Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo.

Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. Here, we use optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes, tailless (tll) and huckebein (hkb), that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, we provide a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.

Contaminants of emerging concern: Silylation procedures, evaluation of the stability of silyl derivatives and associated measurement uncertainty.

Analyte range of gas chromatography-mass spectrometry (GC-MS), widely used in environmental analysis, can be significantly broadened by derivatization. Silyl derivatives have improved volatility and thermal stability, chromatographic and mass spectrometric behaviors, and thus detection, structural elucidation and quantification. However, silylation use is often hindered by the stability of generated derivatives and the need to optimize silylation conditions. In this study, we optimized the derivatization conditions for 70 selected contaminants of emerging concern (CEC) using chemometrics approaches. N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and BSTFA + 1 % trimethylchlorosilane (TMCS) were investigated, among which the latter gave the best yield. CEC were grouped in three derivatization protocols: 60 °C/45 min, 70 °C/90 min, and 70 °C/45 min. The short- and long-term stability of the CEC-trimethylsilyl (TMS) derivatives, i.e. for 28 days and up to 20 weeks were examined in a solvent and artificial wastewater (AWW) extract at 25 °C, 4 °C and - 18 °C, and during repeated five freeze-thaw (F/T) cycles, at two concentration levels: 100 µg/L and 1000 µg/L. Except for TMS derivatives of shikimic acid (SHA), quinic acid (QA) and sulfanilamide (SFA), the remaining derivatized compounds were stable in solvent (EtAc) for 28 days. In AWW extract, TMS derivatives of citric acid (CA), 17ß-estradiol (E2), estriol (E3) and 17α-ethinyl estradiol (EE2) were unstable at 25 °C and 4 °C. Within up to 20 weeks, only the TMS derivatives of CA, meso-erythritol (ERY) and bisphenol BP (BPBP) were unstable. The most significant hydrolytic breakdown was observed during repeated F/T cycles. After three cycles, ≤ 20 % of the initial concentration of six and nine CEC-TMS derivatives had degraded in solvent and AWW extracts, respectively. According to the deep statistical comparison (DSC) approach, the most prominent degradation was observed for TMS derivatives of E2, CA, 9-hydroxyfluorene (9-HF), estrone (E1) and trans-3'-hydroxycotinine (T3HC) in solvent; E2, CA, 9-HF, E3 and E1 in AWW extracts and ERY, E2, CA, 9-HF and E1 in both matrices. Finally, the sample concentration of CEC accounted for most of the measurement uncertainty (MU). Based on our findings, we recommend the derivatized samples to be stored at -18 °C for up to 20 weeks to ensure the stability of their TMS derivatives. Sample freezing and thawing of not more than twice is allowed to maintain ≥80 % of the initial CEC-TMS concentration.

Representing creative thought: A representational similarity analysis of creative idea generation and evaluation.

Dual process theories of creativity suggest that creative thought is supported by both a generation phase, where unconstrained ideas are generated and combined in novel ways, and an evaluation phase, where those ideas are filtered for usefulness in context. Neurocognitively, both the default mode network (DMN) and the executive control network (ECN) have been implicated in generation and evaluation, respectively. Importantly, generating and evaluating ideas implies that the same information, reflected in patterns of neural activity, must be present in both phases, suggesting that information should be 'reinstated' (i.e. multidimensional patterns must reappear) within and/or between network nodes. In the present study, we used representational similarity analysis (RSA) to investigate the extent to which nodes of the DMN and ECN reinstate information between a generation phase, in which participants generated novel or appropriate word associations to single nouns, and an evaluation phase, where we presented the associations back to participants to evaluate them. We showed strong evidence for reinstatement within the ECN dorsal lateral prefrontal cortex during the novel association task, and within the DMN medial prefrontal cortex during the appropriate association task. We additionally showed between network reinstatement between the ECN dorsal lateral prefrontal cortex and the DMN posterior parietal cortex during the novelty task. These results demonstrate the importance of both within- and between-informational reinstatement for generating and evaluating ideas, and implicate both the DMN and ECN in dual process models of creativity.

