Vineyards exposed to wildfire generated smoke can produce wines with elevated levels of lignin derived phenols that have acrid, metallic and smoky aromas and flavour attributes. While a large number of phenols are present in smoke affected wines, the effect of smoke vegetation source on the sensory descriptors has not been reported. Here we report on a descriptive sensory analysis of wines made from grapes exposed to different vegetation sources of smoke to examine: (1) the effect vegetation source has on wine sensory attribute ratings and; (2) associations between volatile and glycoconjugated phenol composition and sensory attributes. Sensory attribute ratings were determined by a trained sensory panel and phenol concentrations determined by gas chromatography-mass spectroscopy. Analysis of variance, principal component analysis and partial least squares regressions were used to evaluate the interrelationships between the phenol composition and sensory attributes. The results showed that vegetation source of smoke significantly affected sensory attribute intensity, especially the taste descriptors. Differences in aroma and taste from smoke exposure were not limited to an elevation in a range of detractive descriptors but also a masking of positive fruit descriptors. Sensory differences due to vegetation type were driven by phenol composition and concentration. In particular, the glycoconjugates of 4-hydroxy-3-methoxybenzaldehyde (vanillin), 1-(4-hydroxy-3-methoxyphenyl)ethanone (acetovanillone), 4-hydroxy-3,5-dimethoxybenzaldehyde  and 1-ethanone (acetosyringone) concentrations were influential in separating the vegetation sources of smoke. It is concluded that the detractive aroma attributes of smoke affected wine, especially of smoke and ash, were associated with volatile phenols while the detractive flavour descriptors were correlated with glycoconjugated phenols. Wines produced from vineyards exposed to bushfire smoke often have unpalatable levels of smoke related attributes including smoky, metallic, bitter, ash and medicinal flavour and aroma sensory descriptors ,2. TheseOur knowledge of smoke taint in grapes and wines has increased considerably in the last five years. For example, we now know that smoke affected wines contain many volatile and glycoconjugated phenols ,4,5,6,7.et al. ) was determined with a leave-one-out cross validation. Contributions of the x-variables to the PLSR model were examined using a variable’s importance for projecting (VIP) the data into x-y score [Data were analysed using a combination of univariate analysis of variance (ANOVA) and multivariate methods  and partial least squares regression (PLSR)). Prior to these analyses, all the data were checked for satisfying the underlying assumptions. For the ANOVA, these involved testing homogeneity of the fuel treatment group variances and normality of each variate. For the PCA and PLSR, multivariate normality was evaluated using the radius test as outlined in GenStat for Windows V17 . The radius test showed all 12 sensory attribute variates examined had a multivariate normal distribution (-y score . Accordim-cresol and p-cresol glycoconjugates.The controlled application of smoke to Merlot grapevines, using vegetation fuel sources from the Margaret River region, has produced wines exhibiting differences in smoke taint aroma and flavour. This study differs from previous studies examining the sensory profile of smoke tainted wine as it has allowed comparisons not confounded by differences in variety and winemaking. The results show smoke taint is not only due to lignin derived phenols exhibiting negative aroma and taste descriptors but also from an elevation of phenols that mitigate the negative descriptors due to their softening effect. While a large number of phenols were found to be elevated in wines made from wildfire exposed vineyards  and may


TEX with limited effector function accumulate in many chronic infections and cancers and are subject to inhibitory signaling mediated by several immune checkpoints . Of note, TEX represent considerable targets for immune-stimulatory therapies and are beginning to be recognized as a major correlate of successful checkpoint blockade approaches targeting the PD-1 pathway. TEX exhibit substantial functional, transcriptomic and epigenomic differences compared to canonical functional T cell subsets . However, phenotypic distinction of TEX from TEFF and TMEM can often be challenging since many molecules expressed by TEX can also be expressed by effector and memory T cell populations. Moreover, significant heterogeneity of TEX has been described, such as subpopulations of exhausted T cells with progenitor-progeny relationships or populations with different degrees of exhaustion or homeostatic potential that may directly inform about disease progression. In addition, TEX subsets have essential clinical implications as they differentially respond to antiviral and checkpoint therapies. The precise assessment of TEX thus requires a high-parametric analysis that accounts for differences to canonical T cell populations as well as for TEX subset heterogeneity. In this review, we discuss how mass cytometry can be used to reveal the role of TEX subsets in humans by combining exhaustion-directed phenotyping with functional profiling. Mass cytometry analysis of human TEX populations is instrumental to gain a better understanding of TEX in chronic infections and cancer. It has important implications for immune monitoring in therapeutic settings aiming to boost T cell immunity, such as during cancer immunotherapy.Mass cytometry has become an important technique for the deep analysis of single cell protein expression required for precision systems immunology. The ability to profile more than 40 markers per cell is particularly relevant for the differentiation of cell types for which low parametric characterization has proven difficult, such as exhausted CD8 However, in addition to this “horizontal” profiling approach, mass cytometry also represents a key tool suitable for deep “vertical” profiling of a given immune cell population and may reveal previously unknown heterogeneity within this population, such as complexity within CD8 T cells . In thisEX) are increasingly recognized as a distinct T cell population with a key role in many chronic infections and cancer. TEX were initially described in chronic viral infection, and many subsequent reports have highlighted the accumulation of TEX in the context of ongoing bacterial and parasitic infection, as well as cancer and autoimmunity , TEX have been perceived long-term as a population of suppressed effector T cells according to a “loss-of-function” model (EFF and memory T cells (TMEM) that is characterized by massive changes in their metabolism, transcriptome, and epigenome  models of acute and chronic viral infection. In these models, genetic differences between acute and chronic LCMV strains are minimal and immunodominant T cell epitopes are identical, facilitating cross-comparison of T cell phenotypes at the level of endogenous responses or by analysis of transferred virus-specific T cells sharing the same T cell receptor. During acute resolving viral infection, viral clearance after induction of functional effector T cells is followed by the establishment of a pool of memory T cells , 18 Fig. In shargoration .EX have been obtained by the study of antigen-specific T cells in chronic infection and cancer, and multiplexed tetramer analysis together with surface and intracellular markers by mass cytometry has allowed important insights into antigen-specific T cells , TEFF, and TMEM and additional subpopulations  cannot reliably distinguish TEX from the effector or effector memory T cell phenotype , while activation markers, such as CD38 are also induced.Many insights into T T cells , 23. Idehenotype . A likelEX biology, such as PD-1  for the assessment of exhausted T cells in chronic infections and cancer . To address these challenges, the characteristic impairment of polyfunctionality with regards to cytokine  but increased chemokine production  can be used to rate individual T cells for their functional chemokine/cytokine exhaustion profile on a single-cell level using a function-passed exhaustion score. Combined with a comprehensive phenotypic exhaustion profiling possible through the use of mass cytometry, the integration of TEX function as a separate metric of T cell exhaustion was able to reliably discriminate TEX from TEFF, TN, and TMEM  , 58, 59.g,” tSNE ], and clg,” tSNE , FlowSOMg,” tSNE , PhenoGrg,” tSNE  and manyg,” tSNE –66. For pression . This apsettings , includisettings . Moreovesettings , 68. Howsettings . As a cocontrols . These ccontrols . Clearly+ T cell subpopulations fit functional properties of T cell exhaustion  while in cancer, TEX more frequently exhibited higher expression of CTLA-4, Lag-3, and CD39. These data suggest a conserved biology of exhausted T cells in chronic infection and cancer but also highlight specific differences in exhaustion programs with potentially large translational relevance. For example, the altered co-expression patterns of immuno-regulatory molecules on different TEX populations across different disease entities or even across different stages of disease suggest that the therapeutic efficacy of combination therapies  could vary according to the TEX subset composition. Importantly, not all subsets of functionally exhausted T cells enrich with disease progression. For example, subsets with expression of PD-1 and co-expression of CD127, TCF-1, and CXCR5 were found enriched in HIV patients with relatively good disease control  and these “health-associated” TEX subsets were also detected in large amounts in tumor-surrounding macroscopically non-infiltrated lung compared to the tumor tissue.Several of the identified exhausted clusters enriched in severe disease contexts, such as severe HIV infection with possible AIDS, or in the tumor microenvironment of lung cancer patients. Phenotypically, these disease-associated exhausted T cells displayed co-expression of exhaustion-specific receptors such as PD-1, CD38 and a transcription factor signature characterized by high Eomes and TOX but low TCF-1 expression . InteresEX subpopulations identified by high-dimensional analysis using mass cytometry are in agreement with reports about severely exhausted T cells co-expressing several inhibitory receptors being linked to severe disease in chronic infection and cancer in HIV, HCV, HBV, and melanoma patients   , 44, 76. therapy , 42, 77. therapy .EX heterogeneity. A model of TEX heterogeneity including potential lineage relationships and the suggested markers required for minimal TEX immunoprofiling via mass cytometry is displayed in Thus, mass cytometry is perfectly suited for the interrogation of the clinically significant TEX are emerging as a central correlate and useful biomarker of successful immune checkpoint blockade therapies. In clinical trials with patients receiving checkpoint therapies, special attention has to be directed to immune-profiling panels, as