowding conditions observers often misreport a flanker as the target44. Such misreport errors led to the proposal of substitution models, according to which increased location uncertainty in the periphery47 renders observers failure to spatially differentiate between the target and the flankers.Proposed models of crowding were often classified to either pooling models48). For example, several studies have proposed a population coding model that codes target and flanker features as a weighted sum within a receptive field and can explain both averaging and misreporting errors of orientation49, color and spatial frequency49. Investigations of these models often use estimation reports in which observers estimated the target feature in a continuous space; for example, reporting the orientation of the target by adjusting the orientation of a probe. In such tasks, observers often reported the flanker instead of the target50. By fitting a probabilistic mixture model with a misreport component , researchers were able to independently assess the precision of reported item and the contribution of the flankers to the distribution of the estimation errors49. This misreport mixture model acts similarly to the population coding model and can, therefore, serve to determine the relative activation of each item in the population49. However, whether the relative contribution, and hence activation, of the outer flanker is different from the inner one is still unclear.In recent years, researchers were able to reconcile these conflicting findings by proposing more advanced pooling models that can predict both averaging and misreporting errors  in which the target (7° eccentricity) appeared alone (uncrowded conditions) or flanked (crowded conditions) by either two (two-flanker condition) or four (four-flanker condition) Gabors. Target and flankers were arranged on the horizontal meridian, either to the left or to the right of fixation. The centre-to-centre distance between two adjacent items was 1.5° .Thirteen undergraduate and graduate students  from The University of Haifa participated in this experiment for course credit or a 40 ILS per hour monetary payment (around $12). On the basis of a priori power analysis, using effect sizes from previous studies51 and presented on a gamma-corrected 21-in CRT monitor  connected with an IMac. Eye movements were monitored with an Eyelink 1000 Plus , by using a chin rest at a 57-cm viewing distance. Observers reported their responses by using the mouse.Stimuli were programmed in Matlab  with the Psychophysics Toolbox extensions2). Each trial began with the presentation of a fixation display. The fixation display consisted of a fixation mark (a centred dot subtending 0.1°) along with two peripheral pairs of place holders, one pair in the right hemifield and one in the left hemifield , which indicated the eccentricity of the upcoming target. Both fixation mark and place holders were white (112 cd/m2).Figure Following observers' fixation for a random duration lasting between 500 and 800 ms, the fixation mark appeared for an interstimulus interval (ISI) of 100 ms. The stimulus display appeared for 400 ms. The stimulus display consisted of the fixation mark along with the target; a sinusoidal grating (Gabors) with a 2D Gaussian spatial envelope (standard deviation 0.55° and 85% contrast); and a spatial frequency of 1.5 cycles per degree (cpd). The target appeared on the horizontal meridian with 7° eccentricity, either in the right or left hemifield. The target could appear alone (uncrowded condition) or along with two flankers (two-flanker crowded condition) or four flankers (four-flanker crowded condition), which were located on the horizontal meridian on either side of the target. The centre-to-centre distance between two adjacent items was 1.5°. In the two-flanker condition, flankers’ eccentricities were 5.5° and 8.5° for the first inner (1I) and first outer (1O) flankers respectively. In the four-flanker condition, flankers’ eccentricities were 4°, 5.5°, 8.5° and 10° for the second inner (2I), first inner (1I), first outer (1O) and second outer (2O) flankers respectively. Thus, all flankers appeared within the window of crowding (< 0.5 of eccentricity). Target and flanker orientation were selected at random from a circular parameter space of 180 values evenly distributed between 1° and 180°, with the restriction that each of the flanker’s orientation differed from that of the target by at least 15°.Following the stimulus display, an additional fixation screen was presented for 200 ms, which was followed by a response display. During the response display, observers were required to use a mouse cursor to adjust the probe Gabor’s orientation to match the orientation of the target by selecting a position on the orientation wheel (marked by a black circle 0.08° thick with an inner radius of 3.8° at the centre of the screen). Following an observer’s response, a blank inter trial interval (ITI) appeared for 400 ms. In each trial, we monitored eye fixation using an eye tracker see “”. TrialsEach observer completed 15 blocks of 40 trials  over a 60-min session. There were 200 trials in each of the three crowding conditions . Crowding conditions were randomly mixed within each block. To reduce location uncertainty, observers were told that the pairs of dots have the same eccentricity as the upcoming target. The experiment began with two practice blocks of 10 trials. Observers were encouraged to take a short rest between blocks.28. We compared among four models:For each trial, we calculated the estimation error for orientation by subtracting the true value of the target from the estimation value, such that zero indicates target value. To assess the contribution of the flankers to the error distribution (i.e. models with misreport components) we calculated flankers’ values by subtracting the true value of the target from the values of the flankers. We analyzed the error distributions by fitting probabilistic mixture models, which were developed from the standard model as well as the standard with misreport model49, has two components: a von Mises (circular) distribution, which describes the probability density of reports around the target’s orientation, and a uniform distribution, which describes the probability of reports unrelated to the target (guessing rate). The model has two free parameters :σ is the von Mises distribution with a standard deviation (variability) σ (the mean is set to zero), and n is the total number of possible values for the target’s feature.The standard mixture model Eq. , as desci flanker. Notice that the von Mises distribution of the estimation errors The standard misreport model Eq.  adds a mTo test for sampling bias over the flankers (differences in sampling weights across flankers), we also fitted models with an independent β component for each flanker.The two-misreport model Eq.  has a seThe four-misreport model Eq.  has a se52. We compared models’ performance by comparing means Akaike information criterion with correction (AICc) to the individual data.We fit the models to the individual data using the MemToolBoxPT) by subtracting the accumulative guessing rate and misreport rate from 1, such that For each fitted model, we calculated target reporting rate  = 28.05, p < 0.001, partial η2 = 0.70, indicating higher precision in the uncrowded condition than in the two-flanker condition, t(12) = 5.30, p < 0.001, Cohen's d = 1.93, and in the two-flanker condition compared to the four-flanker condition, t(12) = 2.18, p = 0.050, Cohen's d = 0.56.Figure t(12) = 4.63, p = 0.001, Cohen's d = 0.72, and t(12) = 2.57, p = 0.025, Cohen's d = 0.64 respectively. In the two-flanker condition, the two-misreport model outperformed the standard misreport model, t(12) = 4.54, p = 0.001, Cohen's d = 0.77. In the four-flanker condition, the four-misreport model outperformed the standard misreport model, t(12) = 2.58, p = 0.024, Cohen's d = 0.56. These results suggest that in crowded conditions observers misreported specific flankers more than others.In uncrowded conditions, the standard mixture model described well the distribution of the errors Fig. A. For eaTo check for averaging errors in the inner–outer asymmetry, we tested whether observers averaged the target with the outer flanker by calculating a mean bias toward the outer flanker . To do tPT) of the best fitted models in each condition. To assess the effect of crowding on performance, we conducted one-way repeated measure analyses of variance (ANOVA) on variability, guessing rate and target reporting rate as dependent variables, with crowding condition as a within subject factor. There was a main effect of crowding condition on guessing rate, F = 5.78, p = 0.009, partial η2 = 0.33, but not on variability, F = 2.37, p = 0.115. There was a main effect of crowding condition on target reporting rate, F = 71.21, p < 0.001, partial η2 = 0.86, revealing higher probability of reporting on the target under the uncrowded condition compared to the two-flanker condition, t(12) = 8.27, p < 0.001, Cohen's d = 2.93, and the four-flanker condition, t(12) = 12.35, p < 0.001, Cohen's d = 4.74. The probability of reporting on the target under the two crowded conditions did not differ significantly, t(12) = − 1.40, p = 0.188.Next, we analyzed the fitted parameters of the best performing model in each condition. Figure t(12) = − 2.59, p = 0.024, Cohen's d = 1.24. In the four-misreport model (the four-flanker condition), we conducted a one-way ANOVA on misreport rate with flanker positions  as within subject factor. Flanker position had a significant effect on misreport rate, F = 5.74, p = 0.003, partial η2 = 0.32, indicating