Application of Optogenetics to Probe the Signaling Dynamics of Cell Fate Decision-Making.

The development of optogenetic control over signaling pathways has provided a unique opportunity to decode the role of signaling dynamics in cell fate programing. Here I present a protocol for decoding cell fates through systematic interrogation with optogenetics and visualization of signaling with live biosensors. Specifically, this is written for Erk control of cell fates using the optoSOS system in mammalian cells or Drosophila embryos, though it is intended to be adapted to apply generally for several optogenetic tools, pathways, and model systems. This guide focuses on calibrating these tools, tricks of their use, and using them to interrogate features which program cell fates.

Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo.

Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal - a gradient of ERK kinase activity - and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn . We find that high or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that an all-or-none stimulus can be 'blurred' through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.

Multi-spectral SWIR lidar for imaging and spectral discrimination through partial obscurations.

We have developed a multi-spectral SWIR lidar system capable of measuring simultaneous spatial-spectral information for imaging and spectral discrimination through partial obscurations. We employ objects in the presence and absence of a series of obscurants to evaluate the capability of the system in classifying the objects of interest based on spectral and range information. We employ a principal component analysis-based algorithm in classifying the objects and quantifying the accuracy of detection under various obscured scenarios. The merits of multi-spectral lidar over hyperspectral imaging are highlighted for target identification in the presence of obscurants.

God in body and space: Investigating the sensorimotor grounding of abstract concepts.

concepts are defined as concepts that cannot be experienced directly through the sensorimotor modalities. Explaining our understanding of such concepts poses a challenge to neurocognitive models of knowledge. One account of how these concepts come to be represented is that sensorimotor representations of grounded experiences are reactivated in a way that is constitutive of the abstract concept. In the present experiment, we investigated how sensorimotor information might constitute GOD-related concepts, and whether a person's self-reported religiosity modulated this grounding. To do so, we manipulated both the state of the body (i.e., kneeling vs. sitting) and the state of stimuli (i.e., spatial position on the screen) in two tasks that required conceptual categorization of abstract words. Linear Mixed Effects model fitting procedures were used to determine which manipulated factors best predicted response latency and accuracy in both tasks. We successfully replicated previous research demonstrating faster categorization of GOD-related words when they were presented at the top of the screen. Importantly, results demonstrated that the kneeling posture manipulation enhanced this effect, as did religiosity, as participants who scored higher in religiosity showed a greater effect of the posture manipulation on the speed with which word categorization occurred when those words were presented in the higher visuospatial presentation condition. Overall, we interpreted our findings to suggest that directly manipulating sensorimotor information can facilitate the categorization of abstract concepts, supporting the notion that this information in part constitutes the representation of abstract concepts.

Context-sensitive computational mechanistic explanation in cognitive neuroscience.

Mainstream cognitive neuroscience aims to build mechanistic explanations of behavior by mapping abilities described at the organismal level via the subpersonal level of computation onto specific brain networks. We provide an integrative review of these commitments and their mismatch with empirical research findings. Context-dependent neural tuning, neural reuse, degeneracy, plasticity, functional recovery, and the neural correlates of enculturated skills each show that there is a lack of stable mappings between organismal, computational, and neural levels of analysis. We furthermore highlight recent research suggesting that task context at the organismal level determines the dynamic parcellation of functional components at the neural level. Such instability prevents the establishment of specific computational descriptions of neural function, which remains a central goal of many brain mappers - including those who are sympathetic to the notion of many-to-many mappings between organismal and neural levels. This between-level instability presents a deep epistemological challenge and requires a reorientation of methodological and theoretical commitments within cognitive neuroscience. We demonstrate the need for change to brain mapping efforts in the face of instability if cognitive neuroscience is to maintain its central goal of constructing computational mechanistic explanations of behavior; we show that such explanations must be contextual at all levels.

Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo.

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.

