The recognition over the past decade that nearly all diseases are associated with changes in the microbiome has raised hope that microbiome-based therapeutics may cure many human ailments. Billions of dollars are being poured into microbiome-oriented biotech companies, and the coming years will undoubtedly witness the approval of the first generation of these products. The recognition over the past decade that nearly all diseases are associated with changes in the microbiome has raised hope that microbiome-based therapeutics may cure many human ailments. Billions of dollars are being poured into microbiome-oriented biotech companies, and the coming years will undoubtedly witness the approval of the first generation of these products. However, significant hurdles remain in expanding the pipeline and advancing these first-generation therapies. In this perspective, I will discuss the challenges related to identifying causal microbes, determining their mechanism of action, and characterizing the specific bacterial molecules required for disease protection. We are approaching these issues through a combination of clinical sampling, animal models, classic microbiology methodologies, and systems-based approaches. The field of microbiome research is on the cusp of being able to identify clinically actionable host-microbe relationships; increasing attention on identifying causal microbes and their bioactive factors will usher in the next generation of microbiome-based therapies. The numerous microbiome-disease associations identified thus far have generated a great deal of hope that understanding relevant host-microbe interactions will open the door to unlimited therapeutic applications . MicrobiClostridioides difficile infection (CDI) , which involves “transplanting” stool from a healthy individual to a diseased patient, with the idea that the “healthy” microbiota will correct whatever derangement may exist in the ill patient and therefore will alleviate symptoms. Fundamentally, this notion is agnostic as to the specific microbial dysbiosis and holds that any healthy microbiota will be curative. Given its success in treating medically recalcitrant on (CDI) , FMT is on (CDI) , which iAlthough FMT offers an important proof of concept that microbiome-based therapies can be effective, treatment is difficult to standardize across large populations because of variability among stool donors and among the endogenous microbiotas of recipients. In addition, FMT is fraught with safety concerns, its mechanism(s) of action is unclear, and its regulatory path forward is largely uncharted. Ultimately, FMT likely represents the prototype of microbiome-based therapies; subsequent generations will include the use of more refined bacterial cocktails, single strains of bacteria, and, ultimately, bacterial factors and/or metabolites as the therapeutic intervention. The field is at the very beginning of this transition phase: several companies have bacterial cocktails, single microbes, and/or prebiotics that are currently in clinical trials , and theHowever, significant hurdles remain in identifying and developing future next-generation microbiome-based therapeutics. Identifying specific microbes causally related to disease protection has largely been serendipitous without a systematic approach for doing so. The challenges associated with finding this proverbial “needle(s) in a haystack” have led to the notion that communities of organisms—not specific microbes—matter most in determining disease susceptibility. This idea is rooted in the fact that the microbiome contains many emergent properties and may not represent just the sum of its constituent parts . However6–My group’s research innovatively integrates gnotobiotic murine models, microbiology, and immunology with the ultimate aim of identifying commensal bacteria that protect against inflammatory and infectious diseases. As part of this research effort, we have worked to make easier the process of identifying causal microbes. In thinking about how to improve microbiome analyses for increased specificity, we noted that microbiome-wide association studies (MWAS) are conceptually modeled after genome-wide association studies (GWAS). The issue confronted in filtering lengthy lists of MWAS results is analogous to the problem with GWAS: the identification of a long list of genes—often with no clear biological rationale—implicated in disease pathogenesis . GWAS beIdentification of causal microbes, particularly single organisms, will open the door to the next frontier in microbiome science: characterizing the mechanism of action from the perspective of both the host and the bacterium. Elucidating the relevant host pathways and cell types required for disease protection will be critical so that the patient population can be appropriately stratified and targeted in future clinical trials. Having a single organism with a clearly defined phenotype will enable identification and characterization of bioactive microbial factors. These advances will not only increase our understanding of host-microbe interactions, but they will also serve to improve our knowledge of the underlying biology of disease pathogenesis. Given that it is not clear how generalizable the findings from these initial proof-of-concept bacteria will be, we and others in the field are beginning to employ a more global, systems-based approach to define a set of guidelines that govern productive host-microbe interactions.My group is approaching these questions by first identifying clinical issues that have significant unmet need and also have suitable animal models. We are applying the principles of microbe-phenotype triangulation to carefully selected clinical samples, isolating the identified taxa using directed culture techniques, and validating a causal microbe-phenotype relationship in animal models . Using tThe medical view of microbes has changed radically, moving from the early-20th-century notion that we are engaged in a constant struggle with microbes—an “us-versus-them” mentality that focused on the necessity of eradicating bacteria—to the more recent understanding that we live in a carefully negotiated state of détente with our commensal organisms. Instead of holding a simple view of microbes as enemies to be eliminated with antibiotics, scientists have increasingly recognized the critical role these organisms play in maintaining human health; loss of these host-microbe interactions in the increasingly sterile environment typical of Western civilization may have predisposed humans to the increased incidence of autoimmune and inflammatory diseases . As a prDespite initial hyperbolic hype and a few false starts, microbiome research now stands at the precipice of an ability to treat the fundamental basis of many diseases. As the field continues to mature, it will need to move beyond correlations and address causation. The identification of causal microbes and their mechanisms of action will create a “microbial toolbox” from which relevant bioactive strains can be chosen on a per-patient basis to correct specific underlying microbial dysbioses. In the near future, our knowledge base regarding the microbiome and its relationship to health and disease will be robust enough that this information can be applied in making important treatment decisions.


Minority Stress Theory posits that specific minority stressors, such as internalized homophobia and outness, predict negative health outcomes in sexual minorities. There has been substantial work in addressing these stressors in young adult samples; however, less in known about older adults. Older sexual minorities were socialized in a time in which same-sex relations were considered deviant and illegal, and therefore, have been exposed to a lifetime of marginalization. Although there is evidence that minority stressors negatively impact health in older adults, many studies exclude a complete lifespan sample. The goal of this study was to collect a sample of individuals in same-sex relationships ranging in age from 18-80  and examine whether age correlates with outness and internalized homophobia. We found that older participants had higher degrees of outness  and less internalized homophobia  resulting in less overall minority stress. We also analyzed different social resources that might explain the age-related decrease in minority stress and found that age was related to higher self-esteem . We tested whether self-esteem moderated the direct relationships between age and the minority stressors and found a significant interaction for internalized homophobia , but not outness. Our findings provide support that older sexual minorities report less minority stress and more research is needed to explain what promotes these trends.


