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Tumors may contain billions of cells, including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that are consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.

The epidermal growth factor receptor (EGFR) plays a role in cell proliferation and differentiation during healthy development and tumor growth; however, its requirement for brain development remains unclear. Here we used a conditional mouse allele for Egfr to examine its contributions to perinatal forebrain development at the tissue level. Subtractive bulk ventral and dorsal forebrain deletions of Egfr uncovered significant and permanent decreases in oligodendrogenesis and myelination in the cortex and corpus callosum. Additionally, an increase in astrogenesis or reactive astrocytes in effected regions was evident in response to cortical scarring. Sparse deletion using mosaic analysis with double markers (MADM) surprisingly revealed a regional requirement for EGFR in rostrodorsal, but not ventrocaudal glial lineages including both astrocytes and oligodendrocytes. The EGFR-independent ventral glial progenitors may compensate for the missing EGFR-dependent dorsal glia in the bulk Egfr-deleted forebrain, potentially exposing a regenerative population of gliogenic progenitors in the mouse forebrain.

The human cortex contains inhibitory interneurons derived from the medial ganglionic eminence (MGE), a germinal zone in the embryonic ventral forebrain. How this germinal zone generates sufficient interneurons for the human brain remains unclear. We found that the human MGE (hMGE) contains nests of proliferative neuroblasts with ultrastructural and transcriptomic features that distinguish them from other progenitors in the hMGE. When dissociated hMGE cells are transplanted into the neonatal mouse brain, they reform into nests containing proliferating neuroblasts that generate young neurons that migrate extensively into the mouse forebrain and mature into different subtypes of functional interneurons. Together, these results indicate that the nest organization and sustained proliferation of neuroblasts in the hMGE provide a mechanism for the extended production of interneurons for the human forebrain.

Electronic Nicotine Delivery Systems (ENDS) contain numerous volatile aerosol constituents (e.g., nicotine, propylene glycol, flavors, etc.). Past work clearly indicates the temporal and chemical dynamics of ENDS aerosol requires consideration of these volatile constituents when measuring the particle size distribution. An MSP-135-8 Mini MOUDI™ and Electrical Low Pressure Impactor (ELPI®+) were used to measure the particle size distribution of two JUUL ENDS products. Volatile chemicals were measured from each cascade impactor's exhaust airflow to assess their effect on collection efficiency. Similar mass median aerodynamic diameters were obtained for both ENDS products by both cascade impactors, however the geometric standard deviation from the ELPI®+ measurements were larger for both products than measurements using the Mini MOUDI™ impactor. Although the measured mass of volatile chemicals was greater in the exhaust from the Mini MOUDI™ impactor, a greater variety of volatile chemicals were found within the exhaust of the ELPI®+. The greater variety of volatile chemicals correlated with more room air sampling by the ELPI®+. The reduced amount of volatile chemicals measured in the exhaust of the ELPI®+ may be due to their collection by the vacuum oil used in the sintered collection plates of the ELPI®+. Accounting for the measured volatile chemicals improved the recovery efficiency of the Mini MOUDI™ impactor by 2.9–7.5% with the average recovery efficiency exceeding 82% for the two JUUL ENDS Products. In comparison, accounting for the measured volatile constituents increased the recovery efficiency of the ELPI®+ impactor by 0.4% or less, which did not narrow the recovery efficiency range, that based upon the estimated dilution, consistently exceeded the measured mass loss from both JUUL ENDS products.

Adult hippocampal neurogenesis was originally discovered in rodents. Subsequent studies identified the adult neural stem cells and found important links between adult neurogenesis and plasticity, behavior, and disease. However, whether new neurons are produced in the human dentate gyrus (DG) during healthy aging is still debated. We and others readily observe proliferating neural progenitors in the infant hippocampus near immature cells expressing doublecortin (DCX), but the number of such cells decreases in children and few, if any, are present in adults. Recent investigations using dual antigen retrieval find many cells stained by DCX antibodies in adult human DG. This has been interpreted as evidence for high rates of adult neurogenesis, even at older ages. However, most of these DCX-labeled cells have mature morphology. Furthermore, studies in the adult human DG have not found a germinal region containing dividing progenitor cells. In this Dual Perspectives article, we show that dual antigen retrieval is not required for the detection of DCX in multiple human brain regions of infants or adults. We review prior studies and present new data showing that DCX is not uniquely expressed by newly born neurons: DCX is present in adult amygdala, entorhinal and parahippocampal cortex neurons despite being absent in the neighboring DG. Analysis of available RNA-sequencing datasets supports the view that DG neurogenesis is rare or absent in the adult human brain. To resolve the conflicting interpretations in humans, it is necessary to identify and visualize dividing neuronal precursors or develop new methods to evaluate the age of a neuron at the single-cell level.

