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1.  Intracellular and Extracellular Redox Status and Free Radical Generation in Primary Immune Cells from Children with Autism 
Autism Research and Treatment  2011;2012:986519.
The modulation of the redox microenvironment is an important regulator of immune cell activation and proliferation. To investigate immune cell redox status in autism we quantified the intracellular glutathione redox couple (GSH/GSSG) in resting peripheral blood mononuclear cells (PBMCs), activated monocytes and CD4 T cells and the extracellular cysteine/cystine redox couple in the plasma from 43 children with autism and 41 age-matched control children. Resting PBMCs and activated monocytes from children with autism exhibited significantly higher oxidized glutathione (GSSG) and percent oxidized glutathione equivalents and decreased glutathione redox status (GSH/GSSG). In activated CD4 T cells from children with autism, the percent oxidized glutathione equivalents were similarly increased, and GSH and GSH/GSSG were decreased. In the plasma, both glutathione and cysteine redox ratios were decreased in autistic compared to control children. Consistent with decreased intracellular and extracellular redox status, generation of free radicals was significantly elevated in lymphocytes from the autistic children. These data indicate primary immune cells from autistic children have a more oxidized intracellular and extracellular microenvironment and a deficit in glutathione-mediated redox/antioxidant capacity compared to control children. These results suggest that the loss of glutathione redox homeostasis and chronic oxidative stress may contribute to immune dysregulation in autism.
PMCID: PMC3420377  PMID: 22928106
2.  Volatile anesthetics attenuate oxidative stress-reduced activity of glutamate transporter type 3 
Anesthesia and analgesia  2009;109(5):1506-1510.
Volatile anesthetics enhance the activity of glutamate transporter type 3 (also called excitatory amino acid transporter type 3, EAAT3), the major neuronal EAAT. In addition to glutamate, EAAT3 can also uptake L-cysteine, the rate-limiting substrate for the synthesis of glutathione. Our previous study showed that oxidative stress inhibited glutamate-induced EAAT3 activity. We determined whether oxidative stress would reduce L-cysteine-induced EAAT3 activity and whether this reduction would be attenuated by volatile anesthetics.
Rat EAAT3 was expressed in Xenopus oocytes. L-glutamate- and L-cysteine-induced membrane currents were recorded using the two-electrode voltage clamp technique. The peak current was quantified to reflect the amount of transported substrates because transport of substrates via EAATs is electrogenic.
Exposure of oocytes to 5 mM tert-butyl hydroperoxide, an organic oxidant, for 10 min reduced the Vmax, but did not affect the Km, of EAAT3 for L-cysteine. The volatile anesthetics isoflurane, sevoflurane and desflurane at concentrations from 1 to 3% attenuated the tert-butyl hydroperoxide-reduced EAAT3 activity for L-glutamate and L-cysteine.
Our results suggest that volatile anesthetics preserve EAAT3 function to transport L-glutamate and L-cysteine under oxidative stress, which may be a mechanism for the neuroprotective effects of volatile anesthetics.
PMCID: PMC2773695  PMID: 19843789
3.  Habituation of exploratory behaviour in VPA rats: animal model of autism 
Interdisciplinary Toxicology  2013;6(4):222-227.
Autism is a neurodevelopmental disorder with multifactorial aetiology, represented as impairment in social behaviour, communication and the occurrence of repetitive activities, which can be observed in the early life. The core features are frequently accompanied by other manifestations, including limited environmental exploration. The aim of the presented study, realised on an animal model of autism – VPA rats, i.e. animals prenatally affected with valproic acid on gestation day 12.5, was to investigate the habituation process of exploratory activity (manifested by a gradual decrease in the intensity of locomotor activity), which reflects the stage of the central nervous system. VPA rats were tested in open-field in three developmental periods – weaning (postnatal day 21 – PND 21), puberty (PND 42) and adulthood (PND 72). In each period of ontogenesis, the rapidity of habituation was evaluated by using the method of linear regression. Compared to controls, VPA rats showed a significant decrease in the intensity and an increase in the rapidity of exploratory activity habituation during puberty and adulthood. Our results indicate that the animal model of autism, i.e. VPA rats, showed disabilities in the development of the nervous system. These findings can help confirm not only the validity of this animal model of autism but can also help better understand neuronal changes in humans with autism.
PMCID: PMC3945762  PMID: 24678262
autism; animal model; VPA rats; exploratory behaviour; habituation
4.  Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism 
Autism is a behaviorally-defined neurodevelopmental disorder usually diagnosed in early childhood that is characterized by impairment in reciprocal communication and speech, repetitive behaviors, and social withdrawal. Although both genetic and environmental factors are thought to be involved, none have been reproducibly identified. The metabolic phenotype of an individual reflects the influence of endogenous and exogenous factors on genotype. As such, it provides a window through which the interactive impact of genes and environment may be viewed and relevant susceptibility factors identified. Although abnormal methionine metabolism has been associated with other neurologic disorders, these pathways and related polymorphisms have not been evaluated in autistic children. Plasma levels of metabolites in methionine transmethylation and transsulfuration pathways were measured in 80 autistic and 73 control children. In addition, common polymorphic variants known to modulate these metabolic pathways were evaluated in 360 autistic children and 205 controls. The metabolic results indicated that plasma methionine and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), an indicator of methylation capacity, were significantly decreased in the autistic children relative to age-matched controls. In addition, plasma levels of cysteine, glutathione, and the ratio of reduced to oxidized glutathione, an indication of antioxidant capacity and redox homeostasis, were significantly decreased. Differences in allele frequency and/or significant gene-gene interactions were found for relevant genes encoding the reduced folate carrier (RFC 80G>A), transcobalamin II (TCN2 776G>C), catechol-O-methyltransferase (COMT 472G>A), methylenetetrahydrofolate reductase (MTHFR 677C>T and 1298A>C), and GST M1. We propose that an increased vulnerability to oxidative stress (endogenous or environmental) may contribute to the development and clinical manifestations of autism.