Psychotic illness is associated with cognitive control deficits and abnormal recruitment of neural circuits subserving cognitive control. It is unclear to what extent this dysfunction underlies the development and/or maintenance of positive and negative symptoms typically observed in schizophrenia. In this study we compared fMRI activation on a standard Stroop task and its relationship with positive and negative symptoms in early psychosis  and chronic schizophrenia  patients. CHR-SZ patients showed reduced frontal, striatal, and parietal activation across incongruent and congruent trials compared to EP patients. Higher positive symptom severity was associated with reduced activation across both trial types in supplementary motor area (SMA), middle temporal gyrus and cerebellum in EP, but not CHR-SZ patients. Higher negative symptom severity was associated with reduced cerebellar activation in EP, but not in CHR-SZ patients. A negative correlation between negative symptoms and activation in SMA and precentral gyrus was observed in EP patients and in CHR-SZ patients. The results suggest that the neural substrate of positive symptoms changes with illness chronicity, and that cognitive control related neural circuits may be most relevant in the initial development phase of positive symptoms. These findings also highlight a changing role for the cerebellum in the development and later maintenance of both positive and negative symptoms. Abnormal cognitive control in particular has been proposed as a unifying theory of cognitive deficits in schizophrenia4 given its importance in a wider array of higher cognitive domains implicated in the illness, including episodic and working memory, flexible learning, task switching, response inhibition, and emotion regulation10. Moreover, symptoms of psychosis can be understood in terms of concepts relevant to cognitive control, namely deficits in the flexible, adaptive control of thoughts and behaviour, as well as a difficulty in separating relevant from irrelevant environmental stimuli. Empirically, impaired behaviour on cognitive control tasks has been shown to correlate with symptoms of psychosis15.Deficits in executive functions are a core characteristic of psychotic disorders. Patients with psychosis typically display impaired performance on tasks involving executive functions such as cognitive control, working memory, planning, and cognitive flexibility18. It has been suggested that abnormal recruitment of these neural circuits, particularly in the context of cognitive control processes such as response inhibition, contributes to the presence of both positive19 and negative20 symptoms. However, several studies have found no relationship between abnormal activation during response inhibition and symptoms22, and thus the degree of specificity of the neural substrate of symptoms to cognitive control related processing remains unclear.Functional imaging studies have demonstrated that cognitive control deficits in psychosis are accompanied by altered neural activation in relevant brain regions, including anterior cingulate cortex (ACC), supplementary motor area (SMA) and pre-SMA, superior and lateral prefrontal cortex (PFC), parietal cortex and aspects of the basal ganglia23, and thus regions associated with psychotic symptoms are likely to vary through the course of the illness. Such a process may mirror habituation in the processing of perceptual stimuli or motor and cognitive learning, whereby a shift can be observed in the neural regions associated with early developmental and later more settled processing during the same task25. Moreover, these neural mechanisms may become harder to identify as the illness progresses as they are increasingly subject to the confounding effects of illness chronicity, medication exposure, and socio-environmental deprivation; as well as any neuroplasticity related to persistence of these symptoms27. For example, a correlation between disorganization symptoms and dorsolateral PFC activation has typically been observed during cognitive control tasks in drug-naïve schizophrenia patients28, but was not observed in medicated patients21. It has therefore been suggested that antipsychotic medication may alter the association between brain activity and symptoms29. This has implications for studies attempting to pinpoint how symptoms develop; many fMRI studies include chronic patients with schizophrenia who have been medicated for a number of years.A further open question concerns the degree to which the neural underpinnings of symptoms in schizophrenia change over the course of the illness. It is possible that the mechanisms underlying the initial development of symptoms during the early phases of the illness differ from those in the later more chronic maintenance phase29. This method has the advantage of establishing a direct link between dysfunctional cognitive processing and clinical psychopathology. However, it relies on the presence of group differences in average activation, leading to a possible failure to identify associations in regions in which activation in patients does not differ from that in controls. Indeed, by its very nature, symptom heterogeneity can obscure group differences in average activation, such that etiologically relevant correlations between these symptom dimensions and neural activation patterns are not detected. This issue can be resolved by assessing associations between symptoms and neural activation on a whole-brain level independently of healthy control comparisons. The earliest studies took this approach with the use of resting state positron emission tomography (PET) by investigating associations between the severity of various symptom domains and regional cerebral blood flow (rCBF) in schizophrenia patients33 or by assessing the specificity of rCBF abnormalities to distinct symptom subgroups36. Throughout the last two decades, the focus has shifted increasingly toward the use of task-related fMRI, although only few studies report the relationship between brain activation and symptom severity irrespective of activation differences relative to healthy control groups40.A classic approach to identifying symptom-related brain activation is to clarify the regions showing abnormal activation in patients compared to healthy controls, and then to test for correlations with symptoms specifically within these regionsIn the current study, we assess fMRI activation on a verbal Stroop task in a large sample of patients with early psychosis (EP) and a further sample of chronic patients with a diagnosis of schizophrenia (CHR-SZ). Previous studies have stressed the involvement of cognitive control in the development of both positive and negative symptoms; the role in maintenance of symptoms is less clear. Thus, we hypothesise an association between symptoms and activation in cognitive control related regions such as ACC and SMA/pre-SMA in EP patients rather than chronic patients. In an exploratory approach, we compare correlations between activation and symptoms between EP and CHR-SZ groups to address the question of changes in the neural substrate of symptoms throughout the course of illness. If cognitive control related mechanisms are primarily involved in the development, but not maintenance, of symptoms, correlations will no longer be observed in the chronic patient group. We analyse both activation that is specific to response inhibition (incongruent vs. congruent condition) as well as activation averaged across both task conditions, reflecting the more general effects of cognitive control and attentional processing.41 and were thus collapsed into one group. Exclusion criteria for all patients were a history of neurological illness, current major physical illness, and drug dependency over the last six months. Positive and negative symptom severity was assessed by a trained researcher using the Positive and Negative Syndrome Scale (PANSS)42. Sample characteristics are presented in Table Ninety-five medicated early psychosis (EP) patients participated in the study. Inclusion criteria required patients to have experienced a first episode of psychosis within the previous five years, however the mean duration of illness was 1.7 years. In addition, 38 medicated chronic schizophrenia patients (CHR-SZ) were included (mean duration of illness 15.7 years). The CHR-SZ group consisted of treatment resistant and treatment responsive patients  who were previously shown not to differ on the Stroop taskSubjects performed a verbal Stroop paradigm while undergoing functional magnetic resonance imaging. A description of the task as well as imaging procedures and processing can be found in the Supplementary Materials.While behavioural performance was monitored during completion of the task to ensure that participants had understood the task instructions and were not responding randomly, reliable reaction times (RTs) from a number of trials could not be extracted from resulting audio files due to technical issues for some participants. Participants for whom RT data from more than 20 trials were unavailable were excluded from the behavioural analysis, resulting in a behavioural sample of 75 EP and 36 CHR-SZ patients. The reaction time (RT) Stroop effect  as well as the Accuracy Stroop effect  was assessed for each subject and compared between groups using t-tests.43 as implemented by FSL Motion Outliers. Contrasts of interest for the group level analysis were the Stroop contrast (Incongruent > Congruent) and the Task contrast (Incongruent + Congruent). Whole-brain activation was determined by a voxelwise z-threshold of 3.1 (p < 0.001 uncorrected) and a cluster significance threshold of p = 0.05 (whole-brain FWE corrected for multiple comparisons).Three EP subjects were excluded due to technical problems with the neuroimaging data, and a further four EP patients were excluded due to the absence of PANSS data, resulting in a final MRI sample of 88 EP and 38 CHR-SZ patients. The functional MRI data were analysed using the general linear model as implemented in FSL FEAT. For the first level analysis, regressors representing onsets of stimulus presentation for incongruent trials and congruent trials were included into the model. Each regressor was modelled as a delta function and convolved with a canonical hemodynamic response function. Standard and extended motion parameters, as well as a motion artefact confound matrix, which identified motion-corrupted volumes, were added as regressors of no interest. Volumes detected as corrupted were calculated by DVARSWe examined significant activation across both groups as well as group differences in activation for both of these contrasts. Correlations between brain activation and symptoms were initially assessed within each group separately. This was done in order to increase power to detect group-specific associations between symptoms and brain activation. Next, group × symptom interactions were assessed on a whole-brain level as well as within regions of interest (ROIs) defined by the significant group-specific correlation clusters. The ROI approach was taken in order to avoid false interpretations about group differences in correlations . Due to a significant group difference in chlorpromazine (CPZ) equivalent dosage and a marginally significant difference in sex distributions between the two groups, we tested whether regional effects from the group comparison analyses remained significant after controlling for CPZ equivalent dose and sex to exclude the possibility that findings were due to these possible confounds.The