that misreport rate differed across flankers. Planned comparisons revealed that the misreport rate of the first (adjacent) outer flanker , was higher compared to the averaged probability of reporting on the other three flankers , t(12) = 3.43, p = 0.005, Cohen’s d = 1.59. These findings support the view that the inner-outer asymmetry is due to misreport of the first (adjacent) outer flanker as the target.In order to test the contribution of each flanker to crowding errors, we compared the misreport rates of the different flankers. Figure 49. Importantly, the results show that when the target is flanked radially from both sides, observers often misreport the orientation of the outer flanker that is adjacent to the target instead of the target. Observers misreported a flanker that was inner or not adjacent to the target in a much smaller proportion of the trials.The results of “Following the results of “Fourteen undergraduate and graduate students , from The University of Haifa participated in this experiment for a course credit or a 40 ILS per hour monetary payment (around $12). All observers were naive to the purposes of the experiment. All observers reported having normal or corrected-to-normal visual acuity and normal colour vision, and none reported attention deficits or epilepsy. The observers signed a written informed consent form before the experiment. The experimental procedures were approved by The University Committee on Activities Involving Human Subjects at The University of Haifa (No. 373/18).The apparatus was the same as described in “Figure The design was the same as described in “The statistical analyses were the same as described in “p = 0.000, partial η2 = 0.77, indicating higher precision in the uncrowded condition compared to the two-flanker condition, t(13) = 6.13, p = 0.000, Cohen's d = 1.45, and in the two-flanker condition compared to the four-flanker condition, t(13) = 4.19, p = 0.001, Cohen's d = 1.60.Figure t(13) = 1.30, p = 0.216, and between the independent two-misreport model and the standard misreport model, t(13) = 1.25, p = 0.232. This result is explained by small crowding interference in this condition. However, in the four-flanker condition, the standard misreport model outperformed the standard mixture model, t(13) = 4.06, p = 0.001, Cohen's d = 0.69, and the independent four-misreport outperformed the standard misreport model, t(13) = 3.80, p = 0.002, Cohen's d = 0.91. In this condition, misreport rates were higher for some flankers compared to others. As in “In the uncrowded condition, the standard mixture model described well the distribution of the errors Fig. A. For eaPT) as dependent variables. Significant differences were found between the crowding conditions for γ, F = 9.86, p = 0.001, partial η2 = 0.43, σ, F = 15.71, p = 0.001, partial η2 = 0.55, and PT, F = 71.66, p < 0.001, partial η2 = 0.85. Specifically, the probability to report on the target decreased as the number of flankers increased, with significant differences between the uncrowded condition and the crowded conditions (two-flanker: t(13) = 3.76, p = 0.002, Cohen's d = 1.35, four-flanker: t(13) = 21.17, p < 0.001, Cohen's d = 7.17), and between the two crowded conditions, t(13) = 6.36, p = 0.001, Cohen's d = 2.26.Next, we analyzed the fitted parameters of the best performing model  in each condition. Figure p = 0.730). In the four-misreport model (four-flanker condition), we conducted a one-way ANOVA on misreport rate with flanker position  as within subject factor. A significant effect for flanker position was obtained in the four-flanker condition in which the target was located near the outer flanker, F = 12.94, p = 0.000, partial η2 = 0.50. Here, planned comparisons revealed that the misreport rate of the first (adjacent) outer flanker (1O)  was significantly higher compared to the averaged misreport rate of the other flankers , t(13) = 4.41, p = 0.001, Cohen's d = 2.03. These findings are consistent with Experiment’s 1 results, and support the view that observers often misreport the first (adjacent) outer flanker as the target.We assessed the contribution of each flanker to crowding errors by comparing the misreport rates of the different flankers. Figure PT) between the flankers. Furthermore, as shown in previous studies53, in both “The results of “PT) and standard deviation (σ) between “PT analyses revealed that in the uncrowded condition, target reporting rate did not differ significantly, t(25) =− 0.39, p = 0.701, meaning that observers were unaffected by item eccentricity per se. However, significant differences were found between the two experiments in the two crowded conditions. In the two-flanker condition, when observers reported the outer item (“t(25) = − 2.69, p = 0.013, Cohen's d = 1.04. Although we increased the target eccentricity, crowding interference was reduced when observers reported the outer item (“t(25) = 2.40, p = 0.024, Cohen's d = 0.92 (see54 for a similar finding). Although the four-flanker condition in “Experiment Finally, we examined the effect of flanker position on crowding interference. To do so, we conducted an independent t-test for each crowded condition and compared the target reporting rate  er item “”, suggeser item “”, crowdier item “”, t(25) 5 er item49, our results showed that orientation crowding errors reflect reporting a flanker instead of the target (misreport errors). Second, we found that in a radial crowding display  item receives heavier weight than the foveal (inner) item. In terms of population coding models, heavier weight over the peripheral item perhaps can be implemented by assuming that summation weights scale with receptive field size, such that heavier weights are assigned to more peripheral items (larger receptive field). A similar principle was proposed with a Bayesian inference framework and with some assumption regarding the growth of receptive field size in the visual periphery35. This explanation predicts that within the same display , crowding interference will be the same regardless of whether observers are reporting on the middle or the outer item. Thus, the reduction of crowding interference in the two-flanker condition of “26.Our results are inconsistent with the cortical magnification account. According to this view, crowding asymmetry results from cortical mapping in which the outer flanker is closer to the target than the inner one23, it is still unclear whether and how this effect is reflected in a typical symmetrical crowding display across the visual field. Furthermore, our results are limited to orientation errors involved in crowding asymmetry. Since recent studies found dissociation of crowding errors across dimensions, such as orientation, color and spatial frequency49, and between color and motion55, further work is needed to determine whether the current findings apply to other feature dimensions. Moreover, the use of Gabor patches also limited our ability to distinguish between object level and feature level. Due to previous studies that pointed to different processes involved at these levels56, further work should use a different type of stimulus, which would allow a clear distinction between those levels. Finally, this study does not directly test the predictions of simple pooling models of crowding, which predict averaging. Future studies should compare the weighted summations (misreport) model, as proposed here, with weighted averaging models to reconcile between the different pooling models.In this study we investigated the inner-outer asymmetry along the horizontal meridian by using a symmetrical display. Although previous studies found the asymmetry effect along the horizontal, but not along the vertical meridianOur findings reveal that in a typical radial crowding display, observers confuse the target with the nearby outer flanker, but not vice versa. This confusion occurs regardless of the number of flankers and reported item position. Probabilistic mixture models explain that these results as due to heavier weights toward the visual periphery. These findings are consistent with recent models that explain crowding in terms of population coding with weighted summation within receptive fields. Our results demonstrate a counterintuitive phenomenon: within a region of selection, the more eccentric item dominates appearance.Supplementary Information


