Ciernia AV, LaSalle J. The landscape of DNA methylation amid a perfect storm of autism aetiologies » 2016 Jul;17(7):411-23. doi: 10.1038/nrn.2016.41. Epub 2016 May 6. PMC4966286

Increasing evidence points to a complex interplay between genes and the environment in autism spectrum disorder (ASD), including rare de novo mutations in chromatin genes such as methyl-CpG binding protein 2 (MECP2) in Rett syndrome. Epigenetic mechanisms such as DNA methylation act at this interface, reflecting the plasticity in metabolic and neurodevelopmentally regulated gene pathways. Genome-wide studies of gene sequences, gene pathways and DNA methylation are providing valuable mechanistic insights into ASD. The dynamic developmental landscape of DNA methylation is vulnerable to numerous genetic and environmental insults: therefore, understanding pathways that are central to this 'perfect storm' will be crucial to improving the diagnosis and treatment of ASD.

Crawley JN, Heyer WD, LaSalle JM. Autism and Cancer Share Risk Genes, Pathways, and Drug Targets. » Trends Genet. 2016 Mar;32(3):139-46. doi: 10.1016/j.tig.2016.01.001. Epub 2016 Jan 29 PMCID:PMC4769654

Autism is a neurodevelopmental disorder, diagnosed behaviorally by social and communication deficits, repetitive behaviors, and restricted interests. Recent genome-wide exome sequencing has revealed extensive overlap in risk genes for autism and for cancer. Understanding the genetic commonalities of autism(s) and cancer(s), with a focus on mechanistic pathways, could lead to repurposed therapeutics.

Jones KL, Croen LA, Yoshida CK, Heuer L, Hansen R, Zerbo O, DeLorenze GN, Kharrazi M, Yolken R, Ashwood P, Van de Water J. Autism with intellectual disability is associated with increased levels of maternal cytokines and chemokines during gestation. » PMID:27217154

Immune abnormalities have been described in some individuals with autism spectrum disorders (ASDs) as well as their family members. However, few studies have directly investigated the role of prenatal cytokine and chemokine profiles on neurodevelopmental outcomes in humans. In the current study, we characterized mid-gestational serum profiles of 22 cytokines and chemokines in mothers of children with ASD (N=415), developmental delay (DD) without ASD (N=188), and general population (GP) controls (N=428) using a bead-based multiplex technology. The ASD group was further divided into those with intellectual disabilities (developmental/cognitive and adaptive composite score <70) (ASD+ID, N=184) and those without (composite score⩾70) (ASD-noID, N=201). Levels of cytokines and chemokines were compared between groups using multivariate logistic regression analyses, adjusting for maternal age, ethnicity, birth country and weight, as well as infant gender, birth year and birth month. Mothers of children with ASD+ID had significantly elevated mid-gestational levels of numerous cytokines and chemokines, such as granulocyte macrophage colony-stimulating factor, interferon-γ, interleukin-1α (IL-1α) and IL-6, compared with mothers of children with either ASD-noID, those with DD, or GP controls. Conversely, mothers of children with either ASD-noID or with DD had significantly lower levels of the chemokines IL-8 and monocyte chemotactic protein-1 compared with mothers of GP controls. This observed immunologic distinction between mothers of children with ASD+ID from mothers of children with ASD-noID or DD suggests that the intellectual disability associated with ASD might be etiologically distinct from DD without ASD. These findings contribute to the ongoing efforts toward identification of early biological markers specific to subphenotypes of ASD.

Krakowiak P, Walker CK, Tancredi D, Hertz-Picciotto I, Van de Water J. Autism-specific maternal anti-fetal brain autoantibodies are associated with metabolic conditions. » Autism Res. 2016 Jun 17. doi: 10.1002/aur.1657. PMID:27312731