Diagnosis of spinal cord injury (SCI) severity at the ultra-acute stage is of great importance for emergency clinical care of patients as well as for potential enrollment into clinical trials. The lack of a diagnostic biomarker for SCI has played a major role in the poor results of clinical trials. We analyzed global gene expression in peripheral white blood cells during the acute injury phase and identified 197 genes whose expression changed after SCI compared with healthy and trauma controls and in direct relation to SCI severity. Unsupervised coexpression network analysis identified several gene modules that predicted injury severity (AIS grades) with an overall accuracy of 72.7% and included signatures of immune cell subtypes. Specifically, for complete SCIs (AIS A), ROC analysis showed impressive specificity and sensitivity (AUC: 0.865). Similar precision was also shown for AIS D SCIs (AUC: 0.938). Our findings indicate that global transcriptomic changes in peripheral blood cells have diagnostic and potentially prognostic value for SCI severity.

The use of microRNAs as biomarkers has been proposed for many diseases, including the diagnosis of melanoma. Although hundreds of microRNAs have been identified as differentially expressed in melanomas as compared to benign melanocytic lesions, a limited consensus has been achieved across studies, constraining the effective use of these potentially useful markers. In this study, we applied a machine learning-based pipeline to a dataset consisting of genetic features, clinical features, and next-generation microRNA sequencing from micro-dissected formalin-fixed paraffin embedded melanomas and their adjacent benign precursor nevi. We identified patient age and tumor cellularity as variables that frequently confound the measured expression of potentially diagnostic microRNAs. By employing the ratios of microRNAs that were either enriched or depleted in melanoma compared to the nevi as a normalization strategy, we developed a model that classified all the available published cohorts with an area under the receiver operating characteristic curve of 0.98. External validation on an independent cohort classified lesions with 81% sensitivity and 88% specificity and was uninfluenced by the tumor content of the sample or patient age.

Blood vessels in the CNS form a specialized and critical structure, the blood-brain barrier (BBB). We present a resource to understand the molecular mechanisms that regulate BBB function in health and dysfunction during disease. Using endothelial cell enrichment and RNA sequencing, we analyzed the gene expression of endothelial cells in mice, comparing brain endothelial cells with peripheral endothelial cells. We also assessed the regulation of CNS endothelial gene expression in models of stroke, multiple sclerosis, traumatic brain injury and seizure, each having profound BBB disruption. We found that although each is caused by a distinct trigger, they exhibit strikingly similar endothelial gene expression changes during BBB disruption, comprising a core BBB dysfunction module that shifts the CNS endothelial cells into a peripheral endothelial cell-like state. The identification of a common pathway for BBB dysfunction suggests that targeting therapeutic agents to limit it may be effective across multiple neurological disorders.

It is widely assumed that cells must be physically isolated to study their molecular profiles. However, intact tissue samples naturally exhibit variation in cellular composition, which drives covariation of cell-class-specific molecular features. By analyzing transcriptional covariation in 7,221 intact CNS samples from 840 neurotypical individuals, representing billions of cells, we reveal the core transcriptional identities of major CNS cell classes in humans. By modeling intact CNS transcriptomes as a function of variation in cellular composition, we identify cell-class-specific transcriptional differences in Alzheimer's disease, among brain regions, and between species. Among these, we show that PMP2 is expressed by human but not mouse astrocytes and significantly increases mouse astrocyte size upon ectopic expression in vivo, causing them to more closely resemble their human counterparts. Our work is available as an online resource ( http://oldhamlab.ctec.ucsf.edu/ ) and provides a generalizable strategy for determining the core molecular features of cellular identity in intact biological systems.

The neocortex of primates, including humans, contains more abundant and diverse inhibitory neurons compared with rodents, but the molecular foundations of these observations are unknown. Through integrative gene coexpression analysis, we determined a consensus transcriptional profile of GABAergic neurons in mid-gestation human neocortex. By comparing this profile to genes expressed in GABAergic neurons purified from neonatal mouse neocortex, we identified conserved and distinct aspects of gene expression in these cells between the species. We show here that the calcium-binding protein secretagogin (SCGN) is robustly expressed by neocortical GABAergic neurons derived from caudal ganglionic eminences (CGE) and lateral ganglionic eminences during human but not mouse brain development. Through electrophysiological and morphometric analyses, we examined the effects of SCGN expression on GABAergic neuron function and form. Forced expression of SCGN in CGE-derived mouse GABAergic neurons significantly increased total neurite length and arbor complexity following transplantation into mouse neocortex, revealing a molecular pathway that contributes to morphological differences in these cells between rodents and primates.

Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved.