Fear extinction remains an unresolved challenge for behavioral exposure therapy in patients with post-traumatic stress disorder (PTSD). Previous reports have suggested that social support from either familiar or unfamiliar same-sex partners is beneficial to attenuating fear responses during fear extinction and renewal. Despite that, few studies have examined the effects of social support in advance on fear extinction and/or retrieval. It is also not clear whether social company by a receptive mating partner in advance facilitates fear extinction. In the present study, we address these questions by introducing a co-housing method, where fear-conditioned male mice are co-housed with or without a receptive mating partner prior to fear extinction. We found that while co-housing with an ovariectomized female mouse showed little effect on fear extinction or retrieval, social company by a receptive mating partner in advance dramatically facilitates fear extinction. In addition, the number of cFos-positive neurons in the basolateral amygdala (BLA) were also found to be reduced in male mice accompanied with receptive mating partner in response to fear extinction and retrieval, indicating diminished neuronal activation. Electrophysiological studies further showed that the excitability of excitatory neurons in BLA was decreased, which is probably due to the attenuated basal level of excitatory synaptic transmission. Together, our observations demonstrate an effect of social company by a receptive mating partner can facilitate fear extinction and afford a possible cellular mechanism. Post-traumatic stress disorder (PTSD) is a serious mental disorder with lifetime prevalence that ranges from 1.3 to 12.2% . PTSD usIn the last decades, despite efforts from multiple studies, which were designed to elucidate the mental and biological foundations of PTSD, the underlying pathological mechanisms remain obscure . In rodeResults from previous studies have supported the notion that the “social buffering” effect exists not only in humans but also in non-human primates as well as rodents . This phSocial support is available from friends and family members in one’s social environment, especially intimate partners since they may impose significant influence over the symptoms and treatment outcome in PTSD patients . While tIn the present study, we addressed these issues by introducing a co-housing method. We first subjected the adult male mice to intense fear conditioning. After acquiring a strong fear memory, male mice were individually housed either alone , with an ovariectomized female mouse (FC + OVC group), or with a receptive female mouse (FC + Receptive group). Twenty-four hours later, all male mice were individually subjected to fear extinction training as well as a retrieval test on the next day. To examine the underlying mechanisms, we characterized neuronal activation in the basolateral amygdala (BLA) by using cFos staining and recorded neuronal excitability and excitatory synaptic transmission by using a patch clamp technique.Chemicals were purchased from Sigma-Aldrich unless otherwise indicated. Mouse monoclonal anti-cFos and rabbit polyclonal anti-PV primary antibodies were obtained from Santa Cruz  and Swant , respectively.The experiments followed the Guidelines for Animal Care and Use of China, and the experimental protocols were approved by the Animal Ethics Committee of Guangzhou Medical University . Signifiad libitum.Adult C57BL/6J mice aged 9–10 weeks were purchased from Guangdong Medical Laboratory Animal Center. Male mice were individually housed, and female mice were housed in groups of four for at least 1 week prior to experiments. Mice were maintained in a temperature- and humidity-controlled holding facility with 12-h light/dark cycles (lights on from 08:00 to 20:00). Food and water were available Adult female mice were anesthetized with 1% pentobarbital sodium. The ovaries, located in a fat pad beneath the muscles, were exposed through bilateral flank incisions. With the help of forceps, the oviducts were ligated, and ovaries were exteriorized. The muscle of the posterior abdominal wall and the exterior skin incision were then sutured. After the surgery, mice were injected with ketoprofen (5 mg/kg) subcutaneously to deliver analgesia. Mice were returned to their home cages, monitored daily, and provided with a recovery period of at least 7 days before experiments.Receptive female mice were prepared as described previously . BrieflyAll subjects were handled for 3 days before behavioral experiments. Prior to fear conditioning, male mice were individually housed. Paradigms that measure tone-cued fear memory formation and extinction were chosen according to a recent report in which fear memory was hardly degraded under such paradigms . BrieflyAfter fear conditioning, each mouse was immediately returned to its home cage. They were divided into three groups based on their freezing level in the training phase, ensuring a similar freezing level prior to subsequent manipulations: Group 1: male mouse without a partner ; Group 2: male mouse accompanied with an ovariectomized female mouse (FC + OVC); and Group 3: male mouse accompanied with a receptive female mouse (FC + Receptive). Twenty-four hours later, male mice were subjected to fear extinction procedure.For fear extinction, each male mouse was transported to a chamber with an altered environment  on day 2. The freezing level was measured for 2 min without CS  and in the presence of subsequent 10 CS with an interval time of 90 s between each CS. The formula for deriving the% cued freezing used was  × 100%. For the retrieval test, male mice were placed again in the same chamber as in day 2 (Context B) on day 3 for 2 min prior to CS. Then, a single CS  was presented. Freezing time was recorded in the absence and the presence of CS. Chambers were cleaned with 1% acetic acid solution at the end of each trial.As described previously , male mi2-2H2O, 2.5 mM MgSO4, 1.25 mM NaH2PO4, 7 mM MgCl2, 25 mM NaHCO3, and 10 mM glucose. Brain slices (300 μm) were cut in ice-cold modified ACSF with a VT-1000S vibratome  and subsequently transferred to a storage chamber containing regular ACSF  for a 30-min recovery period at 32°C and followed at room temperature (25 ± 1°C) for an additional 1 h before recording. All solutions were saturated with 95% O2/5% CO2 (vol/vol).Slices containing BLA were prepared as described previously . Briefly2, 4 mM Mg-ATP, 0.3 mM Na-GTP, 10 mM disodium phosphocreatine, and 0.2 mM EGTA, pH 7.4 with KOH, 285–300 mOsm. Excitatory neurons in the BLA region were identified by their pyramidal shape and spike frequency adaptation induced by a prolonged depolarizing current injection. To assess the neuronal excitability, action potentials (APs) were measured by injecting a series of depolarizing pulses  under a current-clamp mode. Resting membrane potential and membrane input resistance were also calculated in response to a series of hyperpolarizing pulses. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from the excitatory neurons at holding potential of −70 mV and in the presence of 20 μM GABAa receptor blocker-bicuculline methiodide (BMI). Data were filtered at 1 kHz and sampled at 10 kHz. Neurons were collected when the series resistance fluctuated within 20% of the initial values (<20 MΩ) and analyzed using pClamp 10.2 software for neuronal excitability and MiniAnalysis for sEPSCs.Slices were placed in the recording chamber that was perfused (2 ml/min) with ACSF at room temperature (25 ± 1°C). Whole-cell patch-clamp recordings of excitatory neurons in BLA were visualized with infrared optics using an upright microscope equipped with an infrared-sensitive CCD camera  and a 40× water immersion lens (Olympus). The pipettes were pulled by a micropipette puller  with a resistance of 3–5 MΩ. Recordings were made with a MultiClamp 700B amplifier and 1440A digitizer (Molecular Device). Glass pipettes (3–5 MΩ) were filled with a solution containing 125 mM potassium gluconate, 5 mM KCl, 10 mM HEPES, 1 MgCln.” All statistical analyses were performed using a GraphPad Prism7. The sample size choice was based on previous studies , and the number of experimental animals is indicated by “ studies , and it t-test, “FC + Receptive” vs. “FC alone,” t(14) = 0.4009, P = 0.6946; “FC + Receptive” vs. “FC + OVC,” t(14) = 0.0543, P = 0.9574; “FC + OVC” vs. “FC alone,” t(14) = 0.5926, P = 0.5629), which was suggestive of similar fear expression and the limited effects of co-housing on fear consolidation. In accordance with a previous report  = 0.5238, P = 0.8519; “FC + OVC”: F = 0.8912, P = 0.5383). In addition, the freezing level was not found to be significantly different between these two groups  = 1.67, P = 0.2172). The interaction between group and trial was also found to be not significant  = 0.2375, P = 0.9883). These results suggest that the presence of a non-receptive partner  prior to fear extinction exhibits little effect on fear extinction. However, the freezing levels of male mice in the “FC + Receptive” group were gradually reduced during the extinction procedure  = 11.84, P < 0.001). When compared with those in “FC alone” and “FC + OVC” groups, the freezing levels in the “FC + Receptive” group were dramatically reduced  = 7.116, P = 0.0184; “FC + Receptive” vs. “FC + OVC”: F = 15.23, P = 0.0016). The interactions between group and trial were also significant  = 6.1, P < 0.0001; “FC + Receptive” vs. “FC + OVC”: F = 5.612, P < 0.0001). Collectively, these observations indicate facilitated acquisition of fear extinction in the “FC + Receptive” group. During a retrieval test  = 0.617, P = 0.547). However, the freezing level of male mice in the “FC + Receptive” group was dramatically decreased when compared against “FC alone” and “FC + OVC” groups  = 4.312, P = 0.001; “FC + Receptive” vs. “FC + OVC,” t(14) = 5.899, P < 0.001). Please note that the reasons for the reduced freezing level in the retrieval test are not clear since the differences in acquisition of extinction may underlie subsequent differences in retrieval, and the effects of sexual activity on fear retrieval were not specifically determined in the present study. Nevertheless, these observations demonstrate that company by a receptive mating partner, but not a non-receptive one, in advance facilitates acquisition of fear extinction.During the intense fear conditioning training with six pairs of CS–US , adult ms report , the feaval test , mice int-test, “FC + Receptive” vs. “FC alone,” t(8) = 3.663, P = 0.006; “FC + Receptive” vs. “FC + OVC,” t(8) = 6.354, P < 0.001). This result suggests reduced neuronal activation in the BLA of mice accompanied by a receptive mating partner. We also examined the parvalbumin (PV)-positive interneurons in the BLA region since they are the foremost interneurons in the BLA and modulate excitatory neurons in fear extinction processing  = 0.1414, P = 0.891; “FC + Receptive” vs. “FC + OVC,” t(8) = 1.97, P = 0.0844; “FC + OVC” vs. “FC alone,” t(8) = 