Identification and Phenotypic Characterization of Hsp90 Phosphorylation Sites That Modulate Virulence Traits in the Major Human Fungal Pathogen Candida albicans.

The highly conserved, ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. In human pathogenic fungi, which kill more than 1.6 million patients each year worldwide, Hsp90 governs cellular morphogenesis, drug resistance, and virulence. Yet, our understanding of the regulatory mechanisms governing fungal Hsp90 function remains sparse. Post-translational modifications are powerful components of nature's toolbox to regulate protein abundance and function. Phosphorylation in particular is critical in many cellular signaling pathways and errant phosphorylation can have dire consequences for the cell. In the case of Hsp90, phosphorylation affects its stability and governs its interactions with co-chaperones and clients. Thereby modulating the cell's ability to cope with environmental stress. Candida albicans, one of the leading human fungal pathogens, causes ~750,000 life-threatening invasive infections worldwide with unacceptably high mortality rates. Yet, it remains unknown if and how Hsp90 phosphorylation affects C. albicans virulence traits. Here, we show that phosphorylation of Hsp90 is critical for expression of virulence traits. We combined proteomics, molecular evolution analyses and structural modeling with molecular biology to characterize the role of Hsp90 phosphorylation in this non-model pathogen. We demonstrated that phosphorylation negatively affects key virulence traits, such as the thermal stress response, morphogenesis, and drug susceptibility. Our results provide the first record of a specific Hsp90 phosphorylation site acting as modulator of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitations as antifungal drug targets.

Neural correlates of the production effect: An fMRI study.

Recognition memory is improved for items produced at study (e.g., by reading them aloud) relative to a non-produced control condition (e.g., silent reading). This production effect is typically attributed to the extra elements in the production task (e.g., motor activation, auditory perception) enhancing item distinctiveness. To evaluate this claim, the present study examined the neural mechanisms underlying the production effect. Prior to a recognition memory test, different words within a study list were read either aloud, silently, or while saying "check" (as a sensorimotor control condition). Production improved recognition, and aloud words yielded higher rates of both recollection and familiarity judgments than either silent or control words. During encoding, fMRI revealed stronger activation in regions associated with motor, somatosensory, and auditory processing for aloud items than for either silent or control items. These activations were predictive of recollective success for aloud items at test. Together, our findings are compatible with a distinctiveness-based account of the production effect, while also pointing to the possible role of other processing differences during the aloud trials as compared to silent and control.

Scene context shapes category representational geometry during processing of tools.

Tools are ubiquitous in human environments and to think about them we use concepts. Increasingly, conceptual representation is thought to be dynamic and sensitive to the goals of the observer. Indeed, observer goals can reshape representational geometry within cortical networks supporting concepts. In the present study, we investigated the novel hypothesis that task-irrelevant scene context may implicitly alter the representational geometry of regions within the tool network. Participants performed conceptual judgments on images of tools embedded in scenes that either suggested their use (i.e., a kitchen timer sitting on a kitchen counter with vegetables in a frying pan) or that they would simply be moved (i.e., a kitchen timer sitting in an open drawer with other miscellaneous kitchen items around). We investigated whether representations in the tool network reflect category, grip, and shape information using a representational similarity analysis (RSA). We show that a) a number of regions of the tool network reflect category information about tools and b) category information predicts patterns in supramarginal gyrus more strongly in use contexts than in move contexts. Together, these results show that information about tool category is distributed across different regions of the tool network and that scene context helps shape the representational geometry of the tool network.

Implications for research and clinical use from a Rasch analysis of the HOOS-12 and KOOS-12 instruments.