severity of positive and negative symptoms was somewhat higher in the CHR-SZ group compared to the EP group Table , howeverM = 141.70, SD = 105.05, p = < 0.001) and in CHR-SZ  patients, but this did not differ between groups, p = 0.14. There was also a significant Accuracy Stroop effect in EP  and CHR-SZ  patients, which did not differ significantly between groups, p = 0.17.Behavioural data analyses were conducted on 75 EP and 36 CHR-SZ patients. A significant RT Stroop effect was observed both in EP  contrast, significant activation was observed across both groups in regions classically associated with the Stroop task, including a midline section extending from ACC to supplementary motor area (SMA), bilateral precentral cortices, bilateral striatum and thalamus, and intracalcarine cortex. Significant deactivation was observed in right lateral occipital cortex  contrast, there was more widespread activation in areas including ACC/SMA and bilateral prefrontal cortical regions extending to precentral gyri, bilateral thalamus, putamen, brainstem, superior parietal cortices and cerebellum. Significant deactivations were observed in medial prefrontal cortex, bilateral superior frontal cortex, temporal poles, precuneus and lateral occipital cortices , left middle frontal gyrus , and left putamen   symptom interaction at whole-brain level.There were no significant correlations between Task contrast, EP patients showed a significant negative correlation between activation and positive symptoms in a midline precentral gyrus region including the dorsal SMA as well as left middle temporal gyrus  symptom severity were observed for the Task contrast revealed a significant effect in a cluster extending from the cerebellum to lingual gyrus  , as well as the middle temporal gyrus region , indicating that the correlation between positive symptoms and neural activation differed significantly between groups. While EP showed a negative correlation between symptoms and activation in these regions , a non-significant positive correlation was observed in CHR-SZ patients . The interaction terms in these regions remained significant after controlling for CPZ equivalent medication dose and sex, all ps < 0.05. For negative symptoms, a significant interaction was only observed in the cerebellar cluster . Again, while there was a negative correlation in the EP group , there was a non-significant correlation in the CHR-SZ group . The interaction in this region remained significant after controlling for CPZ equivalent medication dose and sex, p < 0.05. In contrast, no significant group × negative symptom interaction was observed in the SMA or precentral gyrus, indicating that the negative correlations between negative symptom severity and activation in these regions did not differ significantly between EP  and CHR-SZ patients .Next we tested for Group × symptom interactions in the significant correlation clusters observed in the EP group, in order to ascertain whether correlations in these regions specifically were significantly different in the CHR-SZ group. For positive symptoms, there was a significant interaction both in the midline precentral region including the dorsal SMA  patients compared to a sample of chronic schizophrenia (CHR-SZ) patients. Subjects showed overall activation in regions consistent with a cognitive control network including ACC, SMA, bilateral motor cortices, thalamus and striatum. The key finding of this study is a differential correlation between positive symptom severity and activation in SMA, middle temporal gyrus and cerebellum in EP and CHR-SZ patients, as well as between negative symptom severity and activation in the cerebellum in the two groups. Activation was inversely related to symptom severity in EP patients, but was not related to symptoms in chronic patients, with the exception of cerebellar activation, which showed a positive correlation with positive symptoms in CHR-SZ. These results suggest that the extent to which cognitive control related mechanisms are associated with symptoms differs as a function of illness chronicity, as is discussed in more detail below.44. The findings suggest that activity in this functional network may decrease over the illness course, consistent with previous findings that Stroop-related activation is negatively related to number of psychotic episodes experienced45.In the between-group comparison of activation, chronic patients showed reduced overall task activation across both trial types compared to EP patients in left middle frontal gyrus, right superior parietal cortex and left putamen, regions known to be important in cognitive control47 and is also known to be involved in resolution of semantic interference during overt responding48. Recent research suggests that similar mechanisms underlie semantic interference effects and classic Stroop colour-word interference49. Additionally, left middle temporal gyrus has been repeatedly implicated in schizophrenia, with reduced volume observed compared to healthy controls in chronic schizophrenia patients as well as first episode psychosis patients and their unaffected siblings52. Middle temporal gyrus activation has been observed during auditory hallucinations54 and functional as well as structural abnormalities have been associated with the severity of hallucinatory symptoms55. Activation of medial sensorimotor and premotor cortices is commonly observed in the Stroop task and is likely involved in error monitoring when exerting cognitive control56. Interestingly, SMA dysfunction has also been associated with alien limb syndrome, whereby movements are experienced as occurring independently of an individual’s own active control58. This is suggested to result from an inability to suppress implicitly activated motor programmes, normally controlled by SMA regions59. The resulting failure to distinguish internally and externally generated movement is akin to delusions of control commonly observed in psychosis. Our findings suggest that abnormal functioning of the SMA and middle temporal regions in the context of cognitive control may provide an important marker of positive symptom severity during the development stage rather than chronic stage of psychotic illness. We did not observe a correlation between symptoms and the region most classically associated with cognitive control, the anterior cingulate cortex (ACC). This relates to a meta-analytic review of neural activation during executive function tasks in schizophrenia, which showed abnormal ACC activation in patients across all tasks, but more dorsal effects closer to the SMA for the Stroop task specifically1.We observed a significant association between positive symptom severity and reduced activation in medial precentral gyrus including the dorsal SMA and left posterior middle temporal gyrus in EP. The left middle temporal gyrus is relevant for semantic control during language generation60 and several authors have emphasised the role of diminished cognitive control in the failure to learn reward outcomes, ultimately leading to motivational subcomponents of negative symptomatology such as anhedonia and avolition62. Interestingly, the correlation in SMA and precentral gyrus did not differ significantly from that in chronic patients, suggesting that the cortical neural substrate of negative symptoms remains more stable over the illness course, in contrast to that of positive symptoms.With respect to negative symptoms, we observed an inverse relationship with activation in SMA, right precentral gyrus and cerebellum in EP. Similar to the correlations with positive symptoms, this association indicates that EP patients with severe negative symptoms are less able to recruit these regions while exerting cognitive control. Negative symptoms have long been associated with cognitive dysfunction in schizophrenia64. There is an increasing consensus that the cerebellum acts as a coordinator or modulator of cortical activity via a cortico-cerebellar-thalamo-cortical circuit (CCTCC). CCTCC dysfunction in schizophrenia, driven or contributed to by cerebellar abnormalities, has been suggested to result in poorly coordinated mental activity (“cognitive dysmetria”), which may underlie a range of symptoms and cognitive abnormalities66. Underscoring a potential causal role of the cerebellum in symptom development, several recent interventional studies have shown that application of repetitive transcranial magnetic stimulation to the cerebellar vermis results in a significant improvement of negative symptom severity in patients with schizophrenia69.A striking finding in this study is that of reduced cerebellar activation with increasing (positive and negative) symptom severity in EP patients, but increased cerebellar activation with more severe symptoms in the CHR-SZ group. The traditional, limited view of the cerebellum as being primarily involved in motor or associative learning has been revised in recent decades with the accumulation of evidence showing that the cerebellum is involved in a multitude of higher cognitive processes70, but a negative correlation with negative symptoms observed in neuroleptic-naive first episode psychosis (FEP) patients by the same group71. Increased cerebellar activation in conjunction with increased symptom severity was interpreted by the authors as compensatory activity in response to decreases in other regions of the CCTCC. This interpretation is in line with reduced cortical and subcortical activation observed in our study in chronic patients compared to EP patients with relatively preserved task performance. Both hyper- and hypoactivations of the cerebellum have been observed in schizophrenia on a range of tasks including working memory and affective processing74, providing further support for a dynamically changing role of the cerebellum in psychosis.Our findings suggest that the role of the cerebellum with respect to symptoms differs in the early and late stages of psychosis. The observation is partly mirrored in findings from previous PET research showing a positive correlation between cerebellar rCBF and both positive and negative symptoms in chronic schizophrenia patientsTask contrast, which averages over both incongruent and congruent trials. Thus, the observed activation changes associated with this contrast are not specific to response inhibition; however they likely nevertheless reflect effects of cognitive or attentional control more generally. As can be seen in Supplementary Fig. Task contrast is highly similar to that associated with the Stroop contrast, with some additional activation in parietal, occipital and cerebellar regions. While it is reasonable to assume that the Stroop contrast taps into response conflict specifically, it is clear that congruent trials are not free from cognitive control  and as such subtracting these out may obscure effects relevant for control related processes75.It is important to highlight that all neuroimaging effects observed in this study relate to the A limitation of this study is the unequal sample sizes of the EP and CHR-SZ groups. It is possible that the EP sample was more sensitive to correlation effects due to a larger number of participants. However, the CHR-SZ consisted of a reasonably large sample of 38 patients, providing over 80% power to detect a comparable correlation of 0.4 as observed in the EP sample. We are therefore confident that our analyses did not miss a clinically meaningful true correlation between brain activation and symptom severity. In this study we cannot rule out a potential treatment effect caused by