1.985, P = 0.0825), indicating unaltered inhibitory neuron densities in mice accompanied by a receptive mating partner.It has been reported that the amygdala is a vital brain area correlated with the social and fear-related behaviors . In partocessing . As showt-test, for resting membrane potential, “FC + Receptive” vs. “FC alone,” t(46) = 1.031, P = 0.308; “FC + Receptive” vs. “FC + OVC,” t(46) = 0.4653, P = 0.6439; “FC + OVC” vs. “FC alone,” t(46) = 1.317, P = 0.1944; for membrane resistance, “FC + Receptive” vs. “FC alone,” t(46) = 1.957, P = 0.0565; “FC + Receptive” vs. “FC + OVC,” t(46) = 1.099, P = 0.2776; “FC + OVC” vs. “FC alone,” t(46) = 0.7562, P = 0.4534), the firing frequencies in response to the injected depolarizing currents with gradual increments in the intensities were significantly decreased in the “FC + Receptive” group compared with those in the “FC alone” and “FC + OVC” groups  = 56.82, P < 0.001; “FC + Receptive” vs. “FC + OVC,” F = 10.66, P = 0.0012). Note that the firing frequencies in the “FC + OVC” group were also decreased compared with those in the “FC alone” group  = 16.95, P < 0.001), which is probably due to a general social buffering effect (not copulatory behavior) on neuron activity in the amygdala. Nevertheless, these observations suggest attenuated excitability of excitatory neurons in the BLA of mice that were accompanied by a receptive mating partner.It is known that approximately 85% of the neuronal population in the BLA are excitatory neurons . In light-test, “FC + Receptive” vs. “FC alone,” t(38) = 2.4, P = 0.0214; “FC + Receptive” vs. “FC + OVC,” t(38) = 2.828, P = 0.0074). In addition, the amplitude was also reduced in the “FC + Receptive” group compared with that in the “FC alone” groups  = 3.462, P = 0.0013), although there was no significant difference between the “FC + Receptive” group and the “FC + OVC” groups  = 1.543, P = 0.1311). Together, these results suggest an attenuated excitatory synaptic input in the amygdala, which may serve in part as the cause of decreased neuronal excitability and subsequent reduced neuronal activation in mice that is accompanied by a receptive mating partner.To further investigate the mechanisms of decreased neuronal excitability in the BLA excitatory neurons in mice that were accompanied by a receptive mating partner, we recorded sEPSCs . The freIn the present study, we examined the effect of social company by a receptive or non-receptive mating partner on fear extinction. We found that company by non-receptive mating partner exhibited little effect on the fear response, while company by a receptive one significantly reduced the fear response during both fear extinction trining and a retrieval test. The number of cFos-positive cells, but not PV-positive interneurons, was found to be decreased in the BLA of male mice that accompanied by a receptive mating partner. Electrophysiological studies suggested that the excitability of excitatory neurons in the BLA of male mice that accompanied by a receptive mating partner was reduced, which may be due to the attenuated basal level of excitatory synaptic transmission. Altogether, these observations revealed that social company by a receptive mating partner had a beneficial effect on the facilitation of fear extinction.Exposure therapy combined with pharmacotherapy represents an effective treatment for PTSD patients. Nevertheless, there were still a number of patients (39%) who failed to respond positively to this therapy . In addiNumerous studies indicate that the BLA region of the amygdala is critical for acquisition of extinction memory . It has It is not clear how sexual behavior modulates neuronal function in the BLA and thus fear extinction. It is well known that sexual behavior, a deeply rooted physiological condition conserved throughout the species, is regulated by the nervous, endocrine, and genital systems whose interplay ensures its proper occurrence . In the The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Animal Ethics Committee of the Guangzhou Medical University.FG wrote the first draft of the manuscript. X-DS edited the manuscript. X-DS, AX, and FG designed the research. FG, JH, Y-FG, G-BH, W-JL, X-YH, and Z-CQ performed the research. Y-LZ, S-TZ, and JL analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Application of repetitive transcranial magnetic stimulation (rTMS) for treating obsessive-compulsive disorder (OCD) has been promising and approved by the Food and Drug Administration in 2018, but effects differ between patients. Knowledge about clinical predictors of rTMS response may help to increase clinical efficacy but is not available so far.n = 38) or bilateral DLPFC rTMS (n = 27) in case of reporting higher affective and depressive symptoms in addition to the primary OCD symptoms. OCD symptoms and depression/anxiety states were measured at baseline (before the 1st session) and after the 20th session of rTMS. Additionally, we performed a binary logistic regression analysis on the demographic and clinical variables based on the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) 3-factor and 2-factor models and individual items to investigate potential predictors of rTMS response.In a retrospective study, we investigated the efficacy of rTMS over the dorsolateral prefrontal cortex (DLPFC) or supplementary motor area (SMA) in 65 pharmaco-resistant OCD outpatients recruited for rTMS treatment from July 2015 to May 2017. Patients received either SMA rTMS (Patients’ scores in Y-BOCS and Beck anxiety/depression inventories were significantly decreased following rTMS treatment. 46.2% of all patients responded to rTMS, based on the criterion of at least a 30% reduction in Y-BOCS scores. There was no significant difference between response rates of patients in DLPFC and SMA groups. No significant demographic predictors of rTMS efficacy were identified. The factors “obsession severity”, “resistance” and “disturbance” and the “interference due to obsessions” and “resistance against compulsions” items of the Y-BOCS significantly predicted response to rTMS.In patients with less intrusive/interfering thoughts, and low scores in the “obsession severity”, “disturbance”, and “resistance” factors, rTMS might have superior effects. Identifying clinical and non-clinical predictors of response is relevant to personalize and adapt rTMS protocols in pharmaco-resistant OCD patients. Interpretation of rTMS efficacy should be done with caution due to the lack of a sham intervention condition. With a lifetime prevalence of 1–3% . OCD, whPrevious studies in humans and animal models suggest that functional abnormalities of the cortico-striato-thalamo-cortical circuits and supplementary motor area (SMA) might be central pathophysiological components of OCD –13. The Previous rTMS studies reported an average response rate of 35% in OCD patients, defined as a minimum of 25–40% reduction in post-treatment symptoms . Higher While specific stimulation parameters and neurobiological predictors of response to rTMS treatment have been investigated in OCD , 36, theIn the present study, we aimed to investigate the efficacy of rTMS over two potentially involved cortical regions  for reducing OCD symptoms. More importantly, we aimed to identify potential clinical and demographic predictors, that could distinguish between rTMS responders and nonresponders in OCD. Based on previous findings about the efficacy of rTMS in OCD patients , we expeWe retrospectively analyzed a dataset of pharmaco-resistant OCD outpatients who received an rTMS treatment between July 2015 and May 2017. Patients were referred to the Atieh Clinical Neuroscience Center in Tehran, Iran, to receive rTMS. The center admits patients with psychiatric disorders , neurological disorders , and pediatric neurodevelopmental disorders .SD = 10.23, 35 females) were included in this report. 69 patients were initially included, but four patients did not either finish the treatment course without any reported reason or meet the minimum symptom severity to be included. Of 65 patients 38 patients underwent SMA rTMS protocol and 27 patients received DLPFC rTMS protocol  using a 70-mm figure-of-8-shaped coil (air film coil). Active motor threshold (AMT) was defined as the minimum stimulus intensity that produced a liminal motor evoked response during active contraction of the abductor policies brevis muscle (APB) (at about 20% maximum contraction) . Motor tSD = 7.01), which is the cut-off criterion for moderate OCD . Treatment response was defined as a reduction of at least 30% in the Y-BOCS total score, based on several previous studies [All patients underwent a baseline clinical assessment with the Y-BOCS, Beck Anxiety Inventory (BAI)  and Beck studies , 47 and  studies , we chosThe Y-BOCS is the most widely used clinician-rated interview for assessing OCD symptom severity with adequate psychometric characteristics  . It contBoth BAI and BDI-II consist of 21 items, which are rated on a Likert scale ranging from 0 to 3, resulting in raw scores ranging from 0 to 63, and are indicative for the presence of anxiety or depression. The BAI is well suited to monitor anxiety treatment outcomes , and thep < 0.05 was used for all statistical comparisons.Data analyses were conducted using IBM, SPSS (version 24). In order to examine rTMS efficacy, mixed model analysis of variance (ANOVA) was conducted with “protocol” (DLPFC rTMS vs. SMA rTMS) as the between-subject and time (pre-intervention vs. post-intervention) as the within-subject factors. Mauchly’s test was used to evaluate sphericity of the data and in case of violation of sphericity, degrees of freedom were corrected using the Greenhouse–Geisser estimates