A high-throughput cigarette smoke-treated bronchosphere model for disease-relevant phenotypic compound screening.

Cigarette smoking (CS) is the leading cause of COPD, and identifying the pathways that are driving pathogenesis in the airway due to CS exposure can aid in the discovery of novel therapies for COPD. An additional barrier to the identification of key pathways that are involved in the CS-induced pathogenesis is the difficulty in building relevant and high throughput models that can recapitulate the phenotypic and transcriptomic changes associated with CS exposure. To identify these drivers, we have developed a cigarette smoke extract (CSE)-treated bronchosphere assay in 384-well plate format that exhibits CSE-induced decreases in size and increase in luminal secretion of MUC5AC. Transcriptomic changes in CSE-treated bronchospheres resemble changes that occur in human smokers both with and without COPD compared to healthy groups, indicating that this model can capture human smoking signature. To identify new targets, we ran a small molecule compound deck screening with diversity in target mechanisms of action and identified hit compounds that attenuated CSE induced changes, either decreasing spheroid size or increasing secreted mucus. This work provides insight into the utility of this bronchopshere model to examine human respiratory disease impacted by CSE exposure and the ability to screen for therapeutics to reverse the pathogenic changes caused by CSE.

Aralar Sequesters GABA into Hyperactive Mitochondria, Causing Social Behavior Deficits.

Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.

Comparison of brain connectomes by MRI and genomics and its implication in Alzheimer's disease.

BACKGROUND: The human brain is complex and interconnected structurally. Brain connectome change is associated with Alzheimer's disease (AD) and other neurodegenerative diseases. Genetics and genomics studies have identified molecular changes in AD; however, the results are often limited to isolated brain regions and are difficult to interpret its findings in respect to brain connectome. The mechanisms of how one brain region impacts the molecular pathways in other regions have not been systematically studied. And how the brain regions susceptible to AD pathology interact with each other at the transcriptome level and how these interactions relate to brain connectome change are unclear. METHODS: Here, we compared structural brain connectomes defined by probabilistic tracts using diffusion magnetic resonance imaging data in Alzheimer's Disease Neuroimaging Initiative database and a brain transcriptome dataset covering 17 brain regions. RESULTS: We observed that the changes in diffusion measures associated with AD diagnosis status and the associations were replicated in an independent cohort. The result suggests that disease associated white matter changes are focal. Analysis of the brain connectome by genomic data, tissue-tissue transcriptional synchronization between 17 brain regions, indicates that the regions connected by AD-associated tracts were likely connected at the transcriptome level with high number of tissue-to-tissue correlated (TTC) gene pairs (P = 0.03). And genes involved in TTC gene pairs between white matter tract connected brain regions were enriched in signaling pathways (P = 6.08 × 10-9). Further pathway interaction analysis identified ionotropic glutamate receptor pathway and Toll receptor signaling pathways to be important for tissue-tissue synchronization at the transcriptome level. Transcript profile entailing Toll receptor signaling in the blood was significantly associated with diffusion properties of white matter tracts, notable association between fractional anisotropy and bilateral cingulum angular bundles (Ppermutation = 1.0 × 10-2 and 4.9 × 10-4 for left and right respectively). CONCLUSIONS: In summary, our study suggests that brain connectomes defined by MRI and transcriptome data overlap with each other.

Domain-Specific Cognitive Impairments in Humans and Flies With Reduced CYFIP1 Dosage.

BACKGROUND: Deletions encompassing a four-gene region on chromosome 15 (BP1-BP2 at 15q11.2), seen at a population frequency of 1 in 500, are associated with increased risk for schizophrenia, epilepsy, and other common neurodevelopmental disorders. However, little is known in terms of how these common deletions impact cognition. METHODS: We used a Web-based tool to characterize cognitive function in a novel cohort of adult carriers and their noncarrier family members. Results from 31 carrier and 38 noncarrier parents from 40 families were compared with control data from 6530 individuals who self-registered on the Lumosity platform and opted in to participate in research. We then examined aspects of sensory and cognitive function in flies harboring a mutation in Cyfip, the homologue of one of the genes within the deletion. For the fly studies, 10 or more groups of 50 individuals per genotype were included. RESULTS: Our human studies revealed profound deficits in grammatical reasoning, arithmetic reasoning, and working memory in BP1-BP2 deletion carriers. No such deficits were observed in noncarrier spouses. Our fly studies revealed deficits in associative and nonassociative learning despite intact sensory perception. CONCLUSIONS: Our results provide new insights into outcomes associated with BP1-BP2 deletions and call for a discussion on how to appropriately communicate these findings to unaffected carriers. Findings also highlight the utility of an online tool in characterizing cognitive function in a geographically distributed population.

Common variation in the autism risk gene CNTNAP2, brain structural connectivity and multisensory speech integration.

Three lines of evidence motivated this study. 1) CNTNAP2 variation is associated with autism risk and speech-language development. 2) CNTNAP2 variations are associated with differences in white matter (WM) tracts comprising the speech-language circuitry. 3) Children with autism show impairment in multisensory speech perception. Here, we asked whether an autism risk-associated CNTNAP2 single nucleotide polymorphism in neurotypical adults was associated with multisensory speech perception performance, and whether