PMCID: PMC2610366  PMID: 16917939
autism; oxidative stress; genotype; glutathione; methionine
5.  Effect of Maternal Lipopolysaccharide Administration on the Development of Dopaminergic Receptors and Transporter in the Rat Offspring 
PLoS ONE  2013;8(1):e54439.
Epidemiological evidence supports that maternal infection during gestation are notable risk factors for developmental mental illnesses including schizophrenia and autism. In prenatal lipopolysaccharide (LPS) model of immune activation in rats, the offspring exhibit significant impairments in behaviors mediated by central dopamine (DA) system. This study aimed to examine the temporal and regional pattern of postnatal DA development in the male offspring of pregnant Sprague-Dawley rats administered with 100 µg/kg LPS or saline at gestational days 15/16. Using ligand autoradiography, D1 and D2 dopamine receptors (D1R, D2R) and dopamine transporter (DAT) binding levels were measured in the prefrontal cortex (PFC) and sub cortical regions (dorsal striatum and nucleus accumbens core and shell) at pre pubertal (P35) and post pubertal ages (P60). We found a significant decrease in D2R ligand [3H] YM-90151-2 binding in the medial PFC (mPFC) in prenatal LPS-treated animals at P35 and P60 compared to respective saline groups. The decrease in D2R levels was not observed in the striatum or accumbens of maternal LPS-treated animals. No significant changes were observed in [3H] SCH23390 binding to D1R. However, the level of [125I] RTI-121 binding to DAT was selectively reduced in the nucleus accumbens core and shell at P35 in the prenatal LPS group. Immunohistochemical analysis showed that number of D2R immunopositive cells in infralimbic/prelimbic (IL/PL) part of mPFC was significantly reduced in the LPS group at P60. Prenatal LPS treatment did not significantly affect either the total number of mature neurons or parvalbumin (PV)-immunopositive interneurons in this region. However the number of PV and D2R co-labeled neurons was significantly reduced in the IL/PL subregion of PFC of LPS treated animals. Our data suggests D2R deficit in the PFC and PV interneurons may be relevant to understanding mechanisms of cortical dysfunctions described in prenatal infection animal models as well as schizophrenia.
PMCID: PMC3547943  PMID: 23349891
6.  Neonatal Behavioral Changes in Rats With Gestational Exposure to Lipopolysaccharide: A Prenatal Infection Model for Developmental Neuropsychiatric Disorders 
Schizophrenia Bulletin  2010;38(3):444-456.
Exposure to prenatal infections has been widely associated with the increased risk for neuropsychiatric disorders of developmental origin such as schizophrenia and autism. Although several behavioral and cognitive deficits have been detected during adulthood in rodent models of prenatal infections, early behavioral changes have not been well characterized. In a prenatal lipopolysaccharide (LPS) model, we have previously observed significant alterations in the neuronal cytoarchitecture during early postnatal life. In the present study, we aimed to investigate the potential effects of prenatal immune activation on early neurophenotypic presentations using a set of behavioral test battery. Female Sprague-Dawley rats were administered with 100 μg/kg LPS (intraperitoneally) at gestational days 15 and 16. During the first postnatal week, we found no significant effect on maternal behavior or mother-pup interaction by this treatment. Also, no major changes in physical developmental milestones of pups were noted from postnatal (P) days P6 to P16. Importantly, prenatal LPS-exposed pups had a significant decrease in the number and duration of ultrasonic vocalization calls at P3 and P5. Prenatal LPS treatment also led to impairments in nest-seeking behavior and odor-stroke associative learning in neonatal rats at P8 and P9. At the molecular level, we detected significant decrease in the expression of cortical 5HT1A and 5HT1B messenger RNA at P3. These data suggest that prenatal exposure to an immune activator can significantly impair the social/communicative behavior in the neonate offspring, which may be relevant to childhood and premorbid abnormalities reported in autism and schizophrenia subjects.
PMCID: PMC3329978  PMID: 20805287
schizophrenia; immune activation; autism; serotonin; premorbid
7.  Nuclear Factor Erythroid 2-Related Factor 2 Facilitates Neuronal Glutathione Synthesis by Upregulating Neuronal Excitatory Amino Acid Transporter 3 Expression 
The Journal of Neuroscience  2011;31(20):7392-7401.
Astrocytes support neuronal antioxidant capacity by releasing glutathione, which is cleaved to cysteine in brain extracellular space. Free cysteine is then taken up by neurons through excitatory amino acid transporter 3 [EAAT3; also termed Slc1a1 (solute carrier family 1 member 1)] to support de novo glutathione synthesis. Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway by oxidative stress promotes astrocyte release of glutathione, but it remains unknown how this release is coupled to neuronal glutathione synthesis. Here we evaluated transcriptional regulation of the neuronal cysteine transporter EAAT3 by the Nrf2-ARE pathway. Nrf2 activators and Nrf2 overexpression both produced EAAT3 transcriptional activation in C6 cells. A conserved ARE-related sequence was found in the EAAT3 promoter of several mammalian species. This ARE-related sequence was bound by Nrf2 in mouse neurons in vivo as observed by chromatin immunoprecipitation. Chemical activation of the Nrf2-ARE pathway in mouse brain increased both neuronal EAAT3 levels and neuronal glutathione content, and these effects were abrogated in mice genetically deficient in either Nrf2 or EAAT3. Selective overexpression of Nrf2 in brain neurons by lentiviral gene transfer was sufficient to upregulate both neuronal EAAT3 protein and glutathione content. These findings identify a mechanism whereby Nrf2 activation can coordinate astrocyte glutathione release with neuronal glutathione synthesis through transcriptional upregulation of neuronal EAAT3 expression.
PMCID: PMC3339848  PMID: 21593323
8.  Associations of hypomelanotic skin disorders with autism: Do they reflect the effects of genetic mutations and epigenetic factors on vitamin-D metabolism in individuals at risk for autism? 