long-term medication exposure in the chronic patient group, who were also receiving a higher dosage of antipsychotic medication at the time of study. This is a common limitation inherent to research in chronic medicated patients, further emphasising the importance of studying neural correlates of symptoms early on in the illness. However, controlling for current medication dosage did not significantly alter the observed group differences, suggesting that this unlikely confounded our findings. A related issue pertains to the heterogeneity of the studied samples. The chronic cohort consisted of patients with a diagnosis of schizophrenia who had responded to antipsychotic treatment to variable extents. The early psychosis cohort, in contrast, were early on in their illness trajectory, and it is possible that a number of these patients will not go on to receive a diagnosis of schizophrenia. Future research will benefit from within-subject longitudinal follow-up designs in order to address the role of illness chronicity in the brain-symptom relationship more reliably. Finally, given the correlative nature of fMRI, it is not possible to infer a definitive causal relationship between cognitive control related neural mechanisms and symptom development. However, understanding the spatial distribution of the neural correlates of symptoms can provide insights into potential associated mechanisms and thereby offer a basis for future causal investigations such as non-invasive intervention studies. Future research could also usefully examine changes in functional connectivity beyond simple activation in order to establish whether a breakdown of network connectivity is involved in symptom development and maintenance.Overall the results of this study suggest that positive symptoms are associated with abnormalities in cognitive control related circuits in the early stages of psychosis, but once stabilised are no longer tightly coupled with mechanisms underlying cognitive control. In contrast, the cortical neural substrate of negative symptoms appears to remain more stable over the psychotic illness course. Finally, the findings suggest that initial formation and later maintenance of positive and negative symptoms may be associated with opposite mechanisms in the cerebellum. These results bear implications not only for the mechanistic understanding of symptoms but also research into potential targets for novel treatments, since different mechanisms may need to be targeted depending on the stage of psychotic illness.Supplementary material


Cercocebus atys atys) and western chimpanzees (Pan troglodytes verus), we tested what properties of potential partners influenced grooming decisions, including their relative value based on available alternatives and the social relationships of potential partners with bystanders who could observe the outcome of the decision. Across 1529 decision events, multiple partner attributes  influenced choice. Individuals preferred to initiate grooming with partners of similar global rank, but this effect was driven by a bias towards partners with a high rank compared to other locally available options. Individuals also avoided grooming partners who had strong social relationships with at least one bystander. Results indicated flexible decision-making in grooming interactions in both species, based on a partner's value given the local social environment. Viewing partner choice as a value-based decision-making process allows researchers to compare how different species solve similar social problems.Living in permanent social groups forces animals to make decisions about when, how and with whom to interact, requiring decisions to be made that integrate multiple sources of information. Changing social environments can influence this decision-making process by constraining choice or altering the likelihood of a positive outcome. Here, we conceptualized grooming as a choice situation where an individual chooses one of a number of potential partners. Studying two wild populations of sympatric primate species, sooty mangabeys ( To balance short- and long-term costs and benefits, animals have to decide when and how to interact with conspecifics so that the choice yields higher profit than an interaction with a random partner . IncreasPrimate grooming has been interpreted as an important commodity that can be exchanged for other services –17. GrooPrimate grooming interactions thus represent an ideal opportunity for examining the flexibility of the decision-making process underlying partner choice. Market theories predict that individuals adapt their partner choice to the local social environment which determines the supply ,14; for In grooming, bystanders can impact the outcome of interactions by actively disrupting or joining grooming , or by iThe presence and absence of group members can influence grooming interactions by changing the relative value of a commodity a partner has to offer ,36. PrevCercocebus atys atys) and western chimpanzees (Pan troglodytes verus) both live in large multi-male, multi-female societies, but with social systems that should differ in terms of the impact of the social environment on cooperative interactions. Sooty mangabeys are female philopatric, probably matrilineal [Sooty mangabeys  and field assistants for the Taï Chimpanzee Project's long-term database. Dominance ranks of all communities were calculated using a modification of the Elo rating method  with withglobx  with bin = 1529 grooming initiations over n = 8809 potential partners. We tested whether those individuals who were chosen differed significantly from those who were not, and if there were group differences. Model 2, focusing only on the chimpanzees, consisted of n = 1372 grooming initiations and n = 7781 potential partners. We tested whether chimpanzee males and females followed the same patterns when choosing grooming partners.The impact of partner rank can occur on two levels: Individuals have a global rank that describes their status in the community, and a relative rank in the party, capturing shifting partner value based on availability. The global and relative ranks are highly correlated and can therefore not be included in the same model; however, they might still vary in meaningful ways. For example, only 6 out of 52 potential partners in our sample were ever the highest-ranking individual by global rank, but 27 individuals at some point held the highest relative rank in the party. The same was true for the global and relative relationship strength. We fitted both Model 1 and Model 2 twice, once with the global ranks and DDSI of focal individual and partner, and once with their relative ranks and DDSI. We can use differences between global and relative rank/DDSI models to understand whether focal individuals represent potential partners independent of the social environment , or whether they represent individuals flexibly based on the availability of other options . We thus created two models to test the effect of multiple dimensions on grooming partner choice, and fitted each of them twice, using the global and relative variables, respectively. Model 1 consisted of each combination of grooming bouts with all potential partners for N = 32) and potential partners (N = 52), and their dyad combinations (N = 342) in models as random effects [n = 20 cases) and females with young offspring in East (n = 36 cases) led to relative uncertainty in the estimates for these variables in interaction with group identity.We included the identities of the focal individual . In the Global Model, the interaction between the squared global partner rank with global focal rank  and the three-way interaction between global partner rank, global focal rank and group  were significant. Taken together, these interactions revealed that in the mangabeys, there was an attraction to closely ranked partners, especially in high-ranking individuals (a). In the East community, low-ranking initiators targeted medium-ranked partners, while medium- and high-ranking initiators focused on closely ranked partners. In the South community, individuals targeted closely ranked partners, and this effect was stronger in low-ranking individuals. In the Relative Model, no interactions of rank were significant and the main effect of relative partner rank revealed that focal individuals in all three communities targeted partners who were high-ranking within the party .The full null model comparisons of both models using global and relative rank for all three communities were significant . Focal individuals in all groups targeted close social partners for grooming, but this effect was more pronounced in the mangabeys (a). There was also a significant main effect for the reproductive state of the partner in the Global Model  and the Relative Model . In both chimpanzee communities and the mangabeys, focal individuals preferably initiated grooming with females with infants less than 3 months of age .Both models showed a significant interaction between global/relative relationship value and group, respectively . Focal individuals preferably targeted individuals with whom they had an aggression in the 30 min prior.There were no group differences related to the effects of the bystander relationships in either model. The maximum DDSI of partner . The results for global ranks matched those of Model 1 . For the relative rank, male chimpanzees continued to show attraction to individuals with high relative rank, while females showed a bias towards individuals who were close to them in relative rank . Both models showed a significant interaction between the relationship value of the dyad and focal sex , with female chimpanzees targeting partners with whom they had a closer social relationship, while males did not or only weakly show this effect (b). The maximum DDSI of the partner in both models showed no sex differences and results matched those of Model 1. The effect of previous aggressions also matched the results of Model 1. In the Global Model, we found a significant interaction between the sex of the focal and the sex of the partner , with females in both communities preferably initiating grooming with males, but no specific preferences in males. In the Relative Model, there was a significant interaction between the two sex variables and group identity , indicating that while the aforementioned effect persisted in the East community, there were no preferences of focal individuals for either sex in South.The full null model comparisons of both the models using global and relative rank for the two chimpanzee communities were significant (Global Model: s effect b. The ma4.In this study, we conceptualized grooming as a decision-making event where individual chimpanzees and sooty mangabeys selected a partner from a group of available individuals. This allowed us to test the flexibility of grooming partner choice with regard to the number of variables both species integrate. We found that multiple partner attributes had an impact on partner selection, but also that the availability of alternative partners and their relationships influenced partner choice. Individuals avoided grooming partners that had strong social relationships with at least one bystander, suggesting these primates use flexible decision-making that assesses a partner's value given the local social environment. Partners are associated with different benefits and risks, and it seems that individuals in both species flexibly distributed their grooming to maximize benefits and minimize costs.Our results suggest that focal individuals in both species incorporated a number of different variables into their grooming decisions. Females in both species preferably initiated grooming with potential partners with whom they already had a strong social relationship, either due to a preference for bond partners  or as reIn both species, the wider social environment also influenced partner choice. Groomers in mangabeys and South chimpanzees focused their efforts on individuals that were close in global rank, as did medium- and high-ranking individuals in East chimpanzees, while low-ranking individuals in East chose medium-ranked partners. However, locally, focal individuals in both species preferably initiated grooming with individuals that had a high relative rank compared to other potential partners. Only female chimpanzees also preferred partners with a similar relative rank. This divergence between the two rank variables indicates that either, initiators were attracted to the highest-ranking individual available , but assThe presence of close social partners of a grooming target might make it more likely that these individuals intervene into the grooming bout  or that Though we predicted species differences in the bystander variables and the impact of rank on partner choice, due to the differences in hierarchy steepness ,100 and When discussing the cognitive requirements facing cooperating animals, researchers have focused on the challenge of representing previous interactions of the cooperators ,101. Her