OBJECTIVE: To evaluate the structural validity of the 12-item Hip disability and Osteoarthritis Outcome Score (HOOS-12) and 12-item Knee injury and Osteoarthritis Outcome Score (KOOS-12) using Rasch analysis and consider psychometric implications for research and clinical use. METHOD: Individual-level HOOS-12 and KOOS-12 data from the Australian Orthopaedic Association National Joint Replacement Registry, collected before and after primary total hip and knee replacement, were used for this analysis. Using the Rasch analytic approach, overall model fit and item fit were examined, together with potential reasons for misfit including response threshold ordering, differential item functioning, internal consistency, unidimensionality and item targeting. RESULTS: Overall misfit to the Rasch model was evident for both instruments. A degree of item misfit was also observed, although most items demonstrated logical sequencing of response options. Only two items (hip/knee pain frequency and awareness of hip/knee problems) displayed disordered response thresholds. The pain, function, and quality of life domains of the HOOS-12 and KOOS-12 demonstrated excellent internal consistency reliability (person separation index: 0.80-0.93) and unidimensionality. A mismatch between item difficulty and person ability scores at the highest end of the HOOS-12 and KOOS-12 scales contributed to post-operative ceiling effects (mean logit for HOOS-12: 3.57; KOOS-12: 2.58; ≈0 indicates well-targeted scale). CONCLUSION: We found evidence to support the structural validity of the three HOOS-12 and KOOS-12 domains for evaluating joint replacement outcomes. However, there may be missing content in both instruments particularly for high-functioning patients. Minor refinement of some response options may be warranted to improve item performance.

Performance of the HOOS-12 and KOOS-12 instruments for evaluating outcomes from joint replacement surgery.

OBJECTIVE: To evaluate the psychometric properties of the 12-item Hip disability and Osteoarthritis Outcome Score (HOOS-12) and Knee injury and Osteoarthritis Outcome Score (KOOS-12) for use in evaluating outcomes after joint replacement for osteoarthritis. DESIGN: Patient-reported outcomes data collected by the Australian Orthopaedic Association National Joint Replacement Registry were used for this analysis. HOOS-12 and KOOS-12 domain (pain, function, quality of life) and summary impact data were available. The Oxford Hip Score (OHS), Oxford Knee Score (OKS) and EQ-5D-5L were used as comparators. Instruments were administered pre-operatively and at 6 months post-operatively. Internal consistency reliability, floor and ceiling effects, convergent validity, known groups validity, and responsiveness were evaluated using standard psychometric techniques. RESULTS: Baseline HOOS-12 and KOOS-12 data were available for 3,023 patients undergoing primary total hip replacement and 4,010 patients undergoing primary total knee replacement. At baseline, high internal consistency was demonstrated for all domains and summary scores (Cronbach's alpha: HOOS-12 = 0.81-0.93; KOOS-12 = 0.82-0.92). Post-operative ceiling effects (>15% of patients scoring the best possible score) were identified for the HOOS-12 pain (46%), function (39%) and quality of life domains (26%) and summary score (17%), and for the KOOS-12 pain (21%) and function domains (18%). The HOOS-12 and KOOS-12 could differentiate between two known groups (lowest/highest OHS or OKS quartiles post-operatively; p < 0.001) and were highly responsive to change (effect sizes for HOOS-12: 2.20-2.83; KOOS-12: 1.82-2.35). CONCLUSION: The HOOS-12 and KOOS-12 have good psychometric properties for capturing joint replacement outcomes including excellent responsiveness, although ceiling effects may limit monitoring of post-operative improvement.

Cis-effects on gene expression in the human prenatal brain associated with genetic risk for neuropsychiatric disorders.