Approximately 23% of mothers of children with autism spectrum disorder (ASD) produce specific patterns of autoantibodies to fetal brain proteins that have been detected in only 1% of mothers of typically developing children. The biological mechanisms underlying the development of ASD-specific maternal autoantibodies are poorly understood. We sought to determine whether ASD-specific maternal autoantibodies identified postnatally were associated with metabolic conditions (MCs) during gestation. Participants were 227 mothers of 2-5 year old children with confirmed ASD, enrolled in CHARGE (Childhood Autism Risk from Genetics and the Environment) between January 2003 and April 2008, and from whom blood samples were collected and analyzed for anti-fetal brain autoantibodies (Ab+). MCs included diabetes, hypertensive disorders, and prepregnancy obesity or overweight, ascertained from medical records or structured telephone interviews. Log-linear regression models were performed to estimate prevalence ratios and 95% confidence intervals (CI) based on robust standard errors. Fifty-six (25%) mothers were Ab+. Ab+ prevalence was higher among mothers with diabetes, hypertensive disorders, or overweight compared to healthy mothers, but differences were not statistically significant. In a subset of 145 mothers whose children exhibited severe ASD (31 Ab+), those diagnosed with type 2 or gestational diabetes were 2.7-fold more likely to be Ab+ (95% CI 1.1, 6.6), controlling for delivery payer and smoking. Gestational diabetes specifically was associated with a 3.2-fold increased Ab+ prevalence (95% CI 1.2, 8.6). In this exploratory study, mothers whose children had severe ASD and who experienced diabetes were more likely to have anti-fetal brain autoantibodies 2-5 years later.

Martínez-Cerdeño V, Camacho J, Fox E, Miller E, Ariza J, Kienzle D, Plank K, Noctor SC, Van de Water J. Prenatal Exposure to Autism-Specific Maternal Autoantibodies Alters Proliferation of Cortical Neural Precursor Cells, Enlarges Brain, and Increases Neuronal Size in Adult Animals » Cereb Cortex. 2016 Jan;26(1):374-83. doi: 10.1093/cercor/bhu291. PMID: 25535268

Autism spectrum disorders (ASDs) affect up to 1 in 68 children. Autism-specific autoantibodies directed against fetal brain proteins have been found exclusively in a subpopulation of mothers whose children were diagnosed with ASD or maternal autoantibody-related autism. We tested the impact of autoantibodies on brain development in mice by transferring human antigen-specific IgG directly into the cerebral ventricles of embryonic mice during cortical neurogenesis. We show that autoantibodies recognize radial glial cells during development. We also show that prenatal exposure to autism-specific maternal autoantibodies increased stem cell proliferation in the subventricular zone (SVZ) of the embryonic neocortex, increased adult brain size and weight, and increased the size of adult cortical neurons. We propose that prenatal exposure to autism-specific maternal autoantibodies directly affects radial glial cell development and presents a viable pathologic mechanism for the maternal autoantibody-related prenatal ASD risk factor.

Matelski, L., Van De Water, J. Risk factors in autism: Thinking outside the brain. » J Autoimmun. 2016 Feb;67:1-7. doi: 10.1016/j.jaut.2015.11.003. Epub 2015 Dec 22 PMID: 26725748

Autism spectrum disorders (ASD) are complex neurodevelopmental conditions that have been rising markedly in prevalence for the past 30 years, now thought to affect 1 in 68 in the United States. This has prompted the search for possible explanations, and has even resulted in some controversy regarding the "true" prevalence of autism. ASD are influenced by a variety of genetic, environmental, and possibly immunological factors that act during critical periods to alter key developmental processes. This can affect multiple systems and manifests as the social and behavioral deficits that define these disorders. The interaction of environmental exposures in the context of an individual's genetic susceptibilities manifests differently in each case, leading to heterogeneous phenotypes and varied comorbid symptoms within the disorder. This has also made it very difficult to elucidate underlying genes and exposure profiles, but progress is being made in this area. Some pharmaceutical drugs, toxicants, and metabolic and nutritional factors have been identified in epidemiological studies as increasing autism risk, especially during the prenatal period. Immunologic risk factors, including maternal infection during pregnancy, autoantibodies to fetal brain proteins, and familial autoimmune disease, have consistently been observed across multiple studies, as have immune abnormalities in individuals with ASD. Mechanistic research using animal models and patient-derived stem cells will help researchers to understand the complex etiology of these neurodevelopmental disorders, which will lead to more effective therapies and preventative strategies. Proposed therapies that need more investigation include special diets, probiotics, immune modulation, oxytocin, and personalized pharmacogenomic targets. The ongoing search for biomarkers and better treatments will result in earlier identification of ASD and provide much needed help and relief for afflicted families.