Artificial insemination (AI) bulls provide a useful model system because of extensive fertility records, measured as sire conception rates (SCR). Genetic variants with moderate to large effects on fertility can be identified by sequencing the genomes of fertile and subfertile or infertile sires identified with high or low SCR as adult AI bulls or yearling bulls that failed Breeding Soundness Evaluation. Variants enriched in frequency in the sequences of subfertile/infertile bulls, particularly those likely to result in the loss of protein function or predicted to be severely deleterious to genes involved in sperm protein structure and function, semen quality or sperm morphology can be designed onto genotyping assays for validation of their effects on fertility. High throughput conventional and image-based flow cytometry, proteomics and cell imaging can be used to establish the functional effects of variants on sperm phenotypes. Integrating the genetic, fertility and sperm phenotype data will accelerate biomarker discovery and validation, improve routine semen testing in bull studs and identify new targets for cost-efficient AI dose optimization approaches such as semen nanopurification. This will maximize semen output from genetically superior sires and will increase the fertility of cattle. Better understanding of the relationships between male genotype and sperm phenotype may also yield new diagnostic tools and treatments for human male and idiopathic infertility.Whole genome sequencing has identified millions of bovine genetic variants; however, there is currently little understanding about which variants affect male fertility. It is imperative that we begin to link detrimental genetic variants to sperm phenotypes  The United States is among the world’s largest producers of beef and dairy products, two industries with a combined herd size of 93 million animals. The direct, retail value-based annual economic impact of the US cattle industry exceeds $74 billion . ImproveWhile the creation of mouse knock-out or mutant models may reveal if a particular gene is essential for fertility, we can only gain an understanding of individual differences in fertility, particularly in livestock, from studying naturally occurring polymorphisms. This is particularly difficult to accomplish in human because there is no such thing as a man with extensive fertility records. However, large animal models provide a unique opportunity since, for example, most AI services and SCR are recorded for breeding bulls, enabling researchers to link fertility records to genotypes in thousands of bulls. One of the goals of such efforts is to link polymorphisms in genes controlling spermatogenesis, sperm function and pre-implantation embryo development to aberrant sperm phenotypes in bulls used in AI. Accessible animal model cohorts include presumed infertile young sires that failed Breeding Soundness Evaluation (BSE) as well as fertile sires in active AI with highly accurately estimated SCR phenotypes from which phenotypically extreme animals can be sampled for sequencing and genomic data mining. Such stakeholder driven genomic research projects benefit commercial agriculture, human assisted reproductive therapies and the fields of genetics and reproductive biology. Research outcomes have the potential to improve the fertility of the US and worldwide cattle herds through the optimization of selection for bull fertility, based on genomics and semen phenotyping for sperm functionality traits. The direct beneficiaries of research efforts are the stakeholders in all sectors of the beef and dairy cattle industries, but particularly AI organizations, who seek to reduce the number of infertile and subfertile bulls brought into AI, which is a profit-limiting factor.Recently, Select Sires, one of the AI industry leaders, published a list of 36 Holstein AI-sires whose genomes harbor fertility-reducing haplotypes which are not observed in homozygous form in living animals; however, while these haplotypes are presumed to harbor early embryonic lethal mutations, the specific biological cause of reduced fertility associated with most of these multi-SNP haplotypes is not known . ImproveA number of recently completed and ongoing sequencing projects have identified potential loss of function  and non-synonymous amino acid mutations in fertility-related genes . This knSubstantial progress has been achieved in the optimization of reproductive performance of cows and heifers , 2011. WITGB5) and collagen type I alpha 2 chain (COL1A2), have been associated with variation in bull fertility  to enable association analyses to establish phenotype-to-genotype associations. This approach allows the identification of mutations associated with high/low fertility and also the associated sperm phenotypes, including sperm morphology, sperm protein structure, function and localization. However, the process of whole genome sequencing frequently will reveal hundreds of thousands of variants that differ in allele frequency between low and high fertility bulls and these cannot all be cost-effectively genotyped in order to test genotype-to-phenotype associations in large samples of phenotyped individuals. We have participated in the development of the BovineSNP50, BovineHD, PorcineSNP60 and the bovine GGP-F250  assays that query genotypes at from 54,000 to 777,000 SNP loci. These assays typically cost in the vicinity of $1M to develop  and this is beyond the means of most small collaborative projects. However, small numbers of novel variants  can cost effectively be genotyped as an “add-on” to the content of some of the assays commercialized by GeneSeek leading to per sample genotyping costs of $40–$60 enabling several thousand individuals to be genotyped within the budget of R01 or USDA NIFA grants.The goal of collaborative research in our laboratories is to link defective sperm phenotypes prevalent in bulls with low SCR to mutations in individual genes and cellular pathways controlling spermatogenesis, sperm function and the paternal genome influence on early embryo development. Our approach is based on whole-genome sequencing for variant discovery, followed by large-scale genotyping of bulls phenotypically characterized as infertile based upon BSE or with acceptable but varied fertility  in the U.S. Holstein population, as estimated using available sequence data for ~500 Holsteins including the 1000 Bull Genomes Project data . Detected mutations can be characterized in a number of ways, including whether the mutation is located within an annotated gene or a predicted regulatory region, and is likely to change the function of a gene via an amino acid substitution, a premature stop codon, frameshift or deletion. Mutations creating amino acid substitutions can be analyzed using SIFT  Identified mutation causes a predicted loss of function allele, predicted isoform or non-synonymous amino acid sequence change, (2) Mutation is at a moderate frequency  Gening SIFT  to detecing SIFT . Gene exing SIFT .In addition to LOF and amino acid substitution-causing polymorphisms, functionally impaired alleles that are in moderate frequency in cattle and therefore have the greatest impact on fertility will include polymorphisms in non-coding regions, particularly the promoter and untranslated regions of genes and within regulatory regions. Since the regions of the bovine genome that are involved in the regulation of gene function are almost completely unknown, the only way that we can potentially identify regulatory polymorphisms is to identify polymorphisms that lie with regions of the genome that are evolutionarily conserved among those ruminant species that have reference genome sequences. These regions of the genome are postulated to be conserved in sequence between species, because they have regulatory function and consequently strong purifying selection operates on these regions to maintain the integrity of the underlying sequence.via embryonic viability impact both male and female fertility and so are included in the variant filtering/selection process. From this process, polymorphisms can be prioritized for their inclusion in the design of custom SNP genotyping assays, to genotype sires with the highest and lowest SCR phenotypes, and also sires that historically failed BSE and that did not enter active AI service. The produced genotypes will be merged with any available BovineSNP50 and other chip genotypes and used to perform GWAA analysis on SCR and sperm phenotypes determined from the cryopreserved semen of these bulls.The genotyped sires are presumed to differ in their genetic merit for SCR because they individually harbor different numbers of deleterious alleles and/or alleles that differ in their severity of effect on fertility. Recessive lethal alleles that impact fertility This approach identifies variants that are associated with male fertility or sperm phenotypes, which does not imply their causality. Establishing the causality of a DNA variant is analogous to presenting compounded evidence in a criminal trial to conclude guilt beyond a reasonable doubt. If a variant can be shown to have a functional effect on gene expression or the protein encoded by that gene and if the variant is the most strongly associated with a trait of all tested variants in the genomic region in several independent replicated studies, we might consider this to be sufficient evidence of causality. For the purpose of producing genomic estimates of genetic merit, there is no need to establish or even rely upon the causality of effect of a variant. All that is required is that the alleles at the variant consistently predict the animals with enhanced fertility across different populations (same breed in different countries or different breeds) of cattle. This requires the presence of very strong linkage disequilibrium (correlation) between the alleles at the variant and the alleles at the causal variant, and of course, this relationship is perfect when the tested variant is the causal variant. To establish the predictive capability of variants