of sphericity. The normality and homogeneity of the data were confirmed by Shapiro-Wilk and Levin tests, respectively. For identifying demographic and clinical predictors of response to rTMS treatment, participants were split into “responders” and “non-responders” and a binary logistic regression was conducted. To control for potential confounding variables, we added these variables into the model, as the model adjusts itself for potential confounders using adjusted odds-ratio . The gooThe datasets used and/or analyzed for the present study are available from the corresponding author upon reasonable request.N = 30, 16 females) and nonresponders  was 32.67 (9.44), and 31.89 (10.99) years old, respectively. The groups did not differ significantly with respect to demographic variables , comorbidity rate, medication use, baseline depression and applied intervention protocol  , at least a 30% reduction of their Y-BOCS scores after 20 sessions of rTMS was observed. The remaining patients (53.8%) were defined as non-responders. Specifically, in patients that underwent SMA rTMS, 16 out of 38 (42.1%) responded to rTMS treatment, which is equivalent to 53.3% of all responders. From the patients received bilateral DLPFC protocol, 14 out of 27 (51.9%) responded to the rTMS treatment, which is equivalent to 46.7% of all responders  = 1.71, p < 0.19, ηp2 = 0.02) or time × protocol interaction (F(1) = 0.06, p < 0.80, ηp2 = 0.01) emerged, indicating no significant dfference between the protocols  in reducing OCD symtoms. No significant interaction of time × protocol or main effect of protocol were found neither for the BAI scores (F(1) = 0.94, p < 0.33, ηp2 = 0.015; F(1) = 0.43, p < 0.51, ηp2 = 0.007), and depression symptoms measured by the BDI-II (F(1) = 0.01, p < 0.99, ηp2 = 0.001; F(1) = 1.01, p < 0.32, ηp2 = 0.016) (See Table No significant main effect of protocol (F(1) = 53.51, p < 0.01, ηp2 = 0.46), indicating a significant reduction of OCD symptoms after 20 sessions regardless of the stimulation protocol patiens underwent. Bonferroni-corrected post hoc paired t-tests for pre-post values showed significant differences in the Y-BOCS scores . With regard to anxiety and depression states, the ANOVA results similarly showed a significant main effect of time on BAI (F(1) = 34.90, p < 0.01, ηp2 = 0.357), and BDI-II scores (F(1) = 19.97, p < 0.01, ηp2 = 0.241). Again, the Bonferroni-corrected post hoc t-tests for pre-post values showed significant differences in the BAI  and BDI-II  scores.The results of overall ANOVA revealed a significant main effect of time on the Y-BOCS overall score in all patients  with a Nagelkerke’s R2 of 0.45 and prediction success of 80% (28 of 35) and 70% (21 of 30) for non-responders and responders respectively. Regarding the 2-factor model [p < 0.01, df = 1) with a Nagelkerke’s R2 of 0.41 and prediction success of 74.3% (26 of 35) and 76.7% (23 of 30) for non-responders and responders respectively. The Wald criterion revealed the “disturbance” factor  as the only significant clinical factor in predicting rTMS response in OCD.Results of the multicollinearity analysis showed a variance inflation factor (VIF) of higher than 3 for only 2 dependent variables, however, a VIF around 10 is indicative for high multicollinearity . The VIFor model , the ovep < 0.01, df = 2), indicating that clinical predictors significantly distinguished non-responders from responders to rTMS treatment. Nagelkerke’s R2 of 0.46 indicates a relatively moderate relationship between predictors and rTMS response, indicating that the model explained 46% of the variance of rTMS response in OCD patients. Prediction success was 82.9% (29 of 35) and 73.3% (22 of 30) for non-responders and responders, respectively. The Wald criterion identified items 2 (interference due to obsessive thought) and 9 (resistance against compulsions) of the Y-BOCS as the two significant clinical predictors of rTMS treatment response in OCD. In other words, it was shown that an increase of 1 unit in response to items 2 and 9, would increase the risk of not responding to rTMS treatment 3.8 and 2.4 times (Odds ratio), respectively (Table p = 0.83), gender (p = 0.23), and educational level (p = 0.11) as well as other clinical variables such as comorbidity (p = 0.81) and medication use (p = 0.86), significantly predicted response to rTMS in OCD patients.We then conducted a separate binary logistic regression on the Y-BOCS items. The overall model was statistically significant , and a significant reduction of OCD symptoms was observed in the whole patient group, including non-responders (less than 30% symptom reduction). No significant difference was found in treatment efficacy between the intervention protocols . Regarding the predictors of rTMS response, no demographic predictor  was identified. Both obsession and compulsion -related items/factors were related to response rate. “Obsessions severity”, “disturbance”, and “resistance” were the clinical factors that significantly predicted response to rTMS. In this line, items 2 and 9 of the Y-BOCS  were the most relevant clinical predictors of response to rTMS based on individual items regression analysis.The response rate to rTMS in our OCD sample is in line with previous studies. The first meta-analysis in the field included 10 randomized controlled rTMS studies (with ≥25–40% reduction in Y-BOCS scores) and reported a 35% response rate in 120 OCD patients that received rTMS –34. In aThis pattern of results suggests that response to rTMS in OCD patients depends on the pathophysiology of target region/s and the appropriate modulation of the involved regions. OCD is a heterogeneous disorder not only at the symptom, but also the pathophysiological level , 5, 61. The length and number of rTMS sessions can also affect the response rate, with a higher number of sessions providing more symptom reduction , 66. Ourobsession severity, resistance and “disturbance” were the factors with the highest predictive ability in rTMS response and the last two factors  include items related to both obsession and compulsion. Our model based on Y-BOCS items showed a similar pattern of predictors. Specifically, we found that higher scores in “interference due to obsessions” (item 2), which is related to disturbance factor, and “resistance against compulsion” (item 9), related to the resistance factor, determined response failure to rTMS treatment. The common factor underlying both obsession and compulsion related items is the disturbance factor and this implicates that those OCD symptoms (including both obsession and compulsion-related ones) that result in more disturbance/interference are important in predicting response to rTMS.Our finding of the absence of relevant demographic predictors of rTMS response is in line with a recent report of a missing correlation between baseline psychometric factors, including age, and rTMS treatment outcome . Age, hoThe predictive value of these factors and relevant items could have clinical implications for treatment response. Patients with more “severe obsessions” have more intrusive thoughts and experience greater overall difficulties due to obsession interference , 71. Thiinterference or inhibition. An alternative option might be to treat these patients with a treatment approach focused on improving cognitive control strategies over both obsessions and compulsions [Since these aspects of OCD symptoms seem to be of utmost importance for response failure to rTMS treatment, it might on the one hand help to decide about therapeutic options in specific patients. On the other hand, it also suggests that patients with a lower probability for successful rTMS treatment with conventional protocols might require alternative interventions. In this line, one important implication of our findings is the need for adapting rTMS protocols to patients’ symptoms. According to our results, high levels of the interference, disturbance and resistance aspects of OCD symptoms have a negative impact on rTMS response. Patients with prominent symptoms related to these factors might benefit more from protocols that are optimized to have a strong impact on pulsions . For exapulsions –80, mighOur findings are preliminary, and this study has some limitations. The major limitation of our analysis is the retrospective study design. Nevertheless, our data has relatively high ecological validity and provides a realistic picture of rTMS application in clinical settings. Secondly, our control condition is limited to a baseline-control and lacks a sham intervention condition. Unblinded assessment by multiple raters, which could be a source of bias, and inter-rater variability, are other limitations of our work. Although the sample size was large enough for investigating the efficacy of rTMS, it may have been relatively underpowered for obtaining robust results by the regression analysis performed on individual items of the Y-BOCS. Sample size, however, was sufficiently large for factor-based regression analysis, which resulted in similar clinical predictors of rTMS response. The use of 120% of active rather than resting motor threshold could be a limiting factor in determining response rate as rTMS at AMT intensity usually delivers underdosage stimulation compared to RMT . AllocatOur findings identified some important predictors of therapeutic efficacy of rTMS in OCD, which might help to develop adaptive and personalized stimulation protocols in future. Specifically, patients with severe obsession symptoms and higher dysfunctions due to intrusive thoughts, more distress and less control over compulsive behaviors might not be good candidates for receiving DLPFC and/ or SMA rTMS treatment.