Nguyen CT, Krakowiak P, Hansen R, Hertz-Picciotto I, Angkustsiri K. Sociodemographic Disparities in Intervention Service Utilization in Families of Children with Autism Spectrum Disorder. » J Autism Dev Disord. 2016 Sep 17. PMID: 27639855

This study investigates whether sociodemographic factors are associated with utilization of intervention services for children with autism spectrum disorder (ASD) enrolled in the Childhood Autism Risks from Genetics and the Environment Study. Maternal ethnicity, insurance status, and education for 696 families of children with ASD were available. Children of Black mothers entered intervention earlier compared to White mothers (2 vs. 2.6 years; p = 0.001). Having public insurance was associated with receiving <15 h/week of individual services, while having a Bachelor degree was associated with receiving <15 h/week of classroom-based services. These differences suggest that SES may be a factor in utilization of services. Efforts should be made to ensure that interventions offered are culturally and linguistically accessible.

Saldarriaga W, Lein P, González Teshima LY, Isaza C, Rosa L, Poly, Hagerman R, Girirajan S, Silva M, Tassone F. Phenobarbital use and neurological problems in FMR1 premutation carriers. » Neurotoxicology. 2016 Mar;53:141-7. doi: 10.1016/j.neuro.2016.01.008 PMCID:PMC4808401

Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by a CGG expansion in the FMR1 gene located at Xq27.3. Patients with the premutation in FMR1 present specific clinical problems associated with the number of CGG repeats (55-200 CGG repeats). Premutation carriers have elevated FMR1 mRNA expression levels, which have been associated with neurotoxicity potentially causing neurodevelopmental problems or neurological problems associated with aging. However, cognitive impairments or neurological problems may also be related to increased vulnerability of premutation carriers to neurotoxicants, including phenobarbital. Here we present a study of three sisters with the premutation who were exposed differentially to phenobarbital therapy throughout their lives, allowing us to compare the neurological effects of this drug in these patients.

Sirish P, Li N, Timofeyev V, Zhang XD, Wang L, Yang J, Lee KS, Bettaieb A, Ma SM, Lee JH, Su D, Lau VC, Myers RE, Lieu DK, López JE, Young JN, Yamoah EN, Haj F, Ripplinger CM, Hammock BD, Chiamvimonvat N. Molecular Mechanisms and New Treatment Paradigm for Atrial Fibrillation. » 2016 May;9(5). pii: e003721. doi: 10.1161/CIRCEP.115.003721. PMCID: PMC4869994

Atrial fibrillation represents the most common arrhythmia leading to increased morbidity and mortality, yet, current treatment strategies have proven inadequate. Conventional treatment with antiarrhythmic drugs carries a high risk for proarrhythmias. The soluble epoxide hydrolase enzyme catalyzes the hydrolysis of anti-inflammatory epoxy fatty acids, including epoxyeicosatrienoic acids from arachidonic acid to the corresponding proinflammatory diols. Therefore, the goal of the study is to directly test the hypotheses that inhibition of the soluble epoxide hydrolase enzyme can result in an increase in the levels of epoxyeicosatrienoic acids, leading to the attenuation of atrial structural and electric remodeling and the prevention of atrial fibrillation.

For the first time, we report findings that inhibition of soluble epoxide hydrolase reduces inflammation, oxidative stress, atrial structural, and electric remodeling. Treatment with soluble epoxide hydrolase inhibitor significantly reduces the activation of key inflammatory signaling molecules, including the transcription factor nuclear factor κ-light-chain-enhancer, mitogen-activated protein kinase, and transforming growth factor-β.

This study provides insights into the underlying molecular mechanisms leading to atrial fibrillation by inflammation and represents a paradigm shift from conventional antiarrhythmic drugs, which block downstream events to a novel upstream therapeutic target by counteracting the inflammatory processes in atrial fibrillation.


Akintunde ME, Rose M, Krakowiak P, Heuer L, Ashwood P, Hansen R, Hertz-Picciotto I, Van de Water J. Increased production of IL-17 in children with autism spectrum disorders and co-morbid asthma. » J Neuroimmunol. 2015 Sep 15;286:33-41. doi: 10.1016/j.jneuroim.2015.07.003. Epub 2015 Jul 11.PMCID: PMC4548834

Inflammation and asthma have both been reported in some children with autism spectrum disorder (ASD). To further assess this connection, peripheral immune cells isolated from young children with ASD and typically developing (TD) controls and the production of cytokines IL-17, -13, and -4 assessed following ex vivo mitogen stimulation. Notably, IL-17 production was significantly higher following stimulation in ASD children compared to controls. Moreover, IL-17 was increased in ASD children with co-morbid asthma compared to controls with the same condition. In conclusion, children with ASD exhibited a differential response to T cell stimulation with elevated IL-17 production compared to controls.