therapeutic antibodies and staining reagents may compete for the same epitope. While combination stainings with secondary antibodies against the checkpoint reagents are established, these remain to provide challenges for bioinformatic analysis. Moreover, transient permeabilization protocols frequently used for intracellular barcoding in mass cytometry trials can reduce anti-PD-1 staining. Despite these challenges, mass cytometry has been successfully applied in multiple studies profiling CD8+ T cells. Reinvigoration of TEX compared to tumor mass was identified as a major correlate of the clinical response of patients with malign melanoma receiving checkpoint therapy with anti-PD-1 antibodies , while programs of homeostasis and access to anatomic niches can be shared by other TEX populations (including those linked to disease control) with memory T cells. Heterogeneous TEX subsets with different clinical roles have now been described in several translational settings of human chronic infection and cancer and are implied in differential responsiveness to immune checkpoint blockade.The inability to discriminate TEX populations, as the profiling approach outlined above already was designed to maximize utilization of markers informative for differences between TEX and functional T cell subsets. Of note, the combinatorial complexity of high dimensional data generated during immunome analysis by mass cytometry is different from other omics approaches. For example, simultaneous analysis of mass cytometric immune profiling together with transcriptome, microbiome, proteome and metabolome analysis during pregnancy indicated that the mass cytometry dataset was more informative based on modularity analysis  compared to the other omics datasets assessing significantly larger numbers of parameters (Other strategies that allow an in-depth profiling of the heterogeneity of exhausted T cell populations have been described, such as scRNA-Seq, which allows the analysis of T cell transcriptomes with single cell resolution. However, currently, these approaches have several limitations over the described mass cytometry approach as they are limited by the limited sensitivity for lowly expressed genes; low cell numbers that can be practically analyzed and therefore limited ability to identify rare populations and limited scaling toward cohort analysis; lack of information on protein expression missing potential post-transcriptional regulation; and, importantly, lack of testing for T cell function. It is further unclear whether excessively larger datasets of mRNA transcripts on a single cell level will reveal more relevant heterogeneity of Trameters .EX has been performed in murine models of chronic infection or cancer. With detailed resolution of TEX heterogeneity in humans now accessible through the use of specialized mass cytometry analysis, more detailed identification of TEX features associated with specific types of diseases and anatomical locations that will guide understanding of changes in TEX cell populations in chronic infections, cancer and even autoimmunity is expected. The expression of many exhaustion-related proteins involved in many immuno-regulatory pathways amenable to therapeutic intervention also suggests that TEX profiling might be required for adequate selection of combination therapies and could become indispensable for the rational design of personalized therapeutic treatments.To date the most detailed profiling of TEX using mass cytometry is expected to provide further insights into the biology underlying this special T cell differentiation stage and its role in pathogenesis and response to immune therapies in cancer, viral infection and autoimmunity.In sum, deep immune profiling of TFW and BB conceived and wrote the manuscript, revising it critically for important intellectual content.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.


Findings remained robust across subjects and were replicated in validation samples. Identified cortical regions implicate key hubs of the salience and default mode networks as neuroanatomical features of social impairment in ASD. Present results highlight the importance of subject-level markers in ASD, and offer an important step forward in understanding the neurobiology of heterogeneous disorders.The neurobiology of heterogeneous neurodevelopmental disorders such as autism spectrum disorders (ASD) are still unclear. Despite extensive efforts, most findings are difficult to reproduce due to high levels of individual variance in phenotypic expression. To quantify individual differences in brain morphometry in ASD, we implemented a novel subject-level, distance-based method on subject-specific attributes. In a large multi-cohort sample, each subject with ASD  was strictly matched to a control participant . Intrapair Euclidean distance of MRI brain morphometry and symptom severity measures  were entered into a regularised machine learning pipeline for feature selection, with rigorous out-of-sample validation and permutation testing. Subject-specific structural morphometry features significantly predicted individual variation in ASD symptom severity (19 cortical thickness features,  Definitive neurobiological mechanisms underlying ASD or other heterogeneous neurodevelopmental disorders have yet to be clearly delineated due to significant heterogeneity within and between individuals2. Magnetic Resonance Imaging (MRI) offers an in vivo method to assay neurobiological abnormalities, and has led to some promising findings of brain dysfunction in ASD neuroimaging3. However, group differences in brain structure or function in ASD remain frequently misidentified because of high levels of variability between and within individuals, giving rise to poor reliability and reproducibility of findings4.The autism spectrum disorders (ASD) are a group of neurodevelopmental conditions characterised by impairments in social communication and restricted and repetitive behaviours5. For example, age-related whole brain volume alterations6, cortical thinning7, and atypical surface area development8 in ASD are associated with continuous shifts throughout the lifespan. Identifying altered neurodevelopmental trajectories in ASD becomes even more complex as cortical volume can be further delineated into separable sub-components of cortical thickness and cortical surface, each with distinct genetic influences on development9. Previous reports of cortical measures of brain structural morphometry in ASD have been inconsistent, such as increased regional cortical thickness12, decreased13, or with significant cortical thinning in frontal, temporal or parietal regions14. Similarly, surface area in ASD has been reported to be increased15, decreased12, or not significantly different from neurotypical peers16. Alterations in grey matter volume in ASD have also been reported to be driven by the absence of typical age-related cortical thinning17. These mixed findings suggest that the expression of ASD in atypical brain structure is likely to differ between individuals with the condition, and across different age cohorts. Consequently, research efforts to identify consistent differences in the brain in individuals with ASD have remained inconclusive. Given the diverse nature of the condition, there is an increasing need for predictive brain-based markers that are sensitive to heterogeneity in the neurobiology and symptom expression of ASD18.In addition to the large phenotypic variation in individuals with ASD, neuroimaging studies are confounded by a number of methodological factors related to differences in image acquisition sites, anatomical sex, IQ, as well as age-dependent perturbations of neurodevelopment20. As conventional neuroimaging investigations typically rely on broad between-group comparisons without sufficient consideration of subject-specific effects, such approaches have been of limited yield in ASD research. Additionally, high-dimensional neuroimaging data with a large number of, often co-linear, features relative to small sample sizes pose further issues with increased risk of false positives. In particular for such a heterogeneous population as ASD, these challenges suggest that investigations of brain structure and function in ASD should incorporate appropriate subject-level modelling, with adequate consideration for common problems associated with large-scale high-dimensional neuroimaging data analysis22.Emerging work suggests that individual-specific variation in brain architecture may be a critical factor underlying idiosyncrasies in ASD symptom characteristics23, we developed a novel subject-level, distance-based method to test the hypothesis that neuroanatomical differences between subjects can explain individual differences in symptom severity. Using this approach on carefully matched case-control pairs at the individual rather than group level, we compared subject-specific differences in brain structural morphometry on MRI to associated intrapair differences in individual symptom severity. Specifically, we hypothesised that intrapair differences in cortical thickness and surface area features could predict individual variation in ASD symptom severity. By investigating relative individual differences within conservatively matched subjects, confounding effects related to inter-subject or cohort differences such as age, sex, intelligence and image acquisition site are also implicitly controlled for. Importantly, our approach implements well-validated and sophisticated machine learning and feature reduction techniques to ensure reproducibility of findings with reduced likelihood of false positives.Drawing from multi-disciplinary methodologies in ecology and twin modellingR2 = 0.153; p = 0.01, 5000 permutations). Based on 1000 iterations of the training loop, nineteen cortical thickness features were retained as predictors of individual differences in symptom severity  from differences in MRI features Fig. , we applR2 = 0.18, p = 0.006; Fig. The above procedure was repeated for cortical surface area measurements to predict differences in symptom severity. Ten surface area features were selected in the training dataset, with a favourable out-of-sample model fit  by comparison. Comparisons of model fit performance for both approaches are shown in Supplementary Fig. We validated our approach against more conventional methods of group-level prediction less robust to heterogeneity across individuals. As expected, without accounting for subject-level within-pair differences, regression model training to predict symptom severity based on group-level MRI features demonstrated a poor fit in out-of-sample validation tests  that also included females, and utilised a different analysis method with the novel subject-level distance-based approach. The consistent results across different age cohorts and samples suggest that differences in the identified surface area features may be a stable feature of ASD over time.Cortical surface area features identified in the present study were strikingly consistent with previous findings of altered surface