identified as trait-associated in a GWAA, the simplest strategy is to include these variants on the genotyping assays that are now being widely used in the cattle industries to enable the genotyping of hundreds of thousands of animals from different breeds. If the variants consistently predict high SCR bulls using AI data recorded for Holsteins, Jerseys, and Norwegian Red dairy cattle and U.S. Angus and German Fleckvieh cattle, we can conclude that the variant is very likely causal.via a change in the quantity, localization and function of the sperm protein encoded by this gene. By sequencing the genomes of subfertile bulls with sperm phenotypes characterized by cell imaging, proteomic analysis and most conveniently by using high throughput multiplex flow cytometry candidate variants can be identified that underlie abnormal phenotypes. Ultimately, by sequencing sufficient numbers of bulls with aberrant sperm phenotypes, identification of variants that are enriched in frequency in these animals relative to bulls with normal sperm phenotypes will elucidate the risk variants that are responsible for morphological sperm defects. Sperm biomarkers can be directly linked to sperm phenotypes using IBFC of spermatozoa labeled with fluorescently conjugated antibodies against target biomarkers (A deleterious non-synonymous substitution or LOF mutation in a gene controlling spermatogenesis or sperm function could render the carrier sire and some of its male offspring subfertile omarkers . CombiniTo validate a new biomarker, multiplex flow cytometry of previously validated sperm quality biomarkers  can be aBos taurus and over 17 million entries for Bos indicus. Some of these variants create variation in male fertility, however, the specific polymorphisms that affect sperm phenotypes and fertilizing ability, or that cause early embryo loss, are generally unknown. Assuming that polymorphisms in paternally- inherited, fertility associated genes expressed during spermatogenesis and preimplantation embryo development are responsible for male subfertility or infertility, and possibly also for early pregnancy loss, the tools are now available to allow us to link detrimental genetic variants to sperm phenotypes measurable in an individual semen sample that impact male fertility in vivo and in vitro.Whole genome sequencing has revealed that there are more variants present in the bovine genome than in the human genome reflecting the difference in age of the two species. We estimate that over 3000 cattle genomes have now been sequenced worldwide revealing over 100 million polymorphisms. dbSNP build 151 contains over 104 million entries for in vivo, measured by sire conception rate, correlates to some extent with sperm phenotypes, particularly when sperm phenotyping is biomarker-based. To identify genetic differences between fertile and subfertile or infertile sires, whole-genome sequencing can now be inexpensively used to identify polymorphisms with alleles that are differentially enriched in high versus low SCR bulls, or in fertile bulls versus yearling bulls that failed BSE. Assuming that fertile and infertile bulls fall on the opposite ends of the fertility spectrum due to polymorphisms in relatively few fertility-associated genes, these genes, and the responsible functional polymorphisms, can be revealed by sequencing relatively few infertile bulls. Likewise, by sequencing bulls with high versus low SCR and identifying polymorphisms with different alleles enriched between the two groups, alleles which influence semen fertility via semen quality or sperm morphology can also be discovered. Using the discovered polymorphisms, genotyping assays that are routinely used in the beef and dairy industries that undergo periodic redesigns can be redesigned to incorporate polymorphisms expected to impact sperm protein structure and function, as well as SCR variation. To link mutations influencing sire fertility to sperm phenotypes easily measurable in semen samples, high throughput multiplex flow cytometry, proteomics and cell imaging can be used to validate these variants as sperm biomarkers. In addition to polymorphisms in protein coding sequences, attention should also be paid to those encoding sperm-borne small non-coding RNAs that could impact early embryo development.Artificial insemination bulls provide a very useful model system for this purpose because their DNA is publically accessible, they have unparalleled, extensive fertility records from thousands of AI services, and their fertility This approach assumes that deleterious mutations in sperm-relevant genes will alter sperm phenotype and change the quantity, localization and function of sperm proteins, thus influencing a carrier’s fertility. Our approach at this stage is necessarily gene-centric due to the lack of annotation of regulatory regions within the bovine genome. However, mutations within sncRNAs and their targets as well as regulatory regions are likely to be even more important for quantitative trait variation than are mutations within gene coding regions and should be prioritized for future research as these genomic regions are elucidated. The focus on genetic control of bull fertility by no means diminishes the importance of epigenetic factors and natural fluctuations in sperm quality that are due to bull age or seasonal influences. Integrating the genome-wide polymorphisms and AI fertility records with sperm phenotypes and biomarker expression patterns will accelerate biomarker discovery and validation, improve routine semen testing in bull studs and identify new targets for cost-efficient AI dose optimization approaches such as semen nanopurification. This approach will also support industry efforts to maximize AI dose output from valuable sires and does not disregard the value of information provided by conventional semen analysis. As an example of the utility of this approach, nanopurification targeting aberrant sperm surface ligands made it possible to obtain full AI dose-level pregnancy rates when half-AI-doses of nanopurified spermatozoa were used, with no adverse effects in 798 inseminated cows, which gave birth to 466 healthy calves . The tec


ABM patients who displayed greater sustained bilateral amygdalar response in the aftermath of neutral stimuli displayed the least improvement in self-reported (but not clinician-rated) vigilance symptoms. In contrast, amygdalar response did not predict improvement in sham patients. Results suggest that in certain anxious individuals, the amygdala may have a robust protracted response even to subjectively neutral cues, which could make these individuals a poor fit for ABM because of its focus on repeatedly retraining attention toward neutral cues. Findings may help elucidate neural mechanisms of ABM and promote the identification of a subset of anxious patients who would be good candidates for this intervention.Individuals with clinical anxiety demonstrate an attention bias toward threatening information, which is thought to be partially driven by heightened amygdala activity to perceived threat. Attention Bias Modification (ABM) is a computer-based treatment that trains attention toward neutral stimuli and away from threatening stimuli. Alterations in initial processing of threat have been linked to ABM responses, but the impact of protracted processing in the aftermath of neutral and threatening information on ABM outcomes has not been well studied. Our study tested whether sustained activity in the amygdala, which occurred after neutral and threatening stimuli had been removed, could predict which individuals would respond well to ABM. Unmedicated anxious individuals underwent a baseline fMRI assessment during performance of a task sensitive to protracted emotional processing. Afterward, they were randomized to complete eight sessions of ABM ( Some of the most deleterious transdiagnostic symptoms of anxiety involve involuntary orientation toward and perseveration about potential threats 7. Thus, the development of novel mechanistic approaches to reduce vigilance may be an essential next step in advancing the treatment of anxiety disorders.Clinical and subclinical forms of anxiety represent a significant public health burden8. Specifically, anxious individuals orient attention more quickly toward threatening stimuli than neutral stimuli, indicating an AB toward threat9. This observation invited speculation that AB may be a mechanism underlying the development and maintenance of anxiety disorders. Therefore, Attention Bias Modification (ABM) treatment was developed to therapeutically exploit the potentially causal role of AB upon anxiety. ABM treatment seeks to modify AB by training patients to preferentially attend to non-threatening stimuli, rather than threatening stimuli, in the initial stages of attention orientation. In a seminal study, MacLeod and colleagues found that ABM was successful in modifying AB and that this AB modification reduced stress reactivity10. Following this study, a continually growing literature suggests that ABM treatment is effective in reducing anxiety symptoms13. However, subsequent meta-analyses examining the effectiveness of ABM treatment show that its potential beneficial effects on anxiety are inconsistent across individuals and studies14. In addition, anxious individuals as a group show a significant but small AB suggesting that there is clinically-relevant variability in AB across anxious individuals9. Thus, more research is necessary to understand which anxious patients will most likely benefit from ABM and why they do, since anxiety disorders affect a heterogeneous group of individuals.Among anxious individuals (as a transdiagnostic group), vigilance often manifests at the behavioral level in the form of an attentional preference for threatening information, or an attentional bias (AB)9. Thus, an individual differences approach that examines the timing of threat processing may capture critical, clinically relevant information. For example, prior research has shown that patients who exhibit larger baseline transient neural responses to threatening stimuli across a range of cognitive-affective brain regions display the greatest reductions in clinician-rated vigilance following ABM treatment15. This work suggests that patients who display an initial preferential response toward threat in AB-related brain regions are best suited for ABM treatment, which is consistent with prior behavioral work that has shown that initial processing of threat is linked to ABM treatment responses16. However, some anxious individuals also exhibit protracted threat processing even after the stimulus has been removed17. Although it is yet to be established empirically, protracted processing in the aftermath of neutral and threatening information could also potentially impact outcomes following ABM treatment given that ABM is only designed to target AB during early stages of attention and while the threat stimulus is still present on the screen. Matching the timescale of intervention mechanisms is critical because if there is a mismatch between the time frame of AB that is shown by a given individual and the time frame of AB that is targeted with ABM, then the result will likely be a worse clinical outcome.To date, ABM studies have largely focused on group-level observations of whether anxious patients, as a group, benefit from ABM, which can mask considerable within-group heterogeneity linked with ABM treatment outcome. To understand which anxious patients are most likely to benefit from ABM, one important consideration may