Vitamin D is crucial for full functioning in many genes, and vitamin-D deficiency interferes with many processes, including brain development and DNA repair. Several lines of evidence suggest that prenatal and early postnatal vitamin-D deficiency increases risk for autism, probably through multiple effects that include impaired brain development and increased de novo mutations. High rates of autism in several genetically based hypomelanotic skin disorders present a puzzle, because ultraviolet-B (UVB) radiation acting on skin is the major natural source of vitamin D, and lighter skin, which increases UVB penetration, helps protect against vitamin-D deficiency, especially at higher latitudes. Understanding autism’s association with hypomelanosis may elucidate autism’s etiology.
We consider two hypotheses that may help explain autism’s association with hypomelanotic disorders. Hypothesis 1) Because genetic and epigenetic variants that produce hypomelanotic conditions may help protect against vitamin-D deficiency, especially at higher latitudes, these variants may tend to decrease mortality – and increase the fertility – of individuals who also carry genetic or epigenetic factors that increase vulnerability to autism. Hypothesis 2) Children with hypomelanotic conditions will be more likely to develop autism, because children’s photosensitivity and parental concerns about sunburn and skin cancer lead them to excessively reduce children’s sun exposure and resultant vitamin-D levels.
One approach to testing these hypotheses would involve comparing the genomes, epigenetic markers, skin pigmentation, and serum and brain levels of the active form of vitamin D in autistic individuals, with and without co-morbid hypomelanoses, as well as in their relatives and controls. Because availability of UVB radiation varies widely around the world, epidemiological and genetic studies of the co-morbidity in different regions would provide complementary means of testing the hypotheses.
If test results support either hypothesis, they will add important evidence for an etiologic role of vitamin-D deficiency in autism, as well as supporting investigation of whether vitamin-D enhancement may aid treatment and prevention of autism.
PMCID: PMC4109325  PMID: 25067901
9.  Reduced Serotonin Receptor Subtypes in a Limbic and a Neocortical Region in Autism 
Autism is a behaviorally defined, neurological disorder with symptom onset before the age of three. Abnormalities in social-emotional behaviors are a core deficit in autism and are characterized by impaired reciprocal social interaction, lack of facial expressions, and the inability to recognize familiar faces. The posterior cingulate cortex (PCC) and fusiform gyrus (FG) are two regions within an extensive limbic-cortical network that contribute to social-emotional behaviors. Evidence indicates that changes in brains of individuals with autism begin prenatally. Serotonin (5HT) is one of the earliest expressed neurotransmitters, and plays an important role in synaptogenesis, neurite outgrowth, and neuronal migration. Abnormalities in 5HT systems have been implicated in several psychiatric disorders including autism, as evidenced by immunology, imaging, genetics, pharmacotherapy, and neuropathology. Although information is known regarding peripheral 5HT in autism, there is emerging evidence that 5HT systems in the CNS, including various 5HT receptor subtypes and transporters, are affected in autism. The present study demonstrated significant reductions in 5HT1A receptor binding density in superficial and deep layers of the PCC and FG, and in the density of 5HT2A receptors in superficial layers of the PCC and FG. Significant reduction in the density of serotonin transporters (5-HTT) was also found in the deep layers of the FG, but normal levels were demonstrated in both layers of the PCC and superficial layers of the FG. These studies provide potential substrates for decreased 5-HT modulation/innervation in the autism brain, and implicate two 5-HT receptor subtypes as potential neuromarkers for novel or existing pharmacotherapies.
PMCID: PMC3859849  PMID: 23894004
Autism; Serotonin; 5-HT1A receptors; 5-HT2A receptors; 5-HT transporters; pharmacotherapy; Selective Serotonin Re-uptake Inhibitors (SSRIs)
10.  Dietary Intake and Plasma Levels of Choline and Betaine in Children with Autism Spectrum Disorders 
Autism Research and Treatment  2013;2013:578429.
Abnormalities in folate-dependent one-carbon metabolism have been reported in many children with autism. Because inadequate choline and betaine can negatively affect folate metabolism and in turn downstream methylation and antioxidant capacity, we sought to determine whether dietary intake of choline and betaine in children with autism was adequate to meet nutritional needs based on national recommendations. Three-day food records were analyzed for 288 children with autism (ASDs) who participated in the national Autism Intervention Research Network for Physical Health (AIR-P) Study on Diet and Nutrition in children with autism. Plasma concentrations of choline and betaine were measured in a subgroup of 35 children with ASDs and 32 age-matched control children. The results indicated that 60–93% of children with ASDs were consuming less than the recommended Adequate Intake (AI) for choline. Strong positive correlations were found between dietary intake and plasma concentrations of choline and betaine in autistic children as well as lower plasma concentrations compared to the control group. We conclude that choline and betaine intake is inadequate in a significant subgroup of children with ASDs and is reflected in lower plasma levels. Inadequate intake of choline and betaine may contribute to the metabolic abnormalities observed in many children with autism and warrants attention in nutritional counseling.
PMCID: PMC3876775  PMID: 24396597
11.  Prenatal methylmercury exposure hampers glutathione antioxidant system ontogenesis and causes long-lasting oxidative stress in the mouse brain 
Toxicology and applied pharmacology  2007;227(1):147-154.
During the perinatal period, the central nervous system (CNS) is extremely sensitive to metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-induced developmental neurotoxicity remains obscure, several studies point to the glutathione (GSH) antioxidant system as an important molecular target for this toxicant. To extend our recent findings of MeHg-induced GSH dyshomeostasis, the present study was designed to assess the developmental profile of the GSH antioxidant system in the mouse brain during the early postnatal period after in utero exposure to MeHg. Pregnant mice were exposed to different doses of MeHg (1, 3 and 10 mg/L, diluted in drinking water, ad libitum) during the gestational period. After delivery, pups were killed at different time points - postnatal days (PNDs) 1, 11 and 21 - and the whole brain was used for determining biochemical parameters related to the antioxidant GSH system, as well as mercury content and the levels of F2-isoprostane. In control animals, cerebral GSH levels significantly increased over time during the early postnatal period; gestational exposure to MeHg caused a dose-dependent inhibition of this developmental event. Cerebral glutathione peroxidase (GPx) and glutathione reductase (GR) activities significantly increased over time during the early postnatal period in control animals; gestational MeHg exposure induced a dose-dependent inhibitory effect on both developmental phenomena. These adverse effects of prenatal MeHg exposure were corroborated by marked increases in cerebral F2-isoprostanes levels at all time points. Significant negative correlations were found between F2-isoprostanes and GSH, as well as between F2-isoprostanes and GPx activity, suggesting that MeHg-induced disruption of the GSH system maturation is related to MeHg-induced increased lipid peroxidation in the pup brain. In utero MeHg exposure also caused a dose-dependent increase in the cerebral levels of mercury at birth. Even though the cerebral mercury concentration decreased to nearly basal levels at postnatal day 21, GSH levels, GPx and GR activities remained decreased in MeHg-exposed mice, indicating that prenatal exposure to MeHg affects the cerebral GSH antioxidant systems by inducing biochemical alterations that endure even when mercury tissue levels decrease and become indistinguishable from those noted in pups born to control dams. This study is the first to show that prenatal exposure to MeHg disrupts the postnatal development of the glutathione antioxidant system in the mouse brain, pointing to an additional molecular mechanism by which MeHg induces pro-oxidative damage in the developing CNS. Moreover, our experimental observation corroborates previous reports on the permanent functional deficits observed after prenatal MeHg exposure.