area underlying increased grey matter volume in 3-year old boys with ASD25. Similar cortical regions underlying individual differences in ASD were identified in the present study in the surface area of the left caudal middle frontal, left supramarginal, right rostral anterior cingulate gyrus, and cortical thickness of the right inferior temporal gyrus, right cuneus, left middle temporal and right fusiform gyrus.Similarly, Wee and colleagues reported that morphological abnormalities in a set of cortical regions were the most discriminative features for the classification of ASD between 5 to 23 years of age. Using a multi-kernel learning strategy for feature selection, classification based on regional and interregional features in unseen samples achieved a sensitivity of 95.5% and specificity of 97%, with an accuracy of 96.27% and area under receiver operating characteristic curve (AUC) of 0.995, suggesting acceptable predictive utility28, posterior cingulate30, isthmus cingulate32, insula31, rostral middle frontal gyrus29, pars orbitalis32, pars triangularis27, medial orbitofrontal29, middle temporal gyrus33, inferior temporal gyrus33, fusiform gyrus29, inferior parietal lobule29, supramarginal gyrus34, lingual gyrus34, cuneus cortex34, and pericalcarine cortex28. Together with previous reports of clusters of cortical features25 similar to that identified in the present study, convergent results across multiple independent investigations suggest that atypical structural morphometry in these specific regions may be characteristic of altered neurodevelopment in ASD.Other neuroimaging investigations in ASD have also implicated the identified cortical regions in either or both hemispheres in the anterior cingulate36. The right middle temporal gyrus and right insula are part of a distributed cortical network for modulating attention to salient features of the multimodal sensory environment37. The right fusiform and occipital-temporal regions are highly specialised for face perception, recognition, and representation of facial features such as eye gaze and facial expressions that are necessary for social communication40. Notably, the identified cortical regions in the cingulate and insula implicate hub regions of the salience network (SN) and default mode network (DMN) that have been increasingly suspect to be aberrant in ASD41. The SN primarily anchored to the anterior insular and dorsal anterior cingulate cortex contributes to cognitive and affective processes such as social behaviour and communication, and the integration of sensory, emotional and cognitive information42. The DMN comprising the posterior cingulate, medial prefrontal and parietal cortices typically demonstrates reduced activity on task initiation, and functions to support self-referential and introspective states and social cognition43. Altered function of the salience network and DMN are highly consistent with ASD symptomatology, and emerging evidence suggests that specific features of cortical regions comprising these networks may discriminate ASD from neurotypical development3.These distributed cortical regions facilitate key aspects of social, language, and sensory functioning, deficits of which are consistent with clinical features in ASD. For example, the middle temporal and inferior temporal gyri subserve language and semantic processing, and visual perception44. Between-group differences in cortical thickness in ASD have also been reported to become non-significant after controlling for IQ, further highlighting the importance of matching on key confound variables13. Present findings support the hypothesis of Katuwal and colleagues that increasing homegeneity between case and control populations can reduce noise and improve precision in classification. Further, similar cortical regions were also implicated in ASD in cortical thickness of occipital-temporal regions and the anterior cingulate. By modelling individual differences in well-matched subgroups, consistent patterns of abnormalities across cortical regions and subjects may emerge or become more distinct. For example, intrapair differences between cases and controls in structural morphometry of certain regions, such as the anterior cingulate, appear to exceed differences observed in other cortical regions in a large proportion of the sample  to improve inter-subject homogeneity. With the robustness of present findings, such methodological considerations may be important for improved characterisation of heterogeneity in ASD brain morphometry that better reflects the continuous spectrum of symptom severity in this population.Interestingly in the current study, individual differences in cortical thickness between ASD and controls appeared to be more prevalent in the right hemisphere, in contrast to a left hemisphere bias for differences in surface area features. Cortical features that were implicated bilaterally were the surface area of the rostral anterior cingulate and isthmus cingulate, and cortical thickness of the middle temporal, inferior temporal and fusiform gyrus.53. For example, the study of Wee and colleagues noted significantly more abnormalities in the right hemisphere than the left in ASD25, in agreement with previous reports of a right-hemisphere bias in brain structural and functional asymmetry in ASD56. In contrast, a leftward lateralisation of abnormalities in ASD was reported for cortical thickness47 and surface area57. Distinct patterns of lateralisation between different morphological features in ASD may be related to the independent growth trajectories of cortical thickness and cortical surface area, each regulated by discrete genetic mechanisms9. Further, the direction of atypical asymmetry in structural morphometry has been shown to shift throughout development in ASD with decreasing leftward asymmetry with age, or could differ between high and low functioning individuals with ASD57. Mixed findings of structural asymmetry or direction of effects in ASD could thus be due to the diverse aetiology or distinct subtypes in the condition, as well as relative differences to control that vary across developmental stages due to altered developmental trajectories58. While we have shown that individual differences in specific cortical features are strongly implicated in ASD, the complex expression of age-dependent changes in ASD that are dynamic over time requires further investigation beyond cross-sectional studies.ASD has been related to a loss or inversion of typical patterns of brain asymmetry or lateralisation, with abnormal asymmetry in brain morphometry and connectivity associated with symptom deficits in ASD59. The robustness of the subject-level distance-based approach is nevertheless promising. Given that complex neurodevelopment conditions such as ASD likely stem from perturbations of anatomically distributed and interconnected neural systems, future applications of the subject-level distance-based approach to investigations of intrinsic brain networks may reveal more sophisticated insights into atypical neural mechanisms in ASD60. As brain structural connectivity constrains the development of functional networks across the lifespan, validation across different imaging modalities will be necessary to elucidate distinct neurobiological mechanisms, such as network analysis of white matter microstructure and functional connectivity investigations61.Together, present results derived from rigorous testing and validation techniques suggest that subject-level variation in brain properties are important characteristics in the expression of ASD. Present findings were limited to cortical thickness and surface area features, and other properties across different measures of brain structure and function could account for larger proportions of variance in symptom severityWe strongly encourage continued multimodal subject-level distance-based investigations to further challenge this hypothesis in large multi-site cohorts. As we have shown, investigations in ASD must necessarily demonstrate generalisability beyond in-sample modelling, given the high levels of inter- and intra-individual heterogeneity in this population. It is likely that the reliable identification of neural correlates in ASD strongly depends on quantifying individual variation in the phenotypic expression of ASD or the broader autism phenotypes. Such individualised approaches will be important for the development of clinical applications to aid management or personalised intervention strategies for each unique patient at the individual level.The robustness and generalisability of present findings is important progress in the search for neural correlates of heterogeneous disorders such as ASD, and offers promising insights into the neurobiology of ASD symptomatology. Based on present results with out-of-sample predictions, cortical hubs of the salience and DMN networks are likely to be implicated as potential neuroanatomical markers of ASD symptomatology. Increased reliability and validity of evidence for subject-specific alterations in brain structure and function will be necessary to advance current knowledge about the aetiology of ASD, where individual variability should be carefully modelled, rather than discarded as noise.62. Protocols specific to each imaging site for diagnosis, behavioural and cognitive assessment, and Magnetic Resonance Imaging (MRI) acquisition are available for public-access. Data collection and sharing for each site was approved by the respective ethics review board or ethics committee prior to data contribution to ABIDE-II with informed consent for study participation. All procedures were conducted in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki), and approved by The Royal Children’s Hospital Human Research Ethics Committee. The Social Responsiveness Scale (SRS) was used as a phenotype measure of ASD symptom severity63. The SRS instrument has been established to be a reliable and valid quantitative measure of ASD traits, demonstrating convergent validity with the gold standard Autism Diagnostic Observation Schedule and Autism Diagnostic Interview, and is able to discriminate ASD from other psychopathologies65. The instrument is commonly used for both screening and as a tool to aid clinical diagnosis.Data was obtained from the Autism Brain Imaging Database Exchange II (ABIDE-II) cohort across 17 independent imaging sites66, each ASD case was individually matched to the nearest control participant in age and IQ, restricted to a maximum distance of 0.25 standard deviations for each variable within each pair. For categorical variables, exact matching criteria were set for participant sex and image acquisition site. The genetic search algorithm67 aims to achieve optimal balance after matching by finding a set of weights for each covariate of interest. Matching balance was evaluated by univariate and paired t-tests for dichotomous variables and the Kolmogrov-Smirnov test for continuous or multinomial variables. The process selected n = 100 individuals with ASD to 100 controls eligible for inclusion (see Table Based