be the timeframe over which a given anxious individual exhibits AB toward threat. As a group, anxious individuals exhibit vigilance to threat during initial stages of processing 18. For example, Siegle and colleagues found that patients who exhibited greater sustained amygdalar responses in the aftermath of negative stimuli displayed greater improvement following cognitive-behavioral therapy19. Because amygdalar response to emotionally salient stimuli is thought to be a transdiagnostic marker of internalizing disorders21 and prominent clinical phenotypes of anxiety involve protracted forms of attention 23, it is possible that sustained amygdalar response in the aftermath of emotional stimuli could also be utilized to predict the effectiveness of interventions for anxiety, such as ABM.Research examining other internalizing disorders, such as depression, has shown that protracted neural processing in the aftermath of emotional stimuli can be an effective predictor of treatment response19, the amygdala was chosen as it is a well-replicated, key player in the neural circuitry implicated in threat processing in anxiety disorders24. Specifically, the amygdala acts as the central fear processor of the brain and initiates responses that encode the salience and affective properties of the stimuli and promote orientation toward fear-related stimuli25 as well as playing a critical role in protracted, ruminative attention to negative information27. Among anxious individuals, increased activation of the amygdala is broadly implicated in attentional bias towards threat29. Notably, in the context of anxiety, altered patterns of amygdalar response over time are not restricted to negative stimuli alone. Of particular interest here, anxious individuals sometimes exhibit protracted amygdalar responses to both neutral and threatening stimuli, which is thought to reflect an overgeneralized perception of potential threat31. Although it is yet to be tested, taken together, these studies strongly suggest that protracted amygdalar response to negative and/or neutral stimuli could be a successful predictor of ABM treatment outcomes.Building from the identified gaps in previous research, the current study asked specifically whether protracted activity in the amygdala in the aftermath of neutral and threatening stimuli could predict who would be a good candidate for ABM. In addition to its potential effectiveness in predicting treatment response32.The primary aim of the current study was to examine protracted amygdalar responses to both threatening and neutral stimuli as a neural predictor of success in ABM treatment. Of note, this study is one of the first to examine neural predictors of ABM response and is the first of which we are aware to extend the literature to examine amygdalar responses during a protracted emotion processing task. Because prior work has suggested that the amygdala plays a central and specific role in protracted processing of threat, we exclusively examined amygdalar responses. Critically, this decision reduced the need for multiple comparisons correction across the whole brain, which presents challenges for individual differences analyses of slow event-related timeseries data . Together, these factors generated compelling a priori hypotheses specific to the amygdala. To measure protracted amygdalar responses, we utilized a protracted emotion processing task that has been well-validated in the context of depression research33. Using a randomized controlled design, patients with transdiagnostic clinical anxiety were assigned to either active ABM or a sham training. Before the onset of treatment, patients completed the protracted emotion processing task, which quantified amygdalar responses in the aftermath of both neutral and threatening idiographically chosen stimuli in an fMRI scanner with the explicit goal of identifying which subset of anxious patients would be good candidates for ABM. Because ABM is only designed to reduce AB to threat stimuli at transient (rather than protracted) stages and only when threat stimuli are currently presented on the screen, we predicted that patients who exhibited greater protracted amygdalar responses in the aftermath of threat words would be a relatively poor fit for the mechanistic target of ABM and thus would display the least improvement with ABM (as defined by both clinician-rated and self-reported symptoms of vigilance). Similarly, because ABM retrains attention toward neutral stimuli, we hypothesized that patients who exhibited greater protracted amygdalar responses in the aftermath of neutral words would also be a poor mechanistic fit and display the least improvement with ABM.Patients across multiple diagnostic categories were recruited for the current study, as transdiagnostic approaches have become increasingly important in advancing the field of psychiatry by better representing the real-world clinical patient populationn = 49) or a sham training (n = 21) . Uneven allocation to the ABM versus sham conditions was used to allocate a greater proportion of available funds towards characterizing the ABM sample in an effort to enhance statistical power in the active ABM group, as the primary study aims focused on mechanistic predictors of ABM response. The sham condition was included so that we could probe the specificity of our results to active ABM, though power was constrained to detect effects statistically moderated by condition. 94% of randomized patients completed their assigned treatment condition and the post-treatment assessment , word pairs  were presented vertically for 500 ms, followed by a probe (“E” or “F”) in either the upper or lower word location. Patients responded via button press to indicate the probe letter displayed.Patients and clinical assessors were blind to treatment assignment. The ABM and sham conditions were modeled after prior studiesThe ABM condition was identical to the sham condition except for the relation between the probe location and the threat word in the threat-neutral trials. In ABM, for 100% of threat-neutral trials , the probe replaced the neutral word in a threat-neutral pair, thereby shaping attention away from threatening cues through practice. In the sham condition, the probe replaced either the threat or neutral word with equal likelihood.Outcome measures were collected at two timepoints: at a pre-training baseline visit (completed approximately 1–2 weeks prior to the beginning of attention training) and at a post-training visit . Residual symptom scores post-treatment (regressing out pre-treatment scores) were calculated within each treatment group, where lower numbers indicate a greater decrease in symptoms (more favorable outcome) relative to other individuals in the same treatment group. Conversely, a higher score indicates symptoms remained high after treatment relative to other individuals in the sample. To assess primary clinical outcomes, the study employed both self-report and clinician-rated outcome measures of vigilance. See 35. The Anxious Arousal subscale was of principle interest given its capacity to capture clinically relevant symptom patterns of anxious vigilance within transdiagnostic disorders. In the current study, internal consistency for the MASQ anxious arousal subscale was good (α = .88).The primary self-report outcome for the trial was the Anxious Arousal subscale from the Mood and Anxiety Symptoms Questionnaire . The MASQ is a well-validated questionnaire that assesses the severity of anxious symptoms and allows discrimination between anxiety symptoms and general distress, with the latter being common across a range of internalizing and externalizing disorders36, which sums two sub-items assessing frequency and intensity of vigilance . To ensure transdiagnostic relevance of this measure, assessors were trained to provide idiographic examples of vigilance that were relevant to the participant’s principle anxiety domains . To assess inter-rater reliability, a random subset of videotaped interviews (15%) was scored by a second rater, and 100% reliability was obtained.The primary clinician-rated outcome for the trial was the “hypervigilance” item of the well-validated Clinician-Administered PTSD Scale (CAPS-vigilance)n = 2). Data on this fMRI task were not acquired from an additional five subjects due to scanner time constraints. Critically, excluded subjects (n = 7) did not differ significantly from included subjects on any study variable.T2*-weighted images depicting BOLD contrast  were acquired on a 3 T Siemens Trio. Visual stimuli were presented on a rear projection screen connected to a computer running E-Prime and viewed through a mirror attached to a head coil. The patient responded to stimuli using a 5-button glove connected to the computer. Standard preprocessing steps were applied using Analysis of Functional Neuroimaging (AFNI) including slice time correction, motion correction, linear detrending to correct drift, outlier rescaling, temporal smoothing, spatial smoothing, and nonlinear warping to the Montreal Neurological Institute Colin-27 brain set. Patients with excessive motion during the task  were excluded from analysis  and neutral (n = 15) trials were presented, using words drawn from the patient’s idiographic threat and neutral word lists, respectively (a random 50% of the words from each list were repeated a second time after each list had been presented in full)  were considered robust if they persisted for at least four consecutive time points. This time-region was identified using Guthrie and Buchwald’s technique to control for Type 1 error in timeseries data38.We chose two a priori brain regions of interests (ROIs), the left and right amygdala, known to be involved in anxiety and processing of threat. These two ROIs were defined using the Automated Anatomical Labeling atlas (AAL)n = 66) completed their assigned treatment condition and returned for post-treatment assessment. Of these, 59 patients  had usable fMRI data within motion limits. One patient in the ABM condition was excluded due to the onset of psychosis during the treatment phase, leaving 58 patients for analyses. Prior published work from this sample showed pre-to-post treatment symptom decreases across a range of clinician-rated and self-report variables, with effect sizes generally favoring ABM over sham  were good to excellent  was significantly and positively associated with MASQ anxious arousal residual scores. Similarly, right amygdala activity following the onset of the digit memory task  was significantly and positively associated with MASQ anxious arousal residual scores. These findings are depicted in Fig. threat trials was significantly associated with reduced anxious arousal following ABM. Finally, to explore specificity of findings to the ABM group, we repeated these analyses in the sham condition. We used lenient significance test thresholding to offset the reduced power due to smaller sample size in the sham condition . For both the left and right amygdala, there were no significant correlations between amygdala activity and anxious arousal MASQ residual scores across sham patients during the neutral or threat trials. These findings are depicted in Figure Activity in both the left and right amygdala during rs <.32).Neither left nor right amygdala activity during neutral or threat trials was significantly associated with reduced scores on the CAPS (consecutive rs < .32). In addition, ratings regarding levels of worry about neutral and threat words were not significantly correlated with either primary outcome measure .Finally, given prior work from this sample that suggested that transient amygdalar response to threat stimuli significantly predicted ABM responseThe primary aim of the current study was to determine whether individual differences in protracted amygdalar response in the aftermath of threatening and neutral stimuli could predict ABM response. Our hypotheses were partially supported. Contrary to initial expectations, protracted amygdalar responses during threat trials of the Protracted Emotion Processing Task did not predict ABM outcome. However, consistent with our hypotheses, results indicated that sustained bilateral amygdala activity during neutral trials predicted poorer outcomes following ABM treatment in anxious patients, as defined by pre- to post-treatment changes in patient-reported symptoms of vigilance. We found that after a neutral word was presented briefly for 300 ms, a subset of anxious individuals appeared to have protracted bilateral amygdalar response to that information, even when they participated in a subsequent non-emotional distractor task. Critically, protracted processing in the bilateral amygdala was correlated with higher residual symptoms of vigilance following ABM treatment, providing evidence that protracted amygdalar response was a significant neural predictor of poorer ABM outcomes. This effect appeared specific to ABM as amygdalar response did not significantly predict improvement in the sham treatment.who might respond poorly to ABM , prior research can provide context as to why amygdalar response to neutral stimuli may be such a salient predictor of ABM outcome. Specifically, although fMRI research in anxious populations has consistently shown dysfunction in circuits involving the amygdala in response to threat24, there is also evidence to suggest that some anxious individuals exhibit similar disruptions in these circuits while processing neutral information31. The amygdala is an important contributor to potential biases for neutral stimuli among anxious individuals because it is responsible for interpreting and encoding the affective properties of stimuli25. Because ABM focuses on training attention away from threatening stimuli and toward neutral stimuli, it may not be well suited for anxious individuals who exhibit protracted amygdalar response after only a benign stimulus has been presented, as these individuals may react to neutral information in their environment as a salient and/or worrisome threat due to an overly threat-oriented appraisal of the environment.While the current findings provide direct insight into The current study’s Protracted Emotion Processing Task may have been particularly well-suited to assess individual differences in salience detection, as the initial prompt of “Does it worry you?” presented briefly at the start of each trial may have primed those who have difficulty distinguishing between neutral and threatening stimuli to exhibit higher amygdalar responses throughout the neutral trials. Alternatively, the task design may have led some patients to experience increased amygdalar reactivity specifically in response to the word “worry”, during both threat and neutral trials. However, in either case, only amygdalar response during neutral trials predicted ABM outcome, suggesting that individuals who are unable to suppress protracted amygdalar responses in the aftermath of objectively neutral stimuli may exhibit saliency biases that are not well-addressed by ABM. Further, this pattern was only apparent in amygdalar reactivity, and not in subjective responses to the neutral stimuli, suggesting that neural predictors of ABM treatment response may be more sensitive than self-report predictors, perhaps due to the reporting biases inherent to self-report indices. However, it should be noted that the subjective worry ratings made during the task were made using a restricted range and thus likely not especially sensitive to fine-grained subjective differences. Future research would benefit from a more sensitive self-report of worry during the task.31. Although the vast majority of ABM research has focused on transient responses to negative stimuli, the current findings suggest that protracted neural responses to neutral stimuli may limit the effectiveness of ABM as they violate the implicit contingencies of the treatment. Specifically, ABM treatments assume that patients will be able to quickly and accurately discriminate between neutral and threat stimuli given that ABM seeks to reallocate attention toward neutral rather than threatening stimuli in early stages of attention. Thus, individuals who have difficulty discriminating between neutral versus threatening stimuli, at the level of amygdalar reactions, may be less able to learn ABM contingencies, which could limit the effectiveness of the intervention.Notably, the current results further extend research suggesting that neural responses to neutral stimuli play an integral role in the development and maintenance of anxiety disorders16. Second, as seen in the current study, because ABM retrains attention to be systematically directed towards neutral stimuli, individuals who exhibit protracted levels of amygdala activation in the aftermath of benign stimuli are likely to be poor mechanistic candidates for this intervention as ABM does not address processing biases related to neutral stimuli. Of note, findings from Price et al.15. and the current study were conducted using the same sample and thus provide opportunities for direct comparisons of these mechanistic candidates. Whereas the task used in Price et al.15. measured transient and sustained responses that occur during the continuous presentation of threatening and neutral images, the Protracted Emotion Processing Task from the current study measured processing that occurs after a threatening or neutral word has been removed and that persists even during a distracting cognitive task. While larger transient amygdalar responses to threatening images predicted superior ABM outcomes in the Price et al.15. study, protracted amygdalar responses in the aftermath of neutral words predicted worse ABM outcomes in the current study. These two discrete features of neural processing were not significantly correlated within the present sample. Together, these findings suggest that transient responses to threat stimuli and protracted processing in the aftermath of neutral stimuli are two critical and distinct individual characteristics of neural processing that each impact ABM effectiveness.Taken together, the results from the current study and prior work suggest that there are at least two critical mechanistic predictors of ABM. First, because ABM seeks to target initial attentional allocation to threat , patients who exhibit greater early neural and behavioral responses to threatening stimuli display superior outcomes following ABM treatment because their symptoms are well-suited to the mechanistic intervention target14. In the current study, disparate findings for clinician-rated versus self-report primary outcome measures could be in part due to differences in the specific aspects of vigilance assessed by the two measures. Specifically, the self-report measure of vigilance  mainly focused on psychosomatic symptoms that are experienced internally and pervasively in daily life. In contrast, the clinician-rated measure of vigilance  more specifically assessed how the patient monitored threat in the external environment. Because the Protracted Emotion Processing Task focused on internal perseveration about potential threats not yet materialized in the external environment , neural predictors from this task may have been better suited to predict more internally-experienced symptoms of vigilance and/or more generalized patterns of somatic anxiety symptoms, which was best assessed by the self-report measure. Future research will benefit from the addition of both self-report and clinician-rated indices that can simultaneously measure and parse symptoms of vigilance experienced during internal versus external states.It is important to note that the current findings were specific to pre- to post-treatment changes in patients’ self-reported symptoms of vigilance, and our findings did not extend to changes in pre- to post-treatment clinician-rated symptoms of vigilance. These findings were contrary to hypotheses and notable given that clinician-rated measures have shown more reliable and consistent effects of ABM compared to self-reportThere were several limitations of the current study that highlight areas for future research. First, although a strength of the study was the use of random assignment to active ABM versus the sham condition, the uneven allocation protocol that favored ABM assignment reduced power to examine neural predictors of treatment response in the sham condition. Relatedly, sham ABM (as defined in the current study) could be conceptualized as merely a lower dose of active ABM  and thus similar mechanisms may impact outcomes during lower-dose ABM, albeit with weaker effects. Thus, while there is preliminary evidence that the neural predictors of treatment response were specific to the active, full-dose ABM group, future research would benefit from larger samples to further test placebo and dosing effects or spontaneous recovery. Second, we selected and analyzed only two brain regions a priori  and therefore may be overlooking other parts of the brain where there could be significant correlations. While this approach preserves power by limiting the number of multiple comparisons , future, larger studies could employ whole brain analyses to identify dysregulated anxiety circuits that predict ABM outcomes. Finally, while the current study was designed to examine neural predictors of ABM outcomes in a controlled laboratory setting, future work should focus on the translation and dissemination of these findings into the clinical setting, including identification of predictors that do not rely on costly fMRI assessments.14. From a precision medicine standpoint, this type of individual mechanistic assessment may allow for identification of specific subsets of anxious patients who would be good candidates for ABM. This would be particularly useful if fMRI predictors can be translated into a clinically available form39. In addition, existing cognitive intervention approaches could be supplemented or refined to target protracted forms of neutral stimulus processing, which have not been a focus of prior attention retraining efforts, but may represent an opportunity to reduce overgeneralized threat perception biases in anxiety. Together, these strategies could potentially increase response rates and thus improve care for individuals with anxiety disorders.In conclusion, the current study provides preliminary evidence that protracted hyperactivity in the amygdala can be used to predict which anxious patients are most likely to benefit from ABM. Specifically, we found that protracted activity in the amygdala in the aftermath of a briefly presented neutral word, which persisted during a subsequent non-emotional distractor, may interfere with successful outcome in ABM. The results of the current study have both important clinical and research implications. For example, these findings add to a growing body of research that suggest there are critical individual difference factors that impact the efficacy of ABM  and that identifying individual differences that predict ABM response may be the key to understanding the inconsistency of efficacy findings in the ABM literature more broadlySupplement