PMCID: PMC2955629  PMID: 18023834
Methylmercury; prenatal exposure; developmental neurotoxicity; glutathione; oxidative stress; antioxidant enzymes
12.  Animal Models of Autism: An Epigenetic and Environmental Viewpoint 
Autism is a neurodevelopmental disorder of social behavior, which is more common in males than in females. The causes of autism are unknown; there is evidence for a substantial genetic component, but it is likely that a combination of genetic, environmental and epigenetic factors contribute to its complex pathogenesis. Rodent models that mimic the behavioral deficits of autism can be useful tools for dissecting both the etiology and molecular mechanisms. This review discusses animal models of autism generated by prenatal or neonatal environmental challenges, including virus infection and exposure to valproic acid (VPA) or stress. Studies of viral infection models suggest that interleukin-6 can influence fetal development and programming. Prenatal exposure to the histone deacetylase inhibitor VPA has been linked to autism in children, and male VPA-exposed rats exhibit a spectrum of autistic-like behaviors. The experience of prenatal stress produces male-specific behavioral abnormalities in rats. These effects may be mediated by epigenetic modifications such as DNA methylation and histone acetylation resulting in alterations to the transcriptome.
PMCID: PMC3661233  PMID: 23861630
autism; environmental factors; epigenetic processes; experimental animal models
13.  Aberrant Rab11-Dependent Trafficking of the Neuronal Glutamate Transporter EAAC1 Causes Oxidative Stress and Cell Death in Huntington's Disease 
The Journal of Neuroscience  2010;30(13):4552-4561.
Oxidative stress contributes to neurodegeneration in Huntington's disease (HD). However, the origins of oxidative stress in HD remain unclear. Studies in HD transgenic models suggest involvement of mitochondrial dysfunction, which would lead to overproduction of reactive oxygen species (ROS). Impaired mitochondria complexes occur in late stages of HD but not in presymptomatic or early-stage HD patients. Thus, other mechanisms may account for the earliest source of oxidative stress caused by endogenous mutant huntingtin. Here, we report that decreased levels of a major intracellular antioxidant glutathione coincide with accumulation of ROS in primary HD neurons prepared from embryos of HD knock-in mice (HD140Q/140Q), which have human huntingtin exon 1 with 140 CAG repeats inserted into the endogenous mouse huntingtin gene. Uptake of extracellular cysteine through the glutamate/cysteine transporter EAAC1 is required for de novo synthesis of glutathione in neurons. We found that, compared with wild-type neurons, HD neurons had lower cell surface levels of EAAC1 and were deficient in taking up cysteine. Constitutive trafficking of EAAC1 from recycling endosomes relies on Rab11 activity, which is defective in the brain of HD140Q/140Q mice. Enhancement of Rab11 activity by expression of a dominant-active Rab11 mutant in primary HD neurons ameliorated the deficit in cysteine uptake, increased levels of intracellular glutathione, normalized clearance of ROS, and improved neuronal survival. Our data support a novel mechanism for oxidative stress in HD: Rab11 dysfunction slows trafficking of EAAC1 to the cell surface and impairs cysteine uptake, thereby leading to deficient synthesis of glutathione.
PMCID: PMC3842456  PMID: 20357106
14.  Metabolic Imbalance Associated with Methylation Dysregulation and Oxidative Damage in Children with Autism 
Oxidative stress and abnormal DNA methylation have been implicated in the pathophysiology of autism. We investigated the dynamics of an integrated metabolic pathway essential for cellular antioxidant and methylation capacity in 68 children with autism, 54 age-matched control children and 40 unaffected siblings. The metabolic profile of unaffected siblings differed significantly from case siblings but not from controls. Oxidative protein/DNA damage and DNA hypomethylation (epigenetic alteration) were found in autistic children but not paired siblings or controls. These data indicate that the deficit in antioxidant and methylation capacity is specific for autism and may promote cellular damage and altered epigenetic gene expression. Further, these results suggest a plausible mechanism by which pro-oxidant environmental stressors may modulate genetic predisposition to autism.