on the multivariate genetic matching methodhttp://surfer.nmr.mgh.harvard.edu/). Visual inspection for movement artefacts were conducted for every subject to ensure data quality control. We inspected images for characteristic ring artefacts caused by in-scanner head motion, and evaluating grey/white matter and grey matter/CSF boundaries. Briefly, the FreeSurfer cortical surface reconstruction pipeline performs the following steps in sequence: non-uniform intensity correction, skull stripping, segmentation into tissue type and subcortical grey matter structures, cortical surface extraction and parcellation. Manual editing was performed on the white matter mask images to avoid false-positive errors in estimated surfaces and to ensure accurate masking of the dura. We further inspected images for topological errors during cortical reconstruction by inspecting surface outputs and parcellations for each subject. Cortical morphometric statistics based on the Desikan–Killiany–Tourville (DKT) brain anatomical atlas were used to estimate cortical thickness (mm) and surface area measurements (mm2) for each brain region. An advantage of the DKT atlas is that labelling is performed on a per-subject basis rather than the projection of a single parcellation onto every cortex, and is well-suited for the present subject-level analyses. Given that participants with high motion in one scan are also likely to move more in other scans during the same session68, we further evaluated head motion parameters in each subject’s resting-state functional MRI (fMRI) scan using framewise displacement (FD) as an estimate of volume-to-volume head movement. Inter-subject mean FD was not significantly associated with differences in SRS scores within matched subject pairs , suggesting that individual variation in symptom measures were not likely explained by differences in in-scanner head motion.Pre-processing and analysis of T1-weighted MRI was performed using FreeSurfer . The aim of machine learning is to estimate model parameters to make accurate predictions on new, unseen or held-out datasets. Elastic net is an embedded technique that combines machine learning and feature reduction functions by implementing a regularisation framework to obtain a reduced subset of selected features. Elastic net has been previously applied in machine learning for neuroimaging in Alzheimer’s disease classification and treatment outcome predictions in ADHD cohorts22. In regularised regression, λ is a parameter controlling for the strength of regularisation. The higher the value of λ, the more likely coefficients will be estimated towards zero with an increased penalty. α is the mixing parameter (0< α <1) and determines the relative quantities of L2 norm penalised regression (ridge regression) and L1 norm penalised regression . Elastic net is an approach that combines both the L1 and L2 penalties of the LASSO and ridge methods.Within-pair Euclidean distances were computed for every matched pair on clinical (SRS score) and demographic (age and IQ) continuous variables and brain structural morphometry measures . Age and total intracranial volume were regressed out using standardised residual adjustment to adjust for inter-subject disparity in age and head sizek subsamples of equal size. For each fold, one subsample is withheld for internal validation to test the model trained on k-1 subsamples. Each of the k subsamples were used as the validation set once per fold. Results from each fold were averaged to obtain a single estimation. Due to intrinsic randomness of model building, estimated coefficients may vary after each run. To account for stochastic error and to ensure robustness of estimates, the process was repeated for n = 1000 iterations, and the averaged coefficient weights used to generate predictions in the out-of-sample test set.A subset of the matched-pairs sample (33%) was held out as an out-of-sample test set independent of the subject data; that is these data were not used in the cross-validation steps (training set), and only examined as an independent validation of the model (test set). The remaining data was used as a training set to obtain optimal model weights for selected features. In the training set, we employed a strict k-fold cross-validation loop  to train the model to predict differences in symptom severity between matched cases and controls in the out-of-sample test set. The model was trained within a k-fold cross-validation loop (k = 10). The training set is first randomly partitioned into p-value of the initial model fit in the out-of-sample test set was computed as the proportion of iterations in the null distribution with model performance exceeding that of the initial model fit. Application and validation of recommended best practice for the regularised regression protocol are detailed elsewhere73. The entire procedure was repeated for cortical thickness and surface area measures. Final model weights were obtained by fitting selected features on the entire dataset to allow independent model testing. To validate our approach against group-level prediction methods, we repeated the entire machine learning pipeline on the same matched ASD cohort but at the group-level without within-pair distance computations, adjusted for age, sex, site, IQ and intracranial volume effects.Model goodness-of-fit was assessed by constructing a null distribution of symptom severity outcome. To generate a null distribution, the symptom severity (difference) variable was randomised across every sample observation of cortical thickness or surface area features using 5,000 permutations. For each iteration, model parameters were obtained using the same machine learning pipeline with regularised regression with elastic net penalty. The 74 using the MatchIt and boot wrapper tools75. Visualisations were generated with in-house scripts. Visualisation scripts for Fig. https://developmentalimagingmcri.github.io/freesurfer_statsurf_display/.Analyses were performed in the R environmentSupplementary material


ND) using the tracer 18F-FE-PE2I in 47 healthy adult subjects and examined relations between BPND in striatum, including its subregions: caudate, putamen, and ventral striatum (VS), and trait impulsivity  and novelty seeking , controlling for age and sex. DAT BPND in each striatal subregion showed nominal negative associations with total BIS-11 but not TPQ-NS. At the subscale level, VS DAT BPND was significantly associated with BIS-11 motor impulsivity  after correction for multiple comparisons. VS DAT BPND explained 13.2% of the variance in motor impulsivity. Our data demonstrate that DAT availability in VS is negatively related to impulsivity and suggest a particular influence of DAT regulation of dopamine signaling in VS on acting without deliberation (BIS motor impulsivity). While needing replication, these data converge with models of ventral striatal functions that emphasize its role as a key interface linking motivation to action.Impulsivity is a transdiagnostic feature of a range of externalizing psychiatric disorders. Preclinical work links reduced ventral striatal dopamine transporter (DAT) availability with heightened impulsivity and novelty seeking. However, there is a lack of human data investigating the relationship between DAT availability, particularly in subregions of the striatum, and the personality traits of impulsivity and novelty seeking. Here we collected PET measures of DAT availability (BP These psychostimulants have been shown to reduce impulsive symptomology  and increase self-control7. Given DAT’s critical role in striatal dopamine (DA) function and existing theoretical links between DA and impulsivity, individual differences in DAT are of particular interest as a potential influence on impulsivity. Individual differences in striatal DA signaling have been shown to relate to differences in trait impulsivity in human subjects9. Specifically, striatal d-amphetamine-induced DA release has been reported to positively correlate with Barratt Impulsiveness Scale (BIS-1110) scores in healthy individuals8. As d-amphetamine releases DA by blocking reuptake and reversing DAT transport of DA12, individual differences in responses to d-amphetamine could potentially indicate that DAT levels influence degree of impulsivity. While studies addressing this relationship are scarce, they are critically needed given that understanding DAT’s role in trait impulsivity could provide insights into the etiology and treatment of impulsivity in externalizing disorders. However, among preclinical and human studies that have investigated the relationship between DAT and impulsivity specifically, there remain questions about the direction of the observed relationship given the potential varied functions DAT plays in DA signaling across different timescales and disease states.Impulsivity is a transdiagnostic feature of psychopathology and is a particularly prominent symptom of externalizing disorders, including attention-deficit/hyperactivity disorder (ADHD), conduct disorder, antisocial personality disorder, and substance/alcohol use disorders. The dopamine transporter (DAT), along with the norepinephrine transporter  may represent a decreased ability for individuals to limit DA overflow in response to stimulation. Consistent with this model of DAT-binding indexing regulatory capacity, rodents with knockout of DAT show signs of heightened dopaminergic tone as reflected in heightened responses to novelty and spontaneous hyperlocomotion15. This preclinical work would suggest that lower DAT availability in humans would be associated with hyperactivity and novelty responding, which are often correlated with impulsivity and are key behavioral hallmarks of ADHD. It should also be noted that more modest reductions in DAT have sometimes16, but not always, been associated with enhanced impulsivity in rodents18. This inconsistency could be the result of the particular subdivision of the striatum where DAT reduction has occurred. Among the strongest evidence to date for a regulatory deficit model of lowered ventral striatal (VS) DAT causing impulsivity comes from work by Jupp et al. (2013), who found that rats with stably high impulsive (premature responding) performance on the five-choice serial reaction time task had lower DAT availability in the nucleus accumbens shell but not in the core or dorsal striatum as assessed with autoradiography19. Combined with data that high impulsive rats showed enhanced electrically induced DA release in the nucleus accumbens shell20, the authors interpret their result as an anatomically specific under-regulation of DA signaling due to impaired clearance of DA.The conflicting hypotheses about DAT and impulsivity in part reflects a core issue in the interpretation of measured DAT availability in human positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging. One potential interpretation is that measured DAT availability provides an index of DA regulatory capacity. DAT is critical in curtailing the large amount of DA released from phasic DA neuron spiking24. However, although the