The majority of common risk alleles identified for neuropsychiatric disorders reside in noncoding regions of the genome and are therefore likely to impact gene regulation. However, the genes that are primarily affected and the nature and developmental timing of these effects remain unclear. Given the hypothesized role for early neurodevelopmental processes in these conditions, we here define genetic predictors of gene expression in the human fetal brain with which we perform transcriptome-wide association studies (TWASs) of attention deficit hyperactivity disorder (ADHD), autism spectrum disorder, bipolar disorder, major depressive disorder, and schizophrenia. We identify prenatal cis-regulatory effects on 63 genes and 166 individual transcripts associated with genetic risk for these conditions. We observe pleiotropic effects of expression predictors for a number of genes and transcripts, including those of decreased DDHD2 expression in association with risk for schizophrenia and bipolar disorder, increased expression of a ST3GAL3 transcript with risk for schizophrenia and ADHD, and increased expression of an XPNPEP3 transcript with risk for schizophrenia, bipolar disorder, and major depression. For the protocadherin alpha cluster genes PCDHA7 and PCDHA8, we find that predictors of low expression are associated with risk for major depressive disorder while those of higher expression are associated with risk for schizophrenia. Our findings support a role for altered gene regulation in the prenatal brain in susceptibility to various neuropsychiatric disorders and prioritize potential risk genes for further neurobiological investigation.

Tobacco Hornworm (Manduca sexta) caterpillars as a novel host model for the study of fungal virulence and drug efficacy.

The two leading yeast pathogens of humans, Candida albicans and Cryptococcus neoformans, cause systemic infections in >1.4 million patients worldwide with mortality rates approaching 75%. It is thus imperative to study fungal virulence mechanisms, efficacy of antifungal drugs, and host response pathways. While this is commonly done in mammalian models, which are afflicted by ethical and practical concerns, invertebrate models, such as wax moth larvae and nematodes have been introduced over the last two decades. To complement existing invertebrate host models, we developed fifth instar caterpillars of the Tobacco Hornworm moth Manduca sexta as a novel host model. These caterpillars can be maintained at 37°C, are suitable for injections with defined amounts of yeast cells, and are susceptible to the most threatening yeast pathogens, including C. albicans, C. neoformans, C. auris, and C. glabrata. Importantly, fungal burden can be assessed daily throughout the course of infection in a single caterpillar's feces and hemolymph. Infected caterpillars can be rescued by treatment with antifungal drugs. Notably, these animals are large enough for weight to provide a reliable and reproducible measure of fungal disease and to facilitate host tissue-specific expression analyses. M. sexta caterpillars combine a suite of parameters that make them suitable for the study of fungal virulence.

Optogenetic Rescue of a Patterning Mutant.

Animal embryos are patterned by a handful of highly conserved inductive signals. Yet, in most cases, it is unknown which pattern features (i.e., spatial gradients or temporal dynamics) are required to support normal development. An ideal experiment to address this question would be to "paint" arbitrary synthetic signaling patterns on "blank canvas" embryos to dissect their requirements. Here, we demonstrate exactly this capability by combining optogenetic control of Ras/extracellular signal-related kinase (ERK) signaling with the genetic loss of the receptor tyrosine-kinase-driven terminal signaling patterning in early Drosophila embryos. Blue-light illumination at the embryonic termini for 90 min was sufficient to rescue normal development, generating viable larvae and fertile adults from an otherwise lethal terminal signaling mutant. Optogenetic rescue was possible even using a simple, all-or-none light input that reduced the gradient of Erk activity and eliminated spatiotemporal differences in terminal gap gene expression. Systematically varying illumination parameters further revealed that at least three distinct developmental programs are triggered at different signaling thresholds and that the morphogenetic movements of gastrulation are robust to a 3-fold variation in the posterior pattern width. These results open the door to controlling tissue organization with simple optical stimuli, providing new tools to probe natural developmental processes, create synthetic tissues with defined organization, or directly correct the patterning errors that underlie developmental defects.

The role of the motor system in generating creative thoughts.

Neurocognitive research is pertinent to developing mechanistic models of how humans generate creative thoughts. Such models usually overlook the role of the motor cortex in creative thinking. The framework of embodied or grounded cognition suggests that creative thoughts (e.g. using a shoe as a hammer, improvising a piano solo) are partially served by simulations of motor activity associated with tools and their use. The major hypothesis stemming from the embodied or grounded account is that, while the motor system is used to execute actions, simulations within this system also support higher-order cognition, creativity included. That is, the cognitive process of generating creative output, not just executing it, is deeply embedded in motor processes. Here, we highlight a collection of neuroimaging research that implicates the motor system in generating creative thoughts, including some evidence for its functionally necessary role in generating creative output. Specifically, the grounded or embodied framework suggests that generating creative output may, in part, rely on motor simulations of possible actions, and that these simulations may by partially implemented in the motor regions themselves. In such cases, action simulations (i.e. reactivating or re-using the motor system), do not result in overt action but instead are used to support higher-order cognitive goals like generating creative uses or improvising.