PMCID: PMC3342663  PMID: 21519954
Autism; Oxidative stress; Metabolic; Epigenetics; Glutathione; DNA methylation
15.  Epigenetics and Neural Developmental Disorders: Washington DC, September 18 and 19, 2006 
Neural developmental disorders, such as autism, Rett Syndrome, Fragile X syndrome, and Angelman syndrome manifest during early postnatal neural development. Although the genes responsible for some of these disorders have been identified, how the mutations of these genes affect neural development is currently unclear. Emerging evidence suggest that these disorders share common underlying defects in neuronal morphology, synaptic connectivity and brain plasticity. In particular, alterations in dendritic branching and spine morphology play a central role in the pathophysiology of most mental retardation disorders, suggesting that common pathways regulating neuronal function may be affected. Epigenetic modulations, mediated by DNA methylation, RNA-associated silencing, and histone modification, can serve as an intermediate process that imprints dynamic environmental experiences on the “fixed” genome, resulting in stable alterations in phenotypes. Disturbance in epigenetic regulations can lead to inappropriate expression or silencing of genes, causing an array of multi-system disorders and neoplasias. Rett syndrome, the most common form of mental retardation in young girls, is due to l mutation of MECP2, encoding a methylated DNA binding protein that translates DNA methylation into gene repression. Angelman syndrome is due to faulty genomic imprinting or maternal mutations in UBE3A. Fragile X Syndrome, in most cases, results from the hypermethylation of FMR1 promoter, hence the loss of expression of functional FMRP protein. Autism, with its complex etiology, may have strong epigenetic link. Together, these observations strongly suggest that epigenetic mechanisms may play a critical role in brain development and etiology of related disorders. This report summarizes the scientific discussions and major conclusions from a recent conference that aimed to gain insight into the common molecular pathways affected among these disorders and discover potential therapeutic targets that have been missed by looking at one disorder at a time.
PMCID: PMC2700626  PMID: 17965627
Epigenetic; DNA methylation; chromatin; development; Rett Syndrome; Fragile X syndrome; Angelman syndrome; autism; neuronal maturation; synaptogenesis
16.  Age-Dependent Brain Gene Expression and Copy Number Anomalies in Autism Suggest Distinct Pathological Processes at Young Versus Mature Ages 
PLoS Genetics  2012;8(3):e1002592.
Autism is a highly heritable neurodevelopmental disorder, yet the genetic underpinnings of the disorder are largely unknown. Aberrant brain overgrowth is a well-replicated observation in the autism literature; but association, linkage, and expression studies have not identified genetic factors that explain this trajectory. Few studies have had sufficient statistical power to investigate whole-genome gene expression and genotypic variation in the autistic brain, especially in regions that display the greatest growth abnormality. Previous functional genomic studies have identified possible alterations in transcript levels of genes related to neurodevelopment and immune function. Thus, there is a need for genetic studies involving key brain regions to replicate these findings and solidify the role of particular functional pathways in autism pathogenesis. We therefore sought to identify abnormal brain gene expression patterns via whole-genome analysis of mRNA levels and copy number variations (CNVs) in autistic and control postmortem brain samples. We focused on prefrontal cortex tissue where excess neuron numbers and cortical overgrowth are pronounced in the majority of autism cases. We found evidence for dysregulation in pathways governing cell number, cortical patterning, and differentiation in young autistic prefrontal cortex. In contrast, adult autistic prefrontal cortex showed dysregulation of signaling and repair pathways. Genes regulating cell cycle also exhibited autism-specific CNVs in DNA derived from prefrontal cortex, and these genes were significantly associated with autism in genome-wide association study datasets. Our results suggest that CNVs and age-dependent gene expression changes in autism may reflect distinct pathological processes in the developing versus the mature autistic prefrontal cortex. Our results raise the hypothesis that genetic dysregulation in the developing brain leads to abnormal regional patterning, excess prefrontal neurons, cortical overgrowth, and neural dysfunction in autism.
Author Summary
Autism is a disorder characterized by aberrant social, communication, and restricted and repetitive behaviors. It develops clinically in the first years of life. Toddlers and children with autism often exhibit early brain enlargement and excess neuron numbers in the prefrontal cortex. Adults with autism generally do not display enlargement but instead may have a smaller brain size. Thus, we investigated DNA and mRNA patterns in prefrontal cortex from young versus adult postmortem individuals with autism to identify age-related gene expression differences as well as possible genetic correlates of abnormal brain enlargement, excess neuron numbers, and abnormal functioning in this disorder. We found abnormalities in genetic pathways governing cell number, neurodevelopment, and cortical lateralization in autism. We also found that the key pathways associated with autism are different between younger and older autistic individuals. These findings suggest that dysregulated gene pathways in the early stages of neurodevelopment could lead to later behavioral and cognitive deficits associated with autism.
PMCID: PMC3310790  PMID: 22457638
17.  Altered posterior cingulate cortical cyctoarchitecture, but normal density of neurons and interneurons in the posterior cingulate cortex and fusiform gyrus in autism 
Lay Abstract
Autism is a behaviorally defined disorder with increasing prevalence rates globally. The disorder is characterized by deficits in several domains including social behaviors, restricted and repetitive behaviors, and deficits in communication. Two regions thought to contribute to deficits in social behavior are the posterior cingulate cortex (PCC) and fusiform gyrus (FFG). The PCC is involved in processing emotionally salient stimuli, and also has a role in processing faces. The FFG is the area responsible for object and face recognition. A potential imbalance between excitatory and inhibitory processing in the brain may contribute to some of the abnormal social behaviors observed in autism. This is supported by previous work suggesting reduced GABA receptors in the autistic brain. The present study used thionin stained section to qualitatively assess cortical patterning and quantitatively assess the density of neurons. Furthermore, immunohistochemistry was used to determine the density of a subset of GABAergic interneurons. In the autistic brain, the PCC displayed several abnormal cortical patterns including irregularly distributed neurons in specific cortical layers, and the presence of increased white matter neurons. In marked contrast, the FFG appeared normal and there were no significant differences in the density of neurons or interneurons in either region. The present study highlights the presence of abnormal findings in the PCC, which appear to have developmental origins and could affect local processing of social-emotional behaviors as well as the function of interrelated cortical areas.
Scientific Abstract
Autism is a developmental disorder with prenatal origins, currently estimated to affect 1 in 91 children in the United States. Social-emotional deficits are a hallmark of autism and early neuropathology studies have indicated involvement of the limbic system. Imaging studies demonstrate abnormal activation of the posterior cingulate cortex (PCC), a component of the limbic system. Abnormal activation has also been noted in the fusiform gyrus (FFG), a region important for facial recognition and a key element in social interaction. A potential imbalance between excitatory and inhibitory interneurons in the cortex may contribute to altered information processing in autism. Furthermore, reduced numbers of GABA receptors have previously been reported in the autistic brain. Thionin stained sections were used to qualitatively assess cytoarchitectonic patterning and quantitatively determine the density of neurons and immunohistochemistry was used to determine the densities of a subset of GABAergic interneurons utilizing parvalbumin- and calbindin- immunoreactivity. In autism, the PCC displayed altered cytoarchitecture with irregularly distributed neurons, poorly demarcated layers IV and V, and increased presence of white matter neurons. In contrast, no neuropathology was observed in the FFG. There was no significant difference in the density of thionin, parvalbumin, or calbindin interneurons in either region and there was a trend towards a reduced density of calbindin neurons in the PCC. This study highlights the presence of abnormal findings in the PCC, which appear to be developmental in nature and could affect the local processing of social-emotional behaviors as well as functioning of interrelated areas.