This data article describes the dataset of the International COVID-19 Impact on Parental Engagement Study (ICIPES). ICIPES is a collaborative effort of more than 20 institutions to investigate the ways in which, parents and caregivers built capacity engaged with children's learning during the period of social distancing arising from global COVID-19 pandemic. A series of data were collected using an online survey conducted in 23 countries and had a total sample of 4,658 parents/caregivers. The description of the data contained in this article is divided into two main parts. The first part is a descriptive analysis of all the items included in the survey and was performed using tables and figures. The second part refers to the construction of scales. Three scales were constructed and included in the dataset: ‘parental acceptance and confidence in the use of technology’, ‘parental engagement in children's learning’ and ‘socioeconomic status’. The scales were created using Confirmatory Factor Analysis (CFA) and Multi-Group Confirmatory Analysis (MG-CFA) and were adopted to evaluate their cross-cultural comparability  across countries and within sub-groups. This dataset will be relevant for researchers in different fields, particularly for those interested in international comparative education. Specifications Table•The database offers first hand valuable information about parental engagement, school support for parents and children, home-schooling and family life balance and parental acceptance and confidence in the use of technology from 23 countries around the world.•The international database provides a rich environment for examining how parents and caregivers relate to children's learning in this period of social distancing caused by the global COVID-19 outbreak.•The international database offers data comparable on parental practices during the lockdown across 23 countries and five regions , allowing investigations on aspects of specific relevance in each of these geographic regions.•The international dataset contains scales such as parental engagement, parental acceptance and confidence in the use of technology scale and family socioeconomic status, which allow testing hypothesis about the interactions of these and other variables across and within the participating countries.•The international database involves considerable information for the researchers, analysts, policymakers and education stakeholders to take steps and measures to improve the quality of parental engagement in children's education during and after the lockdown period.1With the advent of the detection of the first case of COVID-19 in the late of November in China and later in the beginning of March in the other countries, an urgent governance step has been initiated by the Ministries of National Education to carry on various educational activities remotely since schools have experienced compulsory shut downs until the end of April-June, depending in which country you are in, to prevent spreading the virus across countries Research connects children social and cognitive development to parents' educational practices at home In the current pandemic, parents have spent more time with their children. Moreover, the primary responsibility for enforcing and maintaining young people's educational engagement lies with them. While there is a substantial body of literature which explores parental engagement in education e.g., , the uniThe data provided in this study allows researchers to embark on investigations to the above and other related areas and questions.1.1All ICIPES 2020 data files contain several identification variables that provide information to identify the participants’ important characteristics. The variables do not allow identification of individual parents within countries.IDCNTRYThis variable indicates the country or participating education system; the data refers to an up to six-digit numeric code based on the ISO 3166 classification, with adaptations reflecting the participating education systems. This variable should always be used as the first linking variable whenever files are linked within and across countries.CNTThis variable indicates the participant's three-letter alphanumeric code, based on the ISO 3166-1 coding, with adaptations reflecting the participating country.CNTPARIDThis variable indicates the country's three numeric code, based on the ISO 3166–1 coding, plus a unique identifier for each respondent.REGIDThis variable identifies the specific region that each country belongs to. There are five  geographical regions: 1 Africa, 2 East Asia, 3 Europe, 4 South Asia and 5 America.REGThis variable indicates the participant's three-letter alphanumeric code, based on the ISO 3166- 1 coding, with adaptations reflecting the participating geographical regions.URNThis variable identifies the specific questionnaire that was administered to each parent. This number was automatically provided by the Online Surveys tool.In this study, the online survey was conducted with semi-structured questionnaires. Online survey is one of the best ways to reduce the cost when conducting a study, but it is also an effective way to get real data from the online population The following section provides information about the procedure followed to construct three scales in ICIPES 2020.Social cognitive learning theory Before constructing the three scales, we constructed and implemented normalised weights  (SENWT in the dataset) to make sure that when constructing these three scales, all countries are represented equally regardless of their sample sizes. SENWT can also be used when analysing the pooled sample  to ensure the equal contribution of each country to the results.1.21.2.1The parental engagement scale was constructed using the following questions: Q21_2, Q21_3, Q22_2, Q22_3, and Q22_6 from the data set.•Q21_2 Follow my ideas about what my children need to learn•Q21_3 Mix my own ideas with the school's plan on what my children need to learn•Q22_2 I list and prepare the activities myself before developing them with my child(ren)•Q22_3 My children and I have a set home-schooling timetable.•Q22_6 I develop with my children spontaneous learning activities not necessarily school-related such as cooking, woodwork, online games, physical activities, etc.Always, Often, Occasionally, Rarely, Never (from 0 to 4)1.2.2Socioeconomic status (SES) was constructed using the following questions: Q5, Q7, Q13N, and Q14.Q5 What do you do in your main job? . This was an open question that was recorded into an ordinal variable following the list of occupations described in the one-digit ISCO .Q7 In a normal month, what is your total household income? This variable was recorded by grouping the income level reported in deciles of income within each country.Q13N is composed of How many usable devices are there in the house? .Q14 How many computers per child have you got at home?1.2.3Parental engagement scale was constructed as a second-order construct, with constructs measuring the parents’ level of parental acceptance and confidence in the use of technology as ‘tools’, ‘for social purposes’ and ‘self- perceived capacity’. The items asked parents about the frequency with which they carry out different activities using technology , and how confident they felt carrying out these activities .•tool=Q22_1 + Q24_1 + Q24_5;•social=Q21_4 + Q21_5 + Q21_6 + Q24_12;•capacity=Q24_2 + Q24_3 +Q24_4 + Q24_6 + Q24_7 + Q24_8 + Q24_9 + Q24_10+Q24_11+ Q21_7Parental acceptance and confidence in the use of technology= tool + social + capacity.1.31.3.1Confirmatory Factor Analysis (CFA) was used to estimate the model for the three scales and for each country using maximum likelihood (ML). Missing data was handled with listwise deletion. Model fit was evaluated using the Comparative Fit Index (CFI) and the Tucker-Lewis Index (TLI) as the goodness of fit statistics, and the root-mean-squared error of approximation (RMSEA) and the standardized root mean squared residual (SRMR) as residual fit statistics. Acceptable model fit was guided by the cut-offs  as suggested by Internal ConsistencyAfter constructing three scales, in order to evaluate reliability , we used Cronbach's alpha coefficient Multi-Group Confirmatory Factor Analysis (MG-CFA)In order to evaluate the extent to which the scales can be validly compared across countries and geographical areas, we ran Multi-Group Confirmatory Factor Analysis (MG-CFA) first for the pooled sample including all participating countries, and later for countries within sub-groups  1.4The following tables include important information for potential users to be able to interpret the scales correctly.1.4.1Fig. 11.4.2Table 201.4.3Fig. 21.4.4Table 281.4.5Fig. 31.4.6Table 362lavaan and lavaan.survey packages developed by Rosseel (2012) and Oberski (2014), respectively.The researchers employed an online survey research design to gather data from 2658 respondents from 23 countries all over the world. All countries are Cameroon, Ethiopia, Ghana, Tanzania Zanzibar, China , Japan, Belgium, Italy, Spain, Turkey, United Kingdom, India, Pakistan, Sri Lanka, Chile, Colombia, Costa Rica, El Salvador, Honduras, Mexico, Peru, Uruguay and the United States. The countries then divided into five regions which are Africa, East Asia, Europe, South Asia and America. Data were obtained using a semi-structured questionnaire . The queInformed consent was obtained from all individual participants included in the data collection process. The research ethics committee of the University of Bath provided ethical approval EIRA1–5408.Eliana Maria Osorio-Saez and Andres Sandoval-Hernandez: Conceptualization and Methodology; Nurullah Eryilmaz: Data