Mammalian lungs are comprised of large numbers of tracheobronchial airways that transition from the trachea to alveoli. Studies as wide ranging as pollutant deposition and lung development rely on accurate characterization of these airways. Advancements in CT imaging and the value of computational approaches in eliminating the burden of manual measurement are providing increased efficiency in obtaining this geometric data. In this study, we compare an automated method to a manual one for the first six generations of three Balb/c mouse lungs. We find good agreement between manual and automated methods and that much of the disagreement can be attributed to method precision. Using the automated method, we then provide anatomical data for the entire tracheobronchial airway tree from three Balb/C mice. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:2046-2057, 2017. © 2017 Wiley Periodicals, Inc.

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.

Tissue homeostasis requires the production of newly differentiated cells from resident adult stem cells. Central to this process is the expansion of undifferentiated intermediates known as transit-amplifying (TA) cells, but how stem cells are triggered to enter this proliferative TA state remains an important open question. Using the continuously growing mouse incisor as a model of stem cell-based tissue renewal, we found that the transcriptional cofactors YAP and TAZ are required both to maintain TA cell proliferation and to inhibit differentiation. Specifically, we identified a pathway involving activation of integrin α3 in TA cells that signals through an LATS-independent FAK/CDC42/PP1A cascade to control YAP-S397 phosphorylation and nuclear localization. This leads to Rheb expression and potentiates mTOR signaling to drive the proliferation of TA cells. These findings thus reveal a YAP/TAZ signaling mechanism that coordinates stem cell expansion and differentiation during organ renewal.

[This corrects the article DOI: 10.1371/journal.pgen.1006425.].

Investigations into stem cell-fueled renewal of an organ benefit from an inventory of cell type-specific markers and a deep understanding of the cellular diversity within stem cell niches. Using the adult mouse incisor as a model for a continuously renewing organ, we performed an unbiased analysis of gene co-expression relationships to identify modules of co-expressed genes that represent differentiated cells, transit-amplifying cells, and residents of stem cell niches. Through in vivo lineage tracing, we demonstrated the power of this approach by showing that co-expression module members Lrig1 and Igfbp5 define populations of incisor epithelial and mesenchymal stem cells. We further discovered that two adjacent mesenchymal tissues, the periodontium and dental pulp, are maintained by distinct pools of stem cells. These findings reveal novel mechanisms of incisor renewal and illustrate how gene co-expression analysis of intact biological systems can provide insights into the transcriptional basis of cellular identity.

© 2016 Mitra et al.Sexual dimorphism in common disease is pervasive, including a dramatic male preponderance in autism spectrum disorders (ASDs). Potential genetic explanations include a liability threshold model requiring increased polymorphism risk in fe

Our ability to fight pathogens relies on major histocompatibility complex class I (MHC-I) molecules presenting diverse antigens on the surface of diseased cells. The transporter associated with antigen processing (TAP) transports nearly the entire repertoire of antigenic peptides into the endoplasmic reticulum for MHC-I loading. How TAP transports peptides specific for MHC-I is unclear. In this study, we used cryo-EM to determine a series of structures of human TAP, both in the absence and presence of peptides with various sequences and lengths. The structures revealed that peptides of eight or nine residues in length bind in a similarly extended conformation, despite having little sequence overlap. We also identified two peptide-anchoring pockets on either side of the transmembrane cavity, each engaging one end of a peptide with primarily main chain atoms. Occupation of both pockets results in a global conformational change in TAP, bringing the two halves of the transporter closer together to prime it for isomerization and ATP hydrolysis. Shorter peptides are able to bind to each pocket separately but are not long enough to bridge the cavity to bind to both simultaneously. Mutations that disrupt hydrogen bonds with the N and C termini of peptides almost abolish MHC-I surface expression. Our findings reveal that TAP functions as a molecular caliper that selects peptides according to length rather than sequence, providing antigen diversity for MHC-I presentation. [ABSTRACT FROM AUTHOR]

Microglia maintain homeostasis in the brain, but whether aberrant microglial activation can cause neurodegeneration remains controversial. Here, we use transcriptome profiling to demonstrate that deficiency in frontotemporal dementia (FTD) gene progranulin (Grn) leads to an age-dependent, progressive upregulation of lysosomal and innate immunity genes, increased complement production, and enhanced synaptic pruning in microglia. During aging, Grn(-/-) mice show profound microglia infiltration and preferential elimination of inhibitory synapses in the ventral thalamus, which lead to hyperexcitability in the thalamocortical circuits and obsessive-compulsive disorder (OCD)-like grooming behaviors. Remarkably, deleting C1qa gene significantly reduces synaptic pruning by Grn(-/-) microglia and mitigates neurodegeneration, behavioral phenotypes, and premature mortality in Grn(-/-) mice. Together, our results uncover a previously unrecognized role of progranulin in suppressing aberrant microglia activation during aging. These results represent an important conceptual advance that complement activation and microglia-mediated synaptic pruning are major drivers, rather than consequences, of neurodegeneration caused by progranulin deficiency.

Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex.

The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepithelium shows increased progenitor proliferation, decreased cell death, and precocious neuronal differentiation. mRNA profiling at multiple time points during neurulation uncovers a complex pattern of changing targets over time. Overexpression of one of these targets, Fgf15, in the neuroepithelium of the chick embryo induces precocious neuronal differentiation. Compound mutants between mir-302 and the related mir-290 locus have a synthetic lethal phenotype prior to neurulation. Our results show that mir-302 helps regulate neurulation by suppressing neural progenitor expansion and precocious differentiation. Furthermore, these results uncover redundant roles for mir-290 and mir-302 early in development.

© 2014 Macmillan Publishers Limited. All rights reserved.Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential of radial glia (RG; neural ste

BackgroundMixed lineage leukemia-1 (Mll1) epigenetically regulates gene expression patterns that specify cellular identity in both embryonic development and adult stem cell populations. In the adult mouse brain, multipotent neural stem cells (NSCs) in the subventricular zone generate new neurons throughout life, and Mll1 is required for this postnatal neurogenesis but not for glial cell differentiation. Analysis of Mll1-dependent transcription may identify neurogenic genes useful for the direct reprogramming of astrocytes into neurons.ObjectiveTo identify Mll1-dependent transcriptional modules and to determine whether genes in the neurogenic modules can be used to directly reprogram astrocytes into neurons.MethodsWe performed gene coexpression module analysis on microarray data from differentiating wild-type and Mll1-deleted subventricular zone NSCs. Key developmental regulators belonging to the neurogenic modules were overexpressed in Mll1-deleted cells and cultured cortical astrocytes, and cell phenotypes were analyzed by immunocytochemistry and electrophysiology.ResultsTranscriptional modules that correspond to neurogenesis were identified in wild-type NSCs. Modules related to astrocytes and oligodendrocytes were enriched in Mll1-deleted NSCs, consistent with their gliogenic potential. Overexpression of genes selected from the neurogenic modules enhanced the production of neurons from Mll1-deleted cells, and overexpression of Brn4 (Pou3f4) in nonneurogenic cortical astroglia induced their transdifferentiation into electrophysiologically active neurons.ConclusionOur results demonstrate that Mll1 is required for the expression of neurogenic but not gliogenic transcriptional modules in a multipotent NSC population and further indicate that specific Mll1-dependent genes may be useful for direct reprogramming strategies.

The epigenetic mechanisms that enable specialized astrocytes to retain neurogenic competence throughout adult life are still poorly understood. Here we show that astrocytes that serve as neural stem cells (NSCs) in the adult mouse subventricular zone (SVZ)

Ectopic expression of specific factors such as Oct4, Sox2, and Klf4 (OSK) is sufficient to reprogram somatic cells into induced pluripotent stem cells (iPSCs). In this study, we examine the paths taken by cells during the reprogramming process by following the transcriptional activation of two pluripotent miRNA clusters (mir-290 and mir-302) in individual cells in vivo and in vitro with knockin reporters. During embryonic development and embryonic stem cell differentiation, all cells sequentially expressed mir-290 and mir-302. In contrast, during OSK-induced reprogramming, cells activated the miRNA loci in a stochastic, nonordered manner. However, the addition of Sall4 to the OSK cocktail led to a consistent reverse sequence of locus activation (mir-302 then mir-290) and increased reprogramming efficiency. These results demonstrate that cells can follow multiple paths during the late stages of reprogramming, and that the trajectory of any individual cell is strongly influenced by the combination of factors introduced.

Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C-associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions.

Toxicological evaluations of flavor chemicals for use in inhalation products that utilize heat for aerosol generation are complicated because of the potential effect heat may have on the flavor chemical. The objective was to develop a thermal degradation technique to screen flavor chemicals as part of a toxicological testing program for their potential use in ENDS formulations. Based upon published data for acetaldehyde, acrolein, and glycidol from ENDS products (common thermal degradants of propylene glycol and glycerin), the pyrolizer temperature was adjusted until a similar ratio of acetaldehyde, acrolein, and glycidol was obtained from a 60/40 ratio (v/v) of glycerin/propylene glycol via GC/MS analysis. For each of 90 flavor chemicals, quantitative measurements of acetaldehyde, acrolein, and glycidol, in addition to semiquantitative non-targeted analysis tentatively identifying chemicals from thermal degradation, were obtained. Twenty flavor chemicals transferred at greater than 99% intact, another 26 transferred at greater than 95% intact, and another 15 flavor chemicals transferred at greater than 90% intact. Most flavor chemicals resulted in fewer than 10–12 tentatively identified thermal degradants. The practical approach to the thermal degradation of flavor chemicals provided useful information as part of the toxicological evaluation of flavor chemicals for potential use in ENDS formulations. [ABSTRACT FROM AUTHOR]