PMCID: PMC3110607  PMID: 21360830
18.  Nutritional and metabolic status of children with autism vs. neurotypical children, and the association with autism severity 
The relationship between relative metabolic disturbances and developmental disorders is an emerging research focus. This study compares the nutritional and metabolic status of children with autism with that of neurotypical children and investigates the possible association of autism severity with biomarkers.
Participants were children ages 5-16 years in Arizona with Autistic Spectrum Disorder (n = 55) compared with non-sibling, neurotypical controls (n = 44) of similar age, gender and geographical distribution. Neither group had taken any vitamin/mineral supplements in the two months prior to sample collection. Autism severity was assessed using the Pervasive Development Disorder Behavior Inventory (PDD-BI), Autism Treatment Evaluation Checklist (ATEC), and Severity of Autism Scale (SAS). Study measurements included: vitamins, biomarkers of vitamin status, minerals, plasma amino acids, plasma glutathione, and biomarkers of oxidative stress, methylation, sulfation and energy production.
Biomarkers of children with autism compared to those of controls using a t-test or Wilcoxon test found the following statistically significant differences (p < 0.001): Low levels of biotin, plasma glutathione, RBC SAM, plasma uridine, plasma ATP, RBC NADH, RBC NADPH, plasma sulfate (free and total), and plasma tryptophan; also high levels of oxidative stress markers and plasma glutamate. Levels of biomarkers for the neurotypical controls were in good agreement with accessed published reference ranges. In the Autism group, mean levels of vitamins, minerals, and most amino acids commonly measured in clinical care were within published reference ranges.
A stepwise, multiple linear regression analysis demonstrated significant associations between several groups of biomarkers with all three autism severity scales, including vitamins (adjusted R2 of 0.25-0.57), minerals (adj. R2 of 0.22-0.38), and plasma amino acids (adj. R2 of 0.22-0.39).
The autism group had many statistically significant differences in their nutritional and metabolic status, including biomarkers indicative of vitamin insufficiency, increased oxidative stress, reduced capacity for energy transport, sulfation and detoxification. Several of the biomarker groups were significantly associated with variations in the severity of autism. These nutritional and metabolic differences are generally in agreement with other published results and are likely amenable to nutritional supplementation. Research investigating treatment and its relationship to the co-morbidities and etiology of autism is warranted.
PMCID: PMC3135510  PMID: 21651783
19.  Prenatal Treatment for Serious Neurological Sequelae of Congenital Toxoplasmosis: An Observational Prospective Cohort Study 
PLoS Medicine  2010;7(10):e1000351.
An observational study by Ruth Gilbert and colleagues finds that prenatal treatment of congenital toxoplasmosis could substantially reduce the proportion of infected fetuses that develop serious neurological sequelae.
The effectiveness of prenatal treatment to prevent serious neurological sequelae (SNSD) of congenital toxoplasmosis is not known.
Methods and Findings
Congenital toxoplasmosis was prospectively identified by universal prenatal or neonatal screening in 14 European centres and children were followed for a median of 4 years. We evaluated determinants of postnatal death or SNSD defined by one or more of functional neurological abnormalities, severe bilateral visual impairment, or pregnancy termination for confirmed congenital toxoplasmosis. Two-thirds of the cohort received prenatal treatment (189/293; 65%). 23/293 (8%) fetuses developed SNSD of which nine were pregnancy terminations. Prenatal treatment reduced the risk of SNSD. The odds ratio for prenatal treatment, adjusted for gestational age at maternal seroconversion, was 0.24 (95% Bayesian credible intervals 0.07–0.71). This effect was robust to most sensitivity analyses. The number of infected fetuses needed to be treated to prevent one case of SNSD was three (95% Bayesian credible intervals 2–15) after maternal seroconversion at 10 weeks, and 18 (9–75) at 30 weeks of gestation. Pyrimethamine-sulphonamide treatment did not reduce SNSD compared with spiramycin alone (adjusted odds ratio 0.78, 0.21–2.95). The proportion of live-born infants with intracranial lesions detected postnatally who developed SNSD was 31.0% (17.0%–38.1%).
The finding that prenatal treatment reduced the risk of SNSD in infected fetuses should be interpreted with caution because of the low number of SNSD cases and uncertainty about the timing of maternal seroconversion. As these are observational data, policy decisions about screening require further evidence from a randomized trial of prenatal screening and from cost-effectiveness analyses that take into account the incidence and prevalence of maternal infection.
Please see later in the article for the Editors' Summary
Editors' Summary
Toxoplasmosis is a very common parasitic infection. People usually become infected with Toxoplasma gondii, the parasite that causes toxoplasmosis, by eating raw or undercooked meat that contains the parasite, but it can also be contracted by drinking unfiltered water or by handling cat litter. Most people with toxoplasmosis never know they have the disease. However, if a pregnant woman becomes infected with T. gondii, she can transmit the parasite to her unborn baby (fetus). Overall, about a quarter of women who catch toxoplasmosis during pregnancy transmit the parasite to their fetus. If transmission occurs early during pregnancy, the resultant “congenital toxoplasmosis” increases the risk of miscarriage and the risk of the baby being born with brain damage, epilepsy, deafness, blindness, or developmental problems (“serious neurological sequelae”). In the worst cases, babies may be born dead or die soon after birth. Congenital toxoplasmosis caught during the final third of pregnancy may not initially cause any health problems but eyesight problems often develop later in life.
Why Was This Study Done?