Claire Philippat, Deborah Bennett, Antonia M. Calafat, Irva Hertz Picciotto. Exposure to select phthalates and phenols through use of personal care products among Californian adults and their children. » Environmental Research 140 (2015) 369-376

A recent study on phthalate exposure found that children showed higher levels than adults who were exposed to the same environment. Why is this important? Phthalates are found in a range of scented personal care products and have known negative health effects, including preterm birth, hormone disruption, and the development of some cancers. Under current governmental policy, it is very difficult for families to limit their exposure. Companies use the blanket term “fragrance” in their ingredients to protect their individual fragrance recipes, but the “fragrance” could include hundreds of different chemicals – including phthalates!
Take away message: Pay attention to whether personal care products you buy list “fragrance” as an ingredient - purchase products labeled “Phthalate free” whenever you can!

Claire Philippat, Deborah H. Bennett, Paula Krakowiak, Melissa Rose, Hyun-Min Hwang and Irva Hertz-Picciotto. Phthalate concentrations in house dust in relation to autism spectrum disorder and developmental delay in the CHildhood Autism Risks from Genetics and the Environment (CHARGE) study. » Environmental Health 2015 14:56 DOI: 10.1186/s12940-015-0024-9

In a recent study, there was a relationship between phthalate levels in the home and developmental outcomes for children. Phthalate levels tended to be higher in the homes of children with Developmental Delay. The same was true in the homes of boys diagnosed with ASD or DD with greater attention and impulsive behavior problems. Additionally, typically developing children living in homes with higher phthalate levels tended to score lower on tests of adaptive behaviors, such as communication, daily living, and socialization.
Take away message: Limit exposures to phthalates whenever possible - purchase products labeled “phthalate free” whenever you can!

Fox-Edmiston E,Van de Water J. Maternal Anti-Fetal Brain IgG Autoantibodies and Autism Spectrum Disorder: Current Knowledge and its Implications for Potential Therapeutics. » CNS Drugs. 2015 Sep;29(9):715-24. doi: 10.1007/s40263-015-0279-2. PMCID: PMC4605883

Several studies have found a correlation between the presence of circulating maternal autoantibodies and neuronal dysfunction in the neonate. Specifically, maternal anti-brain autoantibodies, which may access the fetal compartment during gestation, have been identified as one risk factor for developing autism spectrum disorder (ASD). Studies by our laboratory elucidated seven neurodevelopmental proteins recognized by maternal autoantibodies whose presence is associated with a diagnosis of maternal autoantibody-related (MAR) autism in the child. While the specific process of anti-brain autoantibody generation is unclear and the detailed pathogenic mechanisms are currently unknown, identification of the maternal autoantibody targets increases the therapeutic possibilities. The potential therapies discussed in this review provide a framework for possible future medical interventions.

Krakowiak P, Walker CK, Tancredi DJ, Hertz-Picciotto I. Maternal Recall Versus Medical Records of Metabolic Conditions from the Prenatal Period: A Validation Study. » Matern Child Health J. 2015 Sep;19(9):1925-35. doi:10.1007/s10995-015-1723-0. PMID: 25656730