curation and Data Analysis; Nurullah Eryilmaz and Eliana Maria Osorio-Saez: Writing- Original draft preparation; Andres Sandoval-Hernandez: Supervision; Yui-yip Lau: Reviewing and Editing; Eliana Maria Osorio-Saez, Nurullah Eryilmaz, Andres Sandoval-Hernandez, Yui-yip Lau, Elma Barahona, Adil Anwar Bhatti, Godfried Ofoe Caesar, Leví Astul Castro Ordóñez, Artemio Arturo Cortez Ochoa, Rafael Ángel Espinoza Pizarro, Esther Fonseca Aguilar, Maria Magdalena Isac, K.V. Dhanapala, Kalyan Kumar Kameshwara, Ysrael Alberto Martínez Contreras, Geberew Tulu, José Fernando Mejía, Catalina Miranda, Shehe Abdalla Moh'd, Ricardo Morales Ulloa, K. Kayon Morgan, T. Lee Morgan, Sara Mori, Forti Ebenezah Nde, Silvia Panzavolta, Lluís Parcerisa, Carla Leticia Paz, Oscar Picardo, Carolina Piñeros, Pablo Rivera-Vargas, Alessia Rosa,  Lina Maria Saldarriaga, Adrián Silveira Aberastury, YM Tang, Kyoko Taniguchi, Ernesto Treviño, Carolina Valladares Celis, Cristóbal Villalobos, Dan Zhao  and Allison Zionts: Data Collection and survey translation and adaptation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Nelumbo nucifera) seeds are widely consumed as functional food or herbal medicine, of which cotyledon (CL) is the main edible part, and lotus plumule (LP) is commonly utilized in traditional Chinese medicine. However, few studies have been conducted to investigate the chemical components of CL and LP in dry lotus seeds, not to mention the comparison between wild and domesticated varieties. In this study, a widely targeted metabolomics approach based on Ultra Performance Liquid Chromatography-electrospray ionization-Tandem mass spectrometry (UPLC-ESI-MS/MS) was utilized to analyze the metabolites in CL and LP of China Antique  and Jianxuan-17 . A total of 402 metabolites were identified, which included flavonoids (23.08% to 27.84%), amino acids and derivatives (14.18–16.57%), phenolic acids (11.49–12.63%), and lipids (9.14–10.95%). These metabolites were classified into ten clusters based on their organ or cultivar-specific characters. Most of these metabolites were more abundant in LP than in CL for both varieties, except for metabolites belonging to organic acids and lipids. The analysis of differentially accumulated metabolites (DAMs) demonstrated that more than 25% of metabolites detected in our study were DAMs in CL and LP comparing “JX” with “CA”, most of which were less abundant in “JX”, including 35 flavonoids in LP, 23 amino acids and derivatives in CL, 7 alkaloids in CL, and 10 nucleotides and derivatives in LP, whereas all of 11 differentially accumulated lipids in LP were more abundant in “JX”. Together with the fact that the seed yield of “JX” is much higher than that of “CA”, these results indicated that abundant metabolites, especially the functional secondary metabolites , were lost during the process of breeding selection.Lotus ( Nelumbo nucifera Gaertn., N. nucifera) belonging to the Nelumbonaceae family [Sacred lotus  and Jianxuan-17 (“JX”) showed great variation, of which “CA” possess slender shaped seeds, while “JX” seeds are round in shape, and the overall width and length of lotus seeds in “JX” were larger than those of “CA” . Here, aO-neohesperidoside and Isorhamnetin-O-Hexoside-O-rhamnoside-O-rhamnoside) belonging to flavonols were only detected in CALP , including primary metabolites  and secondary metabolites .Based on the MS analysis, a total of 402 different metabolites were identified, including 348, 338, 394, and 388 metabolites in cotyledon of “CA” (“CACL”), cotyledon of “JX” (“JXCL”), lotus plumule of “CA” (“CALP”), and lotus plumule of “JX” (“JXLP”), respectively . Of them in CALP B. Six meThere were 113 different flavonoids accounting for over 23% in all the four samples, which was the major category of the total detected metabolites C. Amino d-Alanyl-d-Alanine and p-coumaroylmalic acid) in this cluster were not detected in the CL of “CA”. Cluster III contained the largest number of metabolites that were mainly accumulated in LP. In this cluster, there were 84 (44.4%) different flavonoids  among different samples in this study, the value of the variable importance in the project (VIP) and foldchange for metabolites were calculated. The metabolites with fold change ≥2 or ≤0.5 and VIP > 1 for each comparison were selected as DAMs. A total of 109, 107, 253 and 266 DAMs were identified in the comparison of “CACL” vs. “JXCL”, “CALP” vs. “JXLP”, “CACL” vs. “CALP”, and “JXCL” and “JXLP”, respectively A. For thC-glucoside, genistein 8-C-glucoside, quercetin-3-O-α-l-rhamnopyranoside, and kaempferol-3-O-galactoside, belonging to flavonoids in “CL”, were more abundant in “JX” than in “CA”, whereas only one (cyanidin-3-O-glucoside) of the 36 DAMs belonging to flavonoids in LP were more abundant in “JX” than in “CA”. Three  glutathione) of the 26 DAMs in CL and three  of the 17 DAMs in LP belonging to amino acids and derivatives were also more abundant in “JX” than in “CA”. All of the 11 DAMs  belonging to lipids were more abundant in “JX” in the LP while two  belonging to lipids were down-accumulated in “JX” in the CL. In the comparison of CL vs. LP, most of the DAMs, except those that belonged to organic acids and lipids, were more abundant in LP than in CL in both “CA” and “JX”, indicating that flavonoids, amino acids and derivatives, nucleotides and derivatives, and phenolic acids had higher content in LP than in CL.The DAMs were classified into 12 categories and the main DAMs belonged to flavonoids, amino acids and derivatives, nucleotides and derivatives, phenolic acids and alkaloids . Eleven To further characterize the DAMs in the two varieties of the CL and LP tissues, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the annotated DAMs. The DAMs between “CALP” and “JXLP” were mainly enriched in flavonoid biosynthesis, purine metabolism, zeatin biosynthesis, glycerophospholipid metabolism, and isoflavonoid biosynthesis A, whereaLotus seed is a popular functional food and is always used to treat some ailments ,22. It cO-glucoside, isorhamnetin-O-rutinoside, isoquercitrin, astragalin, isovitexin, isoorientin, orientin, and luteolin-7-O-rutinoside. These metabolites were also identified in previous composition analysis in lotus seed [Food products derived from plants have abundant metabolites including not only the basic primary metabolites, such as lipids and organic acids, but also secondary metabolites that are beneficial in promoting health . Flavonotus seed ,30,31. Qtus seed , our stutus seed ,34,35, atus seed . Lotus stus seed . It was tus seed . A totaltus seed , thirty-tus seed  and anthtus seed , of whictus seed . MoreovePhaseolus vulgaris) using RNA sequencing technology indicated that nucleotide diversity and corresponding gene expression were decreased [The selection of target phenotypes adapting to environmental conditions and human usage directly influences the genetic structure and allele frequency, which regulates the gene expression and the final chemical products. The composition of metabolites in different species reveals the differential evolutionary process, such as in maize and rice . Multi-oecreased . “CA” isecreased , which aPhenotypic selection is the classical approach for breeding cultivars that adapt to the agricultural system, and molecular marker-assisted strategy has become increasingly popular in modern breeding because of the development of biological techniques. Recent studies have shown that metabolite data could be used to predict the performance of agronomic traits, ultimately assisting crop breeding ,47. Bree“China Antique” (“CA”) is a wild lotus variety, derived from long-term seed viability (1300 years) in Liaoning Province , and “Jihttp://www.anpel.com.cn/ (accessed on 4 January 2019).) before UPLC-MS/MS analysis . For preparing quality control (QC) samples, extracts of the twelve samples were combined into one sample that was further divided into three QC samples, which were injected as every fourth experimental sample and analyzed using the same methods for the experimental samples. The samples were freeze-dried and ground into powder using a mixer mill  with a zirconia bead for 1.5 min at 30 Hz. Then, 0.1 g of the powder was weighed and dissolved in 1.2 mL of 70% methanol solution, followed by incubation at 4 °C overnight, during which it was vortexed 6 times (30 s every 30 min) in total. Following centrifugation at 12,000 rpm for 10 min, the extracts were filtrated ) and MS system ) was employed to analyze the extracts. The experimental procedures were set as follows: UPLC column, Agilent SB-C18 ; the mobile phase system, pure water with 0.1% formic acid (solvent A) and acetonitrile (solvent B); gradient program, 95:5 V(A)/V(B) at 0 min, 5:95 V(A)/V(B) at 9 min and kept for 1min, 95:5 V(A)/V(B) at 11.1 min and kept for 2.9 min; temperature of column oven, 40 °C; flow rate, 0.35 mL/min; and injection volume, 4 µL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS/MS (ESI-Q TRAP-MS/MS). The UPLC-ESI-MS/MS system, including UPLC system , equipped with an ESI Turbo Ion-Spray interface, was used to acquire linear ion trap (LIT) and triple quadrupole (QQQ) scans, which was operated in positive and negative ion mode and controlled by Analyst 1.6.3 software . The ESI ion source was turbo spray; ESI temperature was 550 °C; ion spray voltage of positive and negative ion mode was 5500 V and 4500 V, respectively; ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 50, 60, and 30.0 psi, respectively; the collision induced dissociation (CAD) parameter was set as high. The 10 and 100 μmol/L polypropylene glycol solutions  were applied for instrument tuning, and QQQ and LIT modes were performed for mass calibration. QQQ scans were