Clinicians can find out if a woman has been infected with T. gondii during pregnancy by looking for parasite-specific antibodies (proteins made by the immune system that fight infections) in her blood. If the pattern of antibodies suggests a recent infection, the woman can be given spiramycin or pyrimethamine-sulfonamide, antibiotics that are thought to reduce the risk of transmission to the fetus and the severity of toxoplasmosis in infected fetuses. In some countries where toxoplasmosis is particularly common (for example, France), pregnant women are routinely screened for toxoplasmosis and treated with antibiotics if there are signs of recent infection. But is prenatal treatment an effective way to prevent the serious neurological sequelae or postnatal death (SNSD) associated with congenital toxoplasmosis? In this observational study, the researchers examine this question by studying a group of children identified as having congenital toxoplasmosis by prenatal or neonatal screening in six European countries. An observational study measures outcomes in a group of patients without trying to influence those outcomes by providing a specific treatment.
What Did the Researchers Do and Find?
The researchers followed 293 children in whom congenital toxoplasmosis had been identified by prenatal screening (in France, Austria, and Italy) or by neonatal screening (in Denmark, Sweden, and Poland) for an average 4 years. Two-thirds of the children received prenatal treatment for toxoplasmosis and 23 fetuses (8% of the fetuses) developed SNSD; nine of these cases of SNSD were terminated during pregnancy. By comparing the number of cases of SNSD among children who received prenatal treatment with the number among children who did not receive prenatal treatment, the researchers estimate that prenatal treatment reduced the risk of SNSD by three-quarters. They also estimate that to prevent one case of SNSD after maternal infection at 10 weeks of pregnancy, it would be necessary to treat three fetuses with confirmed infection. To prevent one case of SNSD after maternal infection at 30 weeks of pregnancy, 18 fetuses would need to be treated. Finally, the researchers report that the effectiveness of pyrimethamine-sulfonamide and spiramycin (which is less toxic) was similar, and that a third of live-born infants with brain damage that was detected after birth subsequently developed SNSD.
What Do These Findings Mean?
These findings suggest that prenatal treatment of congenital toxoplasmosis could substantially reduce the proportion of infected fetuses that develop SNDS and would be particularly effective in fetuses whose mothers acquired T. gondii during the first third of pregnancy. These findings should be interpreted with caution, however, because of the small number of affected fetuses in the study and because of uncertainty about the timing of maternal infection. Furthermore, these findings only relate to the relatively benign strain of T. gondii that predominates in Europe and North America; further studies are needed to test whether prenatal treatment is effective against the more virulent strains of the parasite that occur in South America. Finally, because this study is an observational study, its findings might reflect differences between the study participants other than whether or not they received prenatal treatment. These findings need to be confirmed in randomized controlled trials of prenatal screening, therefore, before any policy decisions are made about routine prenatal screening and treatment for congenital toxoplasmosis.
Additional Information
Please access these Web sites via the online version of this summary at
The US Centers for Disease Control and Prevention provides detailed information about all aspects of toxoplasmosis, including toxoplasmosis in pregnant women (in English and Spanish)
The UK National Health Services Choices website has information for patients about toxoplasmosis and about the risks of toxoplasmosis during pregnancy
KidsHealth, a resource maintained by the Nemours Foundation (a not-for-profit organization for children's health), provides information for parents about toxoplasmosis (in English and Spanish)
Tommy's, a nonprofit organization that funds research on the health of babies, also has information on toxoplasmosis
MedlinePlus provides links to other information on toxoplasmosis (in English and Spanish)
EUROTOXO contains reports generated by a European consensus development project
Uptodate provides information about toxoplasmosis and pregnancy
PMCID: PMC2953528  PMID: 20967235
20.  Complex epigenetic regulation of Engrailed-2 (EN-2) homeobox gene in the autism cerebellum 
Translational Psychiatry  2013;3(2):e232-.
The elucidation of epigenetic alterations in the autism brain has potential to provide new insights into the molecular mechanisms underlying abnormal gene expression in this disorder. Given strong evidence that engrailed-2 (EN-2) is a developmentally expressed gene relevant to cerebellar abnormalities and autism, the epigenetic evaluation of this candidate gene was undertaken in 26 case and control post-mortem cerebellar samples. Assessments included global DNA methylation, EN-2 promoter methylation, EN-2 gene expression and EN-2 protein levels. Chromatin immunoprecipitation was used to evaluate trimethylation status of histone H3 lysine 27 (H3K27) associated with gene downregulation and histone H3 lysine 4 (H3K4) associated with gene activation. The results revealed an unusual pattern of global and EN-2 promoter region DNA hypermethylation accompanied by significant increases in EN-2 gene expression and protein levels. Consistent with EN-2 overexpression, histone H3K27 trimethylation mark in the EN-2 promoter was significantly decreased in the autism samples relative to matched controls. Supporting a link between reduced histone H3K27 trimethylation and increased EN-2 gene expression, the mean level of histone H3K4 trimethylation was elevated in the autism cerebellar samples. Together, these results suggest that the normal EN-2 downregulation that signals Purkinje cell maturation during late prenatal and early-postnatal development may not have occurred in some individuals with autism and that the postnatal persistence of EN-2 overexpression may contribute to autism cerebellar abnormalities.
PMCID: PMC3590998  PMID: 23423141
Autism; cerebellum; DNA; EN-2; Epigenetics; histone methylation
21.  Association of hypomelanotic skin disorders with autism: links to possible etiologic role of vitamin-D levels in autism? 