To assess validity of maternally-reported diabetes and hypertensive disorders, and reliability of BMI measurements during periconception and pregnancy compared with medical records when mothers are interviewed 2-5 years after delivery. To investigate whether reporting accuracy differed by child's case status (autism, delays, typical development). Participants were mothers of 2-5 year old children with and without neurodevelopmental disorders from the CHARGE (CHildhood Autism Risks from Genetics and the Environment) Study who had both prenatal/delivery records and telephone interviews. Sensitivity and specificity of self-report in telephone interview was assessed by comparison with medical records; agreement was evaluated by kappa statistics. Deviations in reported BMI were evaluated with Bland-Altman plots and concordance correlation coefficient (CCC). Mothers of children with neurodevelopmental disorders (autism or developmental delay) reported metabolic conditions slightly more accurately than control mothers. For diabetes, sensitivity ranged from 73 to 87% and specificity was ≥98% across groups. For hypertensive disorders, sensitivity ranged from 57 to 77% and specificity from 93 to 98%. Reliability of BMI was high (CCC = 0.930); when grouped into BMI categories, a higher proportion of mothers of delayed children were correctly classified (κ(wt) = 0.93) compared with the autism group and controls (κ(wt) = 0.85 and κ(wt) = 0.84, respectively; P = 0.05). Multiparity was associated with higher discrepancies in BMI and misreporting of hypertensive disorders. For purposes of etiologic studies, self-reported diabetes and hypertensive disorders during periconception and pregnancy show high validity among mothers irrespective of child's case status. Recall of pre-pregnancy BMI is reliable compared with self-reported values in medical records.

Krakowiak P, Goines PE, Tancredi DJ, Ashwood P, Hansen RL, Hertz-Picciotto I, Van de Water J. Neonatal Cytokine Profiles Associated with Autism Spectrum Disorder. » Biol Psychiatry. 2015 Aug 14. pii: S0006-3223(15)00655-1. doi: 10.1016/j.biopsych.2015.08.007. PMCID: PMC47531

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition that can be reliably diagnosed at age 24 months. Immunological phenomena, including skewed cytokine production, have been observed among children with ASD. Little is known about whether immune dysregulation is present before diagnosis of ASD.

We examined neonatal blood spots from 214 children with ASD (141 severe, 73 mild/moderate), 62 children with typical development, and 27 children with developmental delay as control subjects who participated in the Childhood Autism Risks from Genetics and the Environment study, a population-based case-control study. Levels of 17 cytokines and chemokines were compared across groups and in relation to developmental and behavioral domains.

Interleukin (IL)-1β and IL-4 were independently associated with ASD compared with typical development, although these relationships varied by ASD symptom intensity. Elevated IL-4 was associated with increased odds of severe ASD (odds ratio [OR] = 1.40, 95% confidence interval [CI], 1.03, 1.91), whereas IL-1β was associated with increased odds of mild/moderate ASD (OR = 3.02, 95% CI, 1.43, 6.38). Additionally, IL-4 was associated with a higher likelihood of severe ASD versus mild/moderate ASD (OR = 1.35, 95% CI, 1.04, 1.75). In male subjects with ASD, IL-4 was negatively associated with nonverbal cognitive ability (β = -3.63, SE = 1.33, p = .04).

This study is part of a growing effort to identify early biological markers for ASD. We demonstrate that peripheral cytokine profiles at birth are associated with ASD later in childhood and that cytokine profiles vary depending on ASD severity. Cytokines have complex roles in neurodevelopment, and dysregulated levels may be indicative of genetic differences and environmental exposures or their interactions that relate to ASD.

Lyall K, Van de Water J, Ashwood P, Hertz-Picciotto I. Asthma and Allergies in Children With Autism Spectrum Disorders: Results From the CHARGE Study. » Autism Res. 2015 Oct;8(5):567-74. doi: 10.1002/aur.1471. PMID: 25722050

Immune aberrations are often noted in children with autism spectrum disorder (ASD), but whether asthma and allergy are related to ASD is not well defined. This study examined asthma and allergies in association with ASD and phenotypic subsets. Participants were 560 children with confirmed ASD and 391 typically developing children from the CHildhood Autism Risks from Genetics and the Environment study. Maternally reported child asthma and allergy was compared between cases and controls, and in association with cognitive and behavioral test scores. Prevalence of asthma and overall allergies did not differ between cases and controls, but overall allergy in children with ASD was associated with higher stereotypy scores as measured by the Aberrant Behavior Checklist. In addition, reported food allergies were significantly associated with ASD (adjusted odds ratio = 2.23, 95% confidence interval 1.28, 3.89). Our results suggest food allergies and sensitivities may be more common in children with ASD, and that these issues may correlate with other behaviors.