positive relationship between striatal DAT and other presynaptic markers of DA system integrity is robust in those with damaged DA neurons due to Parkinson’s or MPTP exposure, it appears much weaker in individuals with intact DA systems or normal levels of DAT25, likely reflecting the fact that there are substantial influences on DAT trafficking26 and DA synthesis27 that are superimposed on individual differences in the number of striatal DA terminals. It is thus unclear to what extent individual differences in DAT availability reflects overall DA system integrity in neurologically healthy adults.Another potential model for the interpretation of DAT availability is to treat it as a presynaptic index of overall DA system signaling capacity, reflecting a higher amount of DA neuron terminals. This model is often cited in the Parkinson’s Disease literature, which uses DAT availability as a measure of DA system integrity28, this effect was not observed in a large study using a similar tracer29. In addition to the relatively low sensitivity and spatial resolution of SPECT imaging30, a limitation of both of these studies is that they did not address VS DAT, focusing instead on either whole striatal values or caudate and putamen values. This limitation could be important given Jupp’s19 finding that reduced DAT in the nucleus accumbens  of rats is associated with higher impulsivity. Additionally, D2-like receptor reductions in high impulsive animals have been principally observed in the ventral rather than in the dorsal striatum31.Only two published SPECT studies in healthy adults to date have reported on the relationship between DAT availability and trait impulsivity in humans. While one reported that higher striatal DAT availability was positively correlated with trait impulsivity10), which have been found frequently to differentially correlate with a variety of psychiatric disorders33, other measures  of impulsivity33, and electrophysiological34 and neuroimaging markers35. Impulsivity is also closely related to the construct of novelty seeking (NS), which has been also linked to DAT functions. Preclinical work has found that high novelty responding rodents36 have decreased DAT affinity and reduced DA uptake at baseline37. Furthermore, knockdown of DAT is associated with increased hyperactivity in a novel environment and enhanced exploration of a novel object in mice38. In humans, NS is typically measured with the Tridimensional Personality Questionnaire (TPQ-NS)39, and heightened TPQ-NS was hypothesized by Cloninger to be the result of low basal DA tone41. One SPECT study has reported a positive correlation between DAT availability and TPQ-NS scores in predominantly male treatment-seeking alcoholic subjects but not controls42. The interpretation of this finding is complicated by the mixture of subjects with widely varying alcohol use histories. In addition, a recent SPECT study in opioid-dependent individuals found an inverted-U-shaped relationship between striatal DAT and TPQ-NS in male patients but a non-significant, positive linear relationship in controls43. As with the SPECT studies of impulsivity, no examination of the VS was provided in either NS study.Impulsivity is a heterogeneous trait. For instance, factor analysis of the BIS-11 demonstrates the presence of three different factors -N-(3-iodoprop-2-enyl)-2beta-carbofluoroethoxy-3beta-(4’-methyl-phenyl)nortropane). FE-PE2I has been used successfully in both non-human primates45 and human studies47 and has excellent reproducibility of nondisplaceable binding potential (BPND) estimates in striatum48. With the higher spatial resolution afforded by PET, we focused our FE-PE2I analysis on striatal subdivisions , which extends previous SPECT work that did not examine VS DAT43. While the past SPECT studies might suggest an opposing hypothesis, given previous preclinical results38, we predicted a negative relationship between VS DAT and aspects of impulsivity/NS in healthy human subjects.In summary, there is a discrepancy in results between existing animal and human studies of DAT and impulsivity/NS, with the failure to separately assess the VS in the human SPECT studies possibly contributing to this discrepancy. Here we tested for a relationship between the personality traits of impulsivity and NS and DAT availability assessed with the radiotracer -FE-PE2I PET scan as part of a larger ongoing study. Participants were recruited from the Nashville, TN area and all participants completed written informed consent approved by the Vanderbilt University Institutional Review Board. Exclusion criteria included any axis-1 psychiatric disorder, use of psychoactive drugs, illicit drug use, and alcohol consumption ~>5 standard alcoholic drinks/week. All participants underwent a structured clinical interview49 for these exclusionary criteria in addition to a medical physical (with electrocardiogram), a complete blood count panel, and structural magnetic resonance (MR) scans (T1 and T2-FLAIR weighted) to exclude pathology. All participants in the study passed a urine drug screen  for commonly abused drugs, including THC, cocaine, amphetamine, opiates, barbiturates, and benzodiazepines, administered on a separate prescreening/physical examination day.Forty-seven healthy participants  and was synonymous to a previously published preparation50. Serial scan acquisition was started simultaneously with a slow bolus injection  of the DAT tracer [18F]-FE-PE2I (specific activity = 2302 ± 1445 Ci/mmol). CT scans were collected for attenuation correction prior to the emission scan. FE-PE2I data were collected during one 61-min dynamic scan with acquisition times listed in Table 48, we chose the 61 min acquisition time to improve participant comfort given that the correlation between putamen FE-PE2I BPND collected after 61 and 90 min in 4 pilot subjects was extremely high . All PET data were collected on a GE Discovery STE PET scanner .FP-CIT BPND and BIS-1128 could thus result from differences in FP-CIT specificity for DAT relative to SERT versus the greater DAT specificity of the FE-PE2I tracer we used here84. Despite this concern, work in non-human primates suggests that FP-CIT signal in striatum is relatively specific for DAT over SERT85. More work is needed to dissociate the relative signals of the monoamine transporters using PET/SPECT radiotracers and new tracers may solve some of these issues87.An additional source of the discrepant results across studies may arise from the specific properties of the DAT ligands employed. [123I]β-CIT used most prominently in SPECT imaging of DAT has high affinity for the serotonin transporter (SERT)61. Our finding of a decline in striatal DAT with age is consistent with a past PET study using FE-PE2I47. Interestingly, despite a clear decline in striatal DAT with age, the relationship between DAT and personality across individuals was consistent across age groups after accounting for the effect of age. This suggests that trait relationships with DAT are maintained at least into late middle age despite the changes in the dopaminergic system across adulthood.A challenge in studying DA and psychological phenotypes is that there are robust declines in many aspects of the DA system with age4 and increase self-control7. It should be noted, however, that, while DAT is a target for psychostimulants  used to treat ADHD, they also target NET and SERT88. Thus the therapeutic efficacy of psychostimulants on impulsivity may not be at DAT, and thus DAT’s relationship to impulsivity may be independent of the effects of methylphenidate and/or (d-)amphetamine on ADHD symptoms. Studies investigating the relationship between DAT availability and ADHD using PET/SPECT have been inconsistent, with a meta-analysis showing that ADHD medication history may explain this inconsistency89: drug-naive ADHD patients tend to have lower DAT availability compared to controls while ADHD patients treated with psychostimulants display higher DAT availability compared to controls. Indeed, sustained methylphenidate treatment, which reduces ADHD symptomology , has been shown to increase DAT availability90. Importantly, the largest PET study in the meta-analysis that excluded ADHD individuals with past psychostimulant treatment found lower DAT availability in the striatum of ADHD relative to control individuals91. Thus our observation of lower DAT availability being associated with higher trait impulsivity fits with lower DAT observed in treatment-naive individuals with ADHD. Additionally, our work suggests that particular components of impulsivity in ADHD might be associated with DAT levels in specific striatal subregions: with VS contributing more to motor impulsivity (i.e. acting on impulse) and the caudate showing somewhat greater association with attentional impulsivity . However, at least in the present data, this does not appear to be a true double dissociation, which is not entirely surprising given the observed correlations between DAT expression in the striatal regions, and the correlations of the different BIS-11 subscales with each other. Nevertheless, future work should investigate whether the modulation of DAT signaling in specific striatal subregions results in different effects on the motor versus attentional aspects of impulsivity in the context of ADHD and other externalizing phenotypes. If the relationships we observed are confirmed in more detailed preclinical behavioral models, this work may aid in the development of novel treatments for these particular subcomponents of ADHD.If, as we interpret it, measured DAT in healthy young and middle-aged adults is primarily indexing DA regulation capacity, it may seem surprising that we found an inverse relationship between VS DAT availability and motor impulsivity given that psychostimulant treatments that block DAT reduce impulsivityND, and BIS-11 scores 64. The challenge of the structural literature is that there are multiple measures being used to assess different aspects of impulsivity, and this has at times limited the ability to replicate findings across studies and species. The present analyses should not be considered a direct attempt to replicate past findings but do suggest that differences in striatal or VTA gray matter are not driving our observed relationship between striatal FE-PE2I BPND and BIS-11 scores.We conducted exploratory analyses between gray matter volume of striatum and ventral tegmental area (VTA), FE-PE2I BP93. Low DAT availability in this volume transmission model would result in DA signaling across a wider brain circuit than would otherwise be the case. This less focused DA transmission would presumably produce more off-target effects as a result of the wider spatial distribution of DA signaling and could affect overall behavioral patterns in distinct ways94, perhaps by modifying the signal to noise of relevant action-value signals in striatum95. These effects might then manifest as motor impulsivity and lead to the same general finding of lower DAT being associated with heightened trait impulsivity. While we propose a regulatory capacity