acquired as multi-reaction monitoring (MRM) experiments with collision gas (nitrogen) set to 5 psi. The optimized decompression potential (DP) and collision energy (CE) were used for individual MRM transitions and the specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period  (Chen ethttp://www.metware.cn (accessed on 4 January 2019)) was used to identify metabolites in CL and LP of “CA” and “JX”, which has been described in previous studies [+, Na+, NH4+, and other large molecular weight substances were removed. The quantitative analysis of metabolites was conducted according to the MRM mode using triple quadruple-bar mass spectrometry. The software of MultiQuant version 3.0.2  was used to integrate and correct the chromatographic peaks, and chromatographic peak area integrals represented the corresponding relative abundance of metabolites.The self-built widely targeted metabolome database MWDB ) and clustering analysis of metabolites was conducted using R package ‘pheatmap’ [2 (Fold Change) were used to screen differentially accumulated metabolites. The KEGG Compound database (http://www.kegg.jp/kegg/compound/ (accessed on 4 January 2019)) was used to annotate the identified metabolites. The annotated metabolites that mapped to pathways were subjected to metabolite sets enrichment analysis, in which the significance was determined by hypergeometric test’s p-values.The data of metabolites amount were scaled by unit variance, and then the principal component analysis (PCA) was carried out based on statistic function ‘prcomp’ in R software (R Development Core Team 2013; version 3.5.0; heatmap’ . For ortheatmap’ . Volcanoheatmap’ , and Venheatmap’ . The var


Biologic studies have suggested that tea may have neuroprotective activity. However, tea’s protective effect on cognitive function is controversial in human epidemiological studies, and no data, including the middle-aged, are available. The objective of this study was to investigate the association of habit, frequency, and types of tea consumption with incident cognitive impairment in middle-aged and older adults.Data from the Asymptomatic Polyvascular Abnormalities in Community study were used (aged over 40y). We gathered information on tea consumption, including habit, frequency, and types, via a standardized questionnaire and assessed cognitive function by Mini-Mental State Examination (MMSE) and/or Montreal Cognitive Assessment (MoCA). Three thousand eight hundred sixty-eight and 806 participants were selected in MMSE and MoCA subgroups. Multivariate logistic regression models were utilized to examine associations between tea consumption and cognitive impairment in middle-aged and older participants.p < 0.001) and high frequency (p trend < 0.001) of tea intake were associated with a lower risk of cognitive impairment. The risk of cognitive impairment was lower in green tea consumption  than other types . In MoCA analyses, we got similar results.In MMSE analyses, after adjustment for potential confounding factors, habitual (odds ratio (OR) 0.47, [95% confidence interval (CI) 0.33–0.68], Habitual tea consumption, especially high-frequency and green tea consumption, was significantly associated with a lower prevalence of cognitive impairment in middle-aged and older individuals.The online version contains supplementary material available at 10.1186/s12877-020-01848-6. Dementia is a significant public health issue resulting in the aging of population and requires urgent addressing and prevention , 2. DemeCamellia sinensis and different in the manufacturing process [Tea drinking was originated in China, and now, it has since spread throughout the whole world. Green, oolong, and black tea, as generally consumed forms of tea, originated from the plant  process . Green t process . Epidemi process , 9. Expe process , 11. In  process , 13, whe process , 15. Notn = 1503), results of Mini-Mental State Examination (MMSE) test (n = 38) or Montreal Cognitive Assessment (MoCA) (n = 3119), and complete data , leaving 3868 and 806 participants included in MMSE and MoCA subgroups, respectively  study is a cross-sectional, community-based, and observational investigation designed to explore the epidemiology of asymptomatic polyvascular abnormalities in Chinese adults. The APAC study population was a part of the previously described Kailuan cohort , 17, witMMSE and/or MoCA measured cognitive. MMSE is one of the most common and popular cognitive status measurements, but it has a ceiling effect to identify mild cognitive impairment . HoweverWe collected information about tea consumption via a standardized questionnaire. People were asked to report the habit, the average frequency, and the type of tea they consumed in the past 5 years. The habit is divided into “never” and “habitual.” For the frequency of tea consumption, we provided five choices, including “never,” “less than once/month,” “1–3 times/month,” “1–3 times/week,” and “≥ 4 times/week.” The type of tea consumption included “never,” “green tea,” “black tea,” and “other types” .2” “intermediate: 25–29 kg/m2,” and “poor: ≥ 30 kg/m2.” Physical activity was classified by the time of moderate or vigorous activity per week, as “≥ 80 min,” “1–79 min,” and “0 min.” Salt intake was categorized into “< 6 g/day,” “6–10 g/day,” and “> 10 g/day.” [Standardized questionnaires collected demographic information , level of education, lifestyle behaviors , and medical history . Body mass index (BMI) was calculated as weight (kg) divided by height in meters squared (m2), and classified into three categories according to BMI levels, as “ideal: < 25 kg/m g/day.”  Biochemi g/day.” .p < 0.05 were regarded as statistically significant.We conducted statistical analyses of different subgroups, respectively, because we classified participants in two subgroups according to cognitive function measurements. Statistical analyses were performed using SAS software . Continuous variables were presented as mean ± standard deviation (SD) and compared using ANOVA or Kruskal-Wallis. Categorical variables were presented as frequencies and percentages and compared using the chi-squared test. The types of tea consumption were grouped as “never,” “green tea,” or “others (including black tea and other types).” Logistic regression analysis was used to assess associations between habit, frequency, and types of tea consumption and cognitive function. We fit three multivariate logistic models. The crude model was the unadjusted model. Model 1 was adjusted for age and sex. Model 2 was adjusted for age, sex, and education level. Model 3 was adjusted for all the covariates in model 2 plus smoking, alcohol consumption, hypertension, diabetes mellitus, dyslipidemia, plasma concentrations of hs-CRP, BMI, physical activities, and salt intake. During the regression analysis, we divide the frequency of tea consumption into four groups: never, ≤ 3 times/month, 1–3 times/week, or ≥ 4 times/week. The “never” group of tea consumption was treated as the reference group. Odds ratio (OR) and 95% confidence interval CI) were calculated. All the statistical tests were two-sided, and differences with The baseline characteristics of participants, according to MMSE assessment, were presented in Table The ORs for cognitive impairment stratified by habit, frequency, and types of tea consumption were given in Fig. In this large population and community-based study, we investigated the association of habit, frequency, and types of tea consumption with the incidence of cognitive impairment in middle-aged and older adults. We observed that tea consumption was associated with a lower risk of cognitive impairment. Notably, high frequency and green tea consumption significantly decreased the risk of cognitive impairment.This study reported an inverse association between tea consumption and cognitive impairment and provided evidence for the neuroprotective activity of tea. Our findings were in line with previous studies , 13. TeaBesides, this study focused on cognitive function in the middle-aged. With the development of research on cognitive aging, factors affecting brain structure and function, which may result in cognitive impairment, have continuous and cumulative influences throughout the entire life . MoreoveHowever, several studies did not show the association. Some studies with uncommon tea drinking customs of participants , 36, andLimitations of this study need to be addressed. First, this study investigated the association between tea consumption and cognitive function at baseline in the middle-age and the elderly. It cannot draw a direct causal relationship between tea consumption and cognitive impairment. Second, this study utilized MMSE and MoCA to evaluate cognitive function, so we cannot identify diagnosis mild cognitive impairment or dementia. Further longitudinal studies, including daily living function assessments, are needed in order to validate our results. Third, only a few participants completed the MoCA test. We will expand the MoCA group size in future research to confirm the findings. All of these limitations need to be addressed in future researches. In this cross-sectional, community-based, and observational study, we observed that habitual tea consumption, especially high-frequency and green tea consumption, was associated with a lower prevalence of cognitive impairment in middle-aged and older individuals.Additional file 1: Supplemental Table S1. Odds ratio for cognitive impairment stratified by habit of tea consumption. Supplemental Table S2. Odds ratio for cognitive impairment stratified by frequency of tea consumption. Supplemental Table S3. Odds ratio for cognitive impairment stratified by types of tea consumption.