McKean SJ, Bartell SM, Hansen RL, Barfod GH, Green PG, Hertz-Picciotto I. Prenatal mercury exposure, autism, and developmental delay, using pharmacokinetic combination of newborn blood concentrations and questionnaire data: a case control study. » Environ Health. 2015 Jul 22;14:62. doi: 10.1186/s12940-015-0045-4. PMID: 26198445

Methylmercury (MeHg), known for well over a century as a neurotoxin in adults, has more recently been studied for potential detrimental effects during early brain development. While several studies have estimated mercury exposure, they usually rely on either a single biomarker or questionnaire data, each of which has limitations. The goal of this paper was to develop a toxicokinetic model that incorporates both biomarker and questionnaire data to estimate the cumulative exposure to MeHg through seafood consumption using data collected from the Childhood Autism Risks from Genetics and the Environment (CHARGE) study.

We utilized a previously described discrete-time model that estimates blood MeHg concentration given a piecewise-constant ingestion rate and single-compartment pharmacokinetics. We measured newborn bloodspot Hg concentrations and obtained information pertaining to maternal fish consumption using a questionnaire. Using MeHg concentration estimates from the toxicokinetic model, cumulative MeHg exposure was estimated in children with autism, children with developmental delay, and typically developing children. Median estimated cumulative MeHg was compared among diagnostic groups using the Kruskal-Wallis Test. Multinomial logistic regression models were constructed to assess the association between cumulative MeHg concentration and the risk of autism and developmental delay (vs. typical development).

The estimated average MeHg concentration of for all fish species consumed by mothers was 42 ppb. Median cumulative MeHg over gestation was similar across diagnostic groups (p-values raged from 0.91 to 0.98). After adjusting for potential confounding, we found no association between cumulative MeHg exposure and the risk of autism (OR = 0.95, 95% CI: 0.95, 1.12) or developmental delay (OR = 1.00, 95% CI: 0.89, 1.13).

The toxicokinetic model described in this paper yielded fish MeHg concentration estimates that are consistent with fish species containing lower levels of MeHg. Overall, cumulative MeHg exposure does not appear to detectably elevate the risk of autism or developmental delay. Based on the regression standard error for the association between ASD and TD, we would have reported statistical significance for an adjusted odds ratio of 1.09 or larger. This method can easily be extended to other epidemiologic studies in which there is a biomarker measurement and questionnaire data regarding exposure.

Powell WT, LaSalle JM. Epigenetic mechanisms in diurnal cycles of metabolism and neurodevelopment. » Hum Mol Genet. 2015 Oct 15;24(R1):R1-9. doi: 10.1093/hmg/ddv234. PMID: 26105183

The circadian cycle is a genetically encoded clock that drives cellular rhythms of transcription, translation and metabolism. The circadian clock interacts with the diurnal environment that also drives transcription and metabolism during light/dark, sleep/wake, hot/cold and feast/fast daily and seasonal cycles. Epigenetic regulation provides a mechanism for cells to integrate genetic programs with environmental signals in order produce an adaptive and consistent output. Recent studies have revealed that DNA methylation is one epigenetic mechanism that entrains the circadian clock to a diurnal environment. We also review recent circadian findings in the epigenetic neurodevelopmental disorders Prader-Willi, Angelman and Rett syndromes and hypothesize a link between optimal brain development and intact synchrony between circadian and diurnal rhythms.

Zerbo O, Qian Y, Yoshida C, Grether JK, Van de Water J, Croen LA. Maternal Infection During Pregnancy and Autism Spectrum Disorders. » J Autism Dev Disord. 2015 Dec;45(12):4015-25. doi: 10.1007/s10803-013-2016-3. PMID: 24366406

We conducted a nested case-control study including 407 cases and 2,075 frequency matched controls to investigate the association between maternal infections during pregnancy and risk of autism spectrum disorders (ASD). Cases, controls, and maternal infections were ascertained from Kaiser Permanente Northern California clinical databases. No overall association between diagnoses of any maternal infection during pregnancy and ASD was observed [adjusted odds ratio (ORadj) = 1.15, 95 % confidence interval (CI) 0.92-1.43]. However, women with infections diagnosed during a hospital admission (ORadj = 1.48, 95 % CI 1.07-2.04), particularly bacterial infections (ORadj = 1.58, 95 % CI 1.06-2.37), were at increased risk of delivering a child with ASD. Multiple infections during pregnancy were associated with ASD (ORadj = 1.36, 95 % CI 1.05-1.78).