explanation for DAT’s effects on trait impulsivity, the lack of published data in vivo in nonhuman primates makes it difficult to confidently infer a clear mechanistic explanation for the relationship between DAT availability, DA signaling, and impulsivity in humans.While our sample size is larger than many PET studies, our findings of an inverse relationship between DAT availability and trait impulsivity will require replication. In addition, our emphasis on DAT’s role as a regulator of synaptic DA signaling warrants the caveat that DAT may be more important in regulating volume transmission of DA than its synaptic signaling per seIn addition, while we found relationships between trait impulsivity and DAT, our range of impulsivity was limited in this sample of psychiatrically healthy adults. This could have limited our detection of relationships. It is possible, for instance, that the caudate DAT relationship with BIS Attention and Nonplanning impulsivity would have been stronger if the range in these scores were larger. As this study focused on healthy adults, our findings may also be limited in their generalizability to patient populations, including those with ADHD. Investigating relationships between particular facets of impulsivity and DAT availability in clinical populations is needed.We observed an inverse relationship between striatal DAT availability and trait impulsivity in healthy young and middle-aged adults. Motor impulsivity showed a statistically significant relation with VS DAT after correction for multiple comparisons, suggesting potential regional and phenotypic specificity. The findings are consistent with preclinical data and have implications for the role of DA signaling in the impulsive traits observed in externalizing disorders. Future work that integrates DAT with other measures of DA signaling dynamics should allow for a more complete understanding of the role of DA functioning in impulsivity.Supplementary Material


Mesenchymal stem cells (MSCs) have a potently immunosuppressive capacity in both innate and adaptive immune responses. Consequently, MSCs transplantation has emerged as a potential beneficial therapy for autoimmune diseases even though the mechanisms underlying the immunomodulatory activity of MSCs is incompletely understood. Transplanted MSCs from healthy individuals with no known history of autoimmune disease are immunosuppressive in systemic lupus erythematosus (SLE) patients and can ameliorate SLE disease symptoms in those same patients. In contrast, autologous MSCs from SLE patients are not immunosuppressive and do not ameliorate disease symptoms. Recent studies have shown that MSCs from SLE patients are dysfunctional in both proliferation and immunoregulation and phenotypically senescent. The senescent phenotype has been attributed to multiple genes and signaling pathways. In this review, we focus on the possible mechanisms for the defective phenotype and function of MSCs from SLE patients and summarize recent research on MSCs in autoimmune diseases. Systemic autoimmune disease is caused by abnormal immune reactivity and antibody production to self-antigens with subsequent inflammation and damage to host tissues or target organs such as skin, kidney, joints, and muscles . SystemiRecently, mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy in refractory SLE patients because of MSCs’ strong immunosuppressive potential . MSCs arBone marrow-derived MSCs (BM-MSCs) were effective in clinical trials for steroid-resistant graft-versus-host disease (GVHD), a complication in allogeneic bone marrow transplantation . The MSCStem cells are a class of undifferentiated cells in multicellular organisms that are pluripotential and self-replicating. Through unequal cell division, stem cells produce one daughter cell with multiple differentiation potential and one identical daughter cell for self-renewal . There ain vitro and in vivo. The concept that the multipotential stromal precursor cells, MSC, identified by Friedenstein et al., could be used to therapeutic benefit was raised in 1991  that in turn inhibited CD4+ Th effector cell activation  , potent in vivo studies of MSC effects on CIA indicated that MSCs can effectively inhibit CIA inflammation and joint pathology  complex encodes the major histocompatibility complex (MHC) proteins that regulate the immune system in humans . Autoimmin vitro (HLA-G molecules are mainly expressed in human placental tissue . HLA-G gin vitro . The HLAin vitro . HLA-G5 in vitro . MAVS, Iin vitro and present a morphological appearance of senescence characterized by inflated volume, deeply stained nucleolus, and disordered cytoskeletal organization (in vitro proliferative rate is decreased, and the proportion of apoptotic cells increased with MSCs from SLE patients compared with those from healthy, normal individuals.BM-MSCs are gradually gaining attention because of their multidirectional differentiation potential that in turn may offer broad application prospects in clinical treatment of autoimmune diseases  and regenization . The in MSCs from SLE patients also exhibit impaired capabilities for differentiation, migration, and immune regulation . MSCs frMSCs from SLE patients have elevated MAVS, TGF-β, ROS, telomerase activity, DNA damage, and increase expression of senescence associated genes that block the cell cycle . MSCs frAutoimmune diseases create microenvironments of chronic inflammation as a consequence of immunological dysregulation that leads to excessive innate and adaptive immune stimulation. As a classic model systemic autoimmune disease, SLE can be characterized by the loss of peripheral immune tolerance, increased lymphocyte numbers and activation, and other immune cell activation, and autoantibody production, all of which contribute to pathogenic chronic inflammation . The hom+T cells to differentiate into Tregs (+ T cells to become effector cells, Tfh, Th1, and Th17, rather than iTregs further tipping the balance away from the immunosuppressive function of Tregs and loss of peripheral tolerance in SLE patients (In addition to inhibiting the secretion of proinflammatory cytokines, MSCs can also induce naïve CD4to Tregs , indicatto Tregs . In SLE to Tregs . Additiopatients .+ T cell proliferation and differentiation to Th effector cell subsets rather than the anti-inflammatory immunomodulatory function of MSCs not stimulated with TNF-α and IL-1β (The immunophenotype and immunoregulatory function of MSCs may be altered by microenvironments as a consequence of the local pro-inflammatory cytokine milieu . For exand IL-1β . In factnd IL-1β , a key rnd IL-1β .L-NMMA, a specific inhibitor of iNOS, could partially restore the generation of Tfh cells inhibited by MSCs in vitro (IDO, mainly secreted by DCs and macrophages, is an enzyme that mediates tryptophan degradation into immunosuppressive metabolites. Lipopolysaccharide and cytokines especially, IFN-γ, can induce the expression of IDO during inflammation or infection . Studiesin vitro .There is another reason why transplanted autologous MSCs may fail to suppress the excessive and damaging immune reactions in SLE patients. The number of autologous immunosuppressive MSCs present in relevant organs and tissues subject to chronic inflammation after autologous transplantaion may be too low to exert an efficient immunosuppressive effect. Compared with MSCs from healthy individuals, MSCs from SLE patients are morphologically biased toward senescent cells with reduced proliferative and migratory capabilities . WhetherSLE is a systemic autoimmune disorder involving a multitude of autoantibodies that are produced by over-activated B cells that circulate in peripheral blood and deposit in organs . The autTreatment of SLE is challenging because of clinical heterogeneity and unpredictable disease flares. The current guideline for treating moderate to severe lupus nephritis is a two-stage treatment regimen including an initial induction phase and a prolonged maintenance phase . InductiFor patients who respond poorly to conventional therapy, MSC therapy has shown satisfactory efficacy with acceptable treatment-related adverse events . Data frwww.clinicaltrials.gov website reached 212 cases worldwide as of July 4, 2019. Moreover, there are 14 different stem cell-associated products approved for therapy1, most of which are MSCs or HSCs.In recent years, various countries or regions have attached great importance to stem cell research and clinical translation. The number of clinical trials for stem cell-based therapies for autoimmune diseases registered in Anecdotal case reports have reported that total remission can be achieved after HSC transplantation in refractory SLE patients with severe disease and who were resistant to conventional therapies . In a 5 MSCs are non-hematopoietic, multipotent progenitor stem cells that possess immunomodulatory capabilities. Several clinical studies have been performed to evaluate the potential clinical efficacy for MSC transplantation as an alternative therapeutic approach to the current pharmacologic therapy for SLE. Results from those studies indicate that MSC transplantation is a safe and effective therapy  that canin vitro (Our review shows that, although allogeneic MSCs are promising candidates for treating SLE, autologous MSCs may not be therapeutically useful because of their defects in both immunomodulatory function and regenerative characteristics. MSCs from SLE patients present a morphological appearance of senescence with impaired capabilities of differentiation, migration and immune regulation . B cellsin vitro  raising Likely, no single factor can account for the intricate mechanisms of the MSCs-mediated immunosuppressive effect on autoimmune diseases. There are several hypothetical mechanisms why MSCs from SLE patients appear defective see . GWAS hain vitro and had superior suppressive effects in CIA mice compared to non-modified MSCs (In conclusion, murine models and clinical trials have produced evidence for the therapeutic potential of MSCs for SLE. Our review shows that although allogeneic MSCs are promising candidates for treating SLE, autologous MSCs may not be eligible to treat SLE patients because of their defective immunomodulatory function and poor regenerative characteristics. Moreover, whether the immunological rejection of allogeneic stem cell transplantation will influence the efficacy of MSC therapies or have long-term effects on recipients is not known. If the causes of MSC dysfunction for MSCs from SLE patients can be better understood in the future, maybe modification or transformation of SLE patient MSCs to restore immunosuppressive and regenerative function can yield a therapeutically beneficial treatment. In fact, recent research has indicated that MSCs transfected with an etanercept-encoding vector can successfully produce the drug ied MSCs . ProspecR-JC wrote the original manuscript. A-JX and Y-HL organized sections of the manuscript. S-YP and Q-PZ prepared the figures. YZ, YL, and TM revised the manuscript.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.