Vitamin D is crucial for several key physiological processes, including brain development, DNA repair, and regulation of many genes. Much evidence indicates prenatal and early postnatal vitamin-D deficiency increases autism risk, probably through multiple effects, including impaired brain development and increased de novo mutations. High autism rates in several genetically based hypomelanotic skin disorders are puzzling, because ultraviolet-B radiation (UVB) in sunlight acting on skin is a key source of vitamin-D, and lighter skin protects against vitamin-D deficiency, especially at high latitudes. We consider two hypotheses to help explain autism's co-morbidity with hypomelanosis. 1) Because genetic and epigenetic variants that produce hypomelanosis help protect against vitamin-D deficiency, they increase reproductive fitness of individuals who also have other autism risk factors. 2) Hypomelanotic children have increased autism risk because photosensitivity and skin-cancer concerns lead families to excessively reduce children's sun exposure. Hypothesis testing could involve studies comparing genomes, epigenetic markers, skin pigmentation, and vitamin-D levels in autistic individuals with and without hypomelanosis, their relatives and controls. Conducting such studies in samples from regions that differ widely in UVB availability would provide particularly valuable data. Support for either hypothesis would elucidate vitamin-D's role in autism and suggest vitamin-D enhancement may aid treatment and prevention of autism.
PMCID: PMC3177756  PMID: 21949515
22.  A functional polymorphism in the reduced folate carrier gene and DNA hypomethylation in mothers of children with autism 
The biologic basis of autism is complex and is thought to involve multiple and variable gene-environment interactions. While the logical focus has been on the affected child, the impact of maternal genetics on intrauterine microenvironment during pivotal developmental windows could be substantial. Folate-dependent one carbon metabolism is a highly polymorphic pathway that regulates the distribution of one-carbon derivatives between DNA synthesis (proliferation) and DNA methylation (cell-specific gene expression and differentiation). These pathways are essential to support the programmed shifts between proliferation and differentiation during embryogenesis and organogenesis. Maternal genetic variants that compromise intrauterine availability of folate derivatives could alter fetal cell trajectories and disrupt normal neurodevelopment. In this investigation, the frequency of common functional polymorphisms in the folate pathway was investigated in a large population-based sample of autism case-parent triads. In case-control analysis, a significant increase in the reduced folate carrier (RFC1) G allele frequency was found among case mothers, but not among fathers or affected children. Subsequent log linear analysis of the RFC1 A80G genotype within family trios revealed that the maternal G allele was associated with a significant increase in risk of autism whereas the inherited genotype of the child was not. Further, maternal DNA from the autism mothers was found to be significantly hypomethylated relative to reference control DNA. Metabolic profiling indicated that plasma homocysteine, adenosine, and S-adenosylhomocyteine were significantly elevated among autism mothers consistent with reduced methylation capacity and DNA hypomethylation. Together, these results suggest that the maternal genetics/epigenetics may influence fetal predisposition to autism.
PMCID: PMC2943349  PMID: 20468076
Autism; reduced folate carrier; maternal; polymorphism; DNA methylation; epigenetics
23.  Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3) 
PLoS ONE  2014;9(10):e109245.
Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.
PMCID: PMC4183567  PMID: 25275463
24.  Above genetics: lessons from cerebral development in autism 
Translational neuroscience  2011;2(2):106-120.
While a distinct minicolumnar phenotype seems to be an underlying factor in a significant portion of cases of autism, great attention is being paid not only to genetics but to epigenetic factors which may lead to development of the conditions. Here we discuss the indivisible role the molecular environment plays in cellular function, particularly the pivotal position which the transcription factor and adhesion molecule, β-catenin, occupies in cellular growth. In addition, the learning environment is not only integral to postnatal plasticity, but the prenatal environment plays a vital role during corticogenesis, neuritogenesis, and synaptogenesis as well. To illustrate these points in the case of autism, we review important findings in genetics studies (e.g., PTEN, TSC1/2, FMRP, MeCP2, Neurexin-Neuroligin) and known epigenetic factors (e.g., valproic acid, estrogen, immune system, ultrasound) which may predispose towards the minicolumnar and connectivity patterns seen in the conditions, showing how one-gene mutational syndromes and exposure to certain CNS teratogens may ultimately lead to comparable phenotypes. This in turn may shed greater light on how environment and complex genetics combinatorially give rise to a heterogenetic group of conditions such as autism.
PMCID: PMC3331673  PMID: 22523638
beta catenin; minicolumns; neural stem cells; Rett syndrome; fragile X syndrome; tuberous sclerosis; valproic acid; PTEN phosphohydrolase; ultrasonography; cell adhesion molecules; neuronal
25.  Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain 
Translational Psychiatry  2012;2(7):e134-.
Despite increasing evidence of oxidative stress in the pathophysiology of autism, most studies have not evaluated biomarkers within specific brain regions, and the functional consequences of oxidative stress remain relatively understudied. We examined frozen samples from the cerebellum and temporal cortex (Brodmann area 22 (BA22)) from individuals with autism and unaffected controls (n=15 and n=12 per group, respectively). Biomarkers of oxidative stress, including reduced glutathione (GSH), oxidized glutathione (GSSG) and glutathione redox/antioxidant capacity (GSH/GSSG), were measured. Biomarkers of oxidative protein damage (3-nitrotyrosine; 3-NT) and oxidative DNA damage (8-oxo-deoxyguanosine; 8-oxo-dG) were also assessed. Functional indicators of oxidative stress included relative levels of 3-chlorotyrosine (3-CT), an established biomarker of a chronic inflammatory response, and aconitase activity, a biomarker of mitochondrial superoxide production. Consistent with previous studies on plasma and immune cells, GSH and GSH/GSSG were significantly decreased in both autism cerebellum (P<0.01) and BA22 (P<0.01). There was a significant increase in 3-NT in the autism cerebellum and BA22 (P<0.01). Similarly, 8-oxo-dG was significantly increased in autism cerebellum and BA22 (P<0.01 and P=0.01, respectively), and was inversely correlated with GSH/GSSG in the cerebellum (P<0.01). There was a significant increase in 3-CT levels in both brain regions (P<0.01), whereas aconitase activity was significantly decreased in autism cerebellum (P<0.01), and was negatively correlated with GSH/GSSG (P=0.01). Together, these results indicate that decreased GSH/GSSG redox/antioxidant capacity and increased oxidative stress in the autism brain may have functional consequence in terms of a chronic inflammatory response, increased mitochondrial superoxide production, and oxidative protein and DNA damage.
PMCID: PMC3410618  PMID: 22781167
aconitase; mitochondria; neuroinflammation; oxidative stress; 3-chlorotyrosine; 3-nitrotyrosine

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