Evaluation of an x-ray CT polymer gel dosimetry system in the measurement of deformed dose.

This study is an evaluation of the use of a N-isopropylacrylamide (NIPAM)-based x-ray CT polymer gel dosimetry (PGD) system in the measurement of deformed dose. This work also compares dose that is measured by the gel dosimetry system to dose calculated by a novel deformable dose accumulation algorithm, defDOSXYZnrc, that uses direct voxel tracking. Deformable gels were first irradiated using a single 3.5 × 5 cm2 open field and the static dose was compared to defDOSXYZnrc as a control measurement. Gel measurement was found to be in excellent agreement with defDOSXYZnrc in the static case with gamma passing rates of 94.5% using a 3%/3 mm criterion and 93.3% using a 3%/2 mm criterion. Following the static measurements, a deformable gel was irradiated with the same single field under an external compression of 25 mm and then released from this compression for dosimetric read out. The measured deformed dose was then compared to deformed dose calculated by defDOSXYZnrc based on deformation vectors produced by the Velocity AI deformable image registration (DIR) algorithm. In the deformed dose distribution there were differences in the measured and calculated field position of up to 0.8 mm and differences in the measured in calculated field size of up to 11.9 mm. Gamma pass rates were 60.0% using a 3%/3 mm criterion and 56.8% using a 3%/2 mm criterion for the deforming measurements representing a decrease in agreement compared to the control measurements. Further analysis showed that passing rates increased to 86.5% using a 3%/3 mm criterion and 70.5% using a 3%/2 mm criterion in voxels within 5 mm of fiducial markers used to guide the deformable image registration. This work represents the first measurement of deformed dose using x-ray CT polymer gel dosimetry. Overall these results highlight some of the challenges in the calculation and measurement of deforming dose and provide insight into possible strategies for improvement.

Novel Insight Into the Etiology of Autism Spectrum Disorder Gained by Integrating Expression Data With Genome-wide Association Statistics.

BACKGROUND: A recent genome-wide association study (GWAS) of autism spectrum disorder (ASD) (ncases = 18,381, ncontrols = 27,969) has provided novel opportunities for investigating the etiology of ASD. Here, we integrate the ASD GWAS summary statistics with summary-level gene expression data to infer differential gene expression in ASD, an approach called transcriptome-wide association study (TWAS). METHODS: Using FUSION software, ASD GWAS summary statistics were integrated with predictors of gene expression from 16 human datasets, including adult and fetal brains. A novel adaptation of established statistical methods was then used to test for enrichment within candidate pathways and specific tissues and at different stages of brain development. The proportion of ASD heritability explained by predicted expression of genes in the TWAS was estimated using stratified linkage disequilibrium score regression. RESULTS: This study identified 14 genes as significantly differentially expressed in ASD, 13 of which were outside of known genome-wide significant loci (±500 kb). XRN2, a gene proximal to an ASD GWAS locus, was inferred to be significantly upregulated in ASD, providing insight into the functional consequence of this associated locus. One novel transcriptome-wide significant association from this study is the downregulation of PDIA6, which showed minimal evidence of association in the GWAS, and in gene-based analysis using MAGMA. Predicted gene expression in this study accounted for 13.0% of the total ASD single nucleotide polymorphism heritability. CONCLUSIONS: This study has implicated several genes as significantly up/downregulated in ASD, providing novel and useful information for subsequent functional studies. This study also explores the utility of TWAS-based enrichment analysis and compares TWAS results with a functionally agnostic approach.