Long noncoding RNAs (lncRNAs) have multiple regulatory roles and are involved in many human diseases. A potential therapeutic strategy based on targeting lncRNAs was recently developed. To gain insight into the global relationship between small molecule drugs and their affected lncRNAs, we constructed a small molecule lncRNA network consisting of 1206 nodes (1033 drugs and 173 lncRNAs) and 4770 drug-lncRNA associations using LNCmap, which reannotated the microarray data from the Connectivity Map (CMap) database. Based on network biology, we found that the connected drug pairs tended to share the same targets, indications, and side effects. In addition, the connected drug pairs tended to have a similar structure. By inferring the functions of lncRNAs through their co-expressing mRNAs, we found that lncRNA functions related to the modular interface were associated with the mode of action or side effects of the corresponding connected drugs, suggesting that lncRNAs may directly/indirectly participate in specific biological processes after drug administration. Finally, we investigated the tissue-specificity of drug-affected lncRNAs and found that some kinds of drugs tended to have a broader influence (e.g. antineoplastic and immunomodulating drugs), whereas some tissue-specific lncRNAs (nervous system) tended to be affected by multiple types of drugs. Increasing research has shown that long noncoding RNAs (lncRNAs) are pivotal regulators in many biological processes, as well as in the generation and progression of various diseases –3. Many Currently, pharmacotherapy is the most effective strategy in the treatment of some diseases. Small molecules are widely used due to their simple structure. In recent years, small molecules are found to either have therapeutic effects or induce side-effects/toxicity through the regulation of lncRNAs , 7. MeanEnormous array-based expression profile resources have promoted the development of research on small molecules. For example, Connectivity Map (CMap), which is a genome-wide transcriptional expression dataset of selected human cells treated with bioactive small molecules, pioneered the systemic transcriptional response-based approach . CompareTo investigate the global relationships between small molecules (drugs) and lncRNAs, we obtained the lncRNA expression profiles affected by small molecules from the LNCmap. We constructed a global small molecule lncRNA network (SMLN) in which nodes represented drugs or lncRNAs. Starting from the bipartite SMLN, we generated two biologically relevant network projections: the small molecule-small molecule network (SSN), in which nodes represented drugs, and two drugs were connected if they shared at least one lncRNA; and the lncRNA-lncRNA network (LLN), in which nodes were lncRNAs and two lncRNAs were connected if they significantly shared small molecules. Then we (i) investigated the pharmacological similarities of linked small molecule pairs in the SSN, (ii) explored the function of modules of the LLN in the responses to drug treatment, and (iii) analyzed the tissue specificity of lncRNAs after drug treatment.To construct the global SMLN, we retrieved lncRNA expression profiles affected by small molecules from LNCmap . LNCmap 2=0.874 and 0.532, respectively  and 4,770 edges  . All nodectively . Thus, tThe degree of small molecule nodes spanned a wide range from 1 to 87. The highest degree node was trichostatin A (TSA), an organic compound that serves as an antifungal antibiotic and selectively inhibits class I and II mammalian histone deacetylases (HDACs) . TSA canDLEU2 (Deleted in Lymphocytic leukemia 2) is well studied. It encodes a pair of critical pro-apoptotic microRNAs, miR-15a/16-1, which are critical for the increased survival exhibited by chronic lymphocytic leukemia cells . On the other hand, we found that some tissue-specific lncRNAs  were prone to be affected by many kinds of drugs. The highest Jaccard coefficient was found between alimentary tract and metabolism drugs and the nervous system, suggesting that this kind of drug might predominantly affect nervous system-specific lncRNAs. To ensure the robustness of our results, we also calculated the Jaccard coefficients of overlapped lncRNAs between 13 drug classes and 16 tissues by processing the RNA-seq data of 16 normal human individual tissues from the ArrayExpress database (ERP000546) . Th. Th37]. P000546)   code and their affected lncRNAs . Many lnLncRNAs have multiple biological function and are considered as potential therapeutic targets for diseases –42. LncRThen, we constructed a global SMLN consisting of 1206 nodes , and 4770 edges (2628 downregulated and 2142 upregulated). Based on the SMLN, we found that connected drug pairs (two drugs sharing the same lncRNAs) tended to have similar pharmacological properties. Especially, these connected drug pairs tended to have similar structure, indicating the possibility of structure-based combinations between drugs and lncRNAs in our SMLN. Because two drugs may bind to the same "structural motifs" in the corresponding lncRNA, future investigation of lncRNA structures may reveal critical functional RNA motifs of lncRNAs susceptible to small molecule targeting . FurtherTo confirm the validity of our results, we also constructed the SMLN with fold change = 1.5 and fold change = 3 respectively and repeated some of the analyses for the network. We found that the SMLN networks with different fold changes were robust . This reIn summary, we have constructed and analyzed the SMLN that provided a comprehensive picture of global associations between drugs and their affected lncRNAs. A better understanding of the relation between small molecule agents and lncRNAs would not only promote the repositioning and rational clinical use of these agents but also provides new insights into lncRNA-targeting therapeutics.http://bio-bigdata.hrbmu.edu.cn/LncMAP/). The LNCmap extracted drug-affected lncRNA expression profiles by reannotating the microarray data from the CMap database. According to the pipeline of ncFANs [Drugs and affected lncRNAs were obtained from the LNCmap  and small molecules (1005), lncRNAs were connected to each other closely. To improve the specificity and identify the more significant lncRNA pairs, we adopted a hypergeometric test to generate the LLN.Here, we collected m total small molecules in the SMLN, for each two lncRNAs i and j, t was the number of small molecules affected by lncRNA i, and n was the number of small molecules affected by lncRNA j, of which r was overlapped small molecules of the two small molecule sets. After calculating the P-value, we adopted the FDR-corrected q-values to reduce the false positive discovery rate. Significant lncRNA pairs  were obtained to construct the LLN.We collected the drug-indication associations from the study of Yildirim et al . We alsoWe downloaded the drug-target associations from the DrugBank database , which iWe downloaded the drug side effect dataset from a public computer-readable resource, SIDER, which is a freely available database that contains information on marketed medicines and their recorded adverse drug reactions . We collhttp://www.kegg.jp/kegg/drug/). We computed the TC scores of drug pairs using the Chemical Development Kit with default parameters [We downloaded the SMILES files of small molecules in the SSN from the DrugBank database and Kyoto Encyclopedia of Genes and Genomes  to study the tissue-specificity of drug-affected lncRNAs. We first reannotated the microarray dataset of GSE1133 and obtained 176 lncRNAs across 79 healthy tissues; then, we calculated tissue specificity scores for lncRNAs and identified tissue-specific lncRNAs (score >0.8) for each tissue . AccordiSupplementary MaterialsSupplementary FiguresSupplementary TablesSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4


Euprymna scolopes squid and its bioluminescent bacterial symbiont, Vibrio fischeri, is a valuable model system to study a natural, coevolved host-microbe association. Over the past 30 years, researchers have developed and optimized many experimental methods to study both partners in isolation and during symbiosis.The symbiosis between  Euprymna scolopes squid and its bioluminescent bacterial symbiont, Vibrio fischeri, is a valuable model system to study a natural, coevolved host-microbe association. Over the past 30 years, researchers have developed and optimized many experimental methods to study both partners in isolation and during symbiosis. These powerful tools, along with a strong foundational knowledge about the system, position the Vibrio-squid symbiosis at the forefront of host-microbe interactions because this system is uniquely suited to investigation of symbiosis from both host and bacterial perspectives. Moreover, the ability to isolate and characterize different strains of V. fischeri has revealed exciting new insights about how different genotypes evolve to compete for a host niche, including deploying interbacterial weapons early during host colonization. This Perspective explores how interbacterial warfare influences the diversity and spatial structure of the symbiotic population, as well as the possible effects that intraspecific competition might have on the host.The symbiosis between  The competitive exclusion principle states that two species with identical niches cannot stably coexist: eventually, one will exclude the other . Yet todVibrio-squid symbiosis (Vibrio fischeri) compete for colonization of the host light organ. Juvenile squid hatch without their symbionts, which they must acquire from the seawater  to spatially structure the symbiotic population as they establish a mutualistic relationship with their animal host , which represents a contact-dependent interbacterial weapon  the mechanism of lethality for these toxins is largely unknown; and (iii) the strain specificity of this weaponry indicates that when different strains come into physical contact with one another, T6SS2-depedent killing results in the elimination of the less fit strain. Thus, light organ isolates are largely incompatible and unable to coexist in the same space, an observation that is consistent with the competitive exclusion principle. Yet we consistently isolate incompatible strains from the same adult light organ   hybridization chain reaction-fluorescent CR-FISH)  and (ii)enotypes . Furtherenotypes . Speare enotypes  5). The. Thein sVibrio-squid symbiosis is particularly well suited for studying the role of T6SS-dependent competition in a natural system because V. fischeri T6SS2 is active both in the host and in culture. Different strain types can be quantified and visually discriminated within mixed populations using culture-based assays that replicate the competitive interactions observed in the light organ environment (The ironment . These aironment , allowinironment . By usinV. fischeri strains, as well as improved tools for genetic manipulation and transfer of DNA from one strain to another (This symbiosis also exhibits characteristics ideal for investigating how bacteria evolve mechanisms to compete for the host niche. The squid host can be routinely collected from its natural environment in several bays along the coast of Hawaii . Both th another , and the another . TogetheV. fischeri and host cells using established in vitro techniques may provide insight into how the T6SS directly or indirectly impacts the host. For example, proteomics data from host hemocytes that have been coincubated with V. fischeri cells (Although this system has revealed important findings about how closely related organisms compete for a niche, many issues remain whose resolution will benefit from recently developed techniques. For example, the extent to which the host may be impacted by the secretion of antimicrobial toxins during intraspecific competition remains unknown. Moreover, T6SSs in other bacteria have been shown to inject effectors directly into eukaryotic cells where they can alter host cell physiology (ri cells  may reveri cells , which cFinally, although fierce competition among potential symbionts likely reduces the diversity in the light organ, it is also predicted that competition can increase the stability of a system . Future


Coronary artery spasm (CAS) is an underdiagnosed disease especially in heart transplant patients, and in those patients the etiology and pathophysiology remain largely unknown, although it has been associated with cardiac allograft vasculopathy or graft rejection.We report the case of a heart-transplant patient whose cardiac graft experienced two coronary vasospasms: the first before transplantation, and the other at one-month of a postoperative course complicated by primary graft failure.Our case illustrates that a transplanted heart predisposed with coronary vasospasm may suffer from early relapse in the recipient despite of complete post-surgical autonomic denervation. Exacerbated endothelial dysfunction of the donor heart after transplant, with the addition of systemic factors in the recipient may be involved in the genesis of this puzzling phenomenon. Coronary Artery Spasm (CAS) is a condition caused by the vasoconstriction of the epicardial coronary arteries, leading to myocardial ischemia that can play a role in various conditions, from rest angina to acute coronary syndrome. Several responsible mechanisms have been reported: abnormal autonomic nervous system, endothelial dysfunction, hyperreactivity of the coronary smooth muscle and, as recently demonstrated, specific anatomy of the coronary artery and inflammation of perivascular component, with a fundamental role played by inflammation of coronary adventitia and perivascular adipose tissue through Rho-kinase activation, one of the main molecular mediator in the mechanisms of coronary artery spasm in animals and humans –3.CAS in heart transplant has previously been described to be rare . NeverthSince autonomic innervation is not necessary to develop CAS in heart transplant , 10, patFor the diagnosis, ECG changes in ST-T segments during chest symptoms, including good response to nitroderivates, is very important, but the gold standard is represented by Spasm Provocating Tests (SPT), using either Acethylcoline (Ach), either Ergonovine maleate (EM) or a combination of both : the criteria of positivity is a > 90% transient stenosis of a coronary artery with signs/symptoms of myocardial ischemia , 15, 16.Our case illustrates that a transplanted heart predisposed with coronary vasospasm may suffer from early relapse in the recipient despite of complete post-surgical autonomic denervation.A 46 years old man with end-stage hypertrophic cardiomyopathy and electrical storm underwent urgent orthotopic heart transplantation: the donor was a 54 years old woman, known for diabetes, nicotine consumption, morbid obesity, combined ventilation disorder with restriction due to obesity hypoventilation syndrome and suspected chronic obstructive pulmonary disease, and history of thrombophilia (activated protein C resistance) with recurrent deep vein thrombosis and pulmonary embolism in cerebral death due to brain hemorrhage. Pre-transplant cardiac workup showed a 90% stenosis in the middle right coronary artery Fig. , with noThe technically uneventful transplant was completed with a venous coronary artery bypass graft (CABG) on the right coronary artery (RCA), with a total ischemic time of 191 min. After cross-clamp removal and appropriate induction therapy with methylprednisolone 500 mg IV, the heart showed severe global biventricular failure with severe functional mitral regurgitation. In the absence of preformed donor specific HLA antibodies in favor of an acute humoral rejection, primary graft failure was suspected, and mechanical hemodynamic support was immediately initiated with a central veno-arterial extracorporeal membrane oxygenation (ECMO) and intra-aortic balloon pump (IABP), and high dose of cathecolamines  A relatively low troponin release was observed during the first 24 h post-operative (peak at 2386 ng/l), favoring the hypothesis of myocardial stunning over necrosis.A cardiac tamponade on post-operative day 1 led to surgical revision. The intra-operative status was noteworthy for an occlusion of the venous CABG on the RCA. A coronary angiogram was urgently performed in attempt to treat the stenosis in the native vessel. Surprisingly, only a < 50% stenosis could be seen the mid RCA Fig. . It was In stable phase, during his fourth post-transplant week at the intermediate care unit, the patient developed sustained ventricular tachycardia at rest, with a heart rate of 150 bpm and no associated hemodynamic instability or even symptoms. Rapid cardioversion was easily achieved with a single administration of 2 mg magnesium sulfate IV. Nevertheless, the immediate post-cardioversion 12-lead ECG and echocardiography respectively showed severe ST-segment elevation in the inferior leads Fig.  and 4 wiInterestingly, a few days before this coronary vasospasm, the patient complained from recurrent abdominal pain followed by melena. A colonoscopy showed ulcerations in the distal ileum, correlated with reversible signs of bowel ischemia in two consecutive CT scans, without evidence of occlusion or embolization in the mesenteric vessels. We could speculate that systemic factors in the recipient might favor vasospastic events. Because of a previously reported case of segmental mesenteric ischemia related to mycophenolate mofetil , this drThree lessons can be learned from this case. First, vasospasm should never be forgotten as a possible cause of angiographic coronary stenosis, even in the setting of pre-transplant assessment: it may typically develop on the site of an atherosclerotic plaque, with the risk to overestimate the severity of a stenosis. In our case, this led to perform an unnecessary CABG in addition to transplant, which prolonged the ischemic time of these 54 years-old hearts over 3 h, and participated in the genesis of primary graft failure. The execution of provocation tests during the pre-transplant coronary angiogram could be considered for the early identification of this phenomenon in the donor heart: these tests have been proven to be not only safe but also useful to enhance the diagnostic value of a diagnostic angiography in the population suffering from vasospastic angina .Secondly, it shows that coronary vasospasm may manifest very early in the recipient despite of complete autonomic denervation of the heart, and out of any context of graft rejection or cardiac graft vasculopathy. Although this was already reported , the parThirdly, our case shows that full heart recovery may still be possible after 9 days on ECMO support following primary graft failure with severe biventricular failure, which is longer compared to previously reported delays, usually ranging from 72 h to 7 days . This coIn conclusion, coronary vasospasm in heart-transplant patients is a complex and multi-faceted problem that is not limited to the context of graft rejection or cardiac graft vasculopathy. Independently of cardiac autonomic innervation, endothelial dysfunction - possibly induced by immunological, neurohormonal and metabolic factors – is now seen as the main trigger for this phenomenon, and may be exacerbated by ischemia-reperfusion injury in the near post-operative course. Our case identifies pre-existent atherosclerosis and vasospasm in the donor heart as a clear risk factor for early post-transplant vasospasm.Additional file 1. Apical 4-chamber TTE view.Additional file 2. Apical 2-chamber TTE view showing inferior hypokinesia.Additional file 3. Apical 3-chamber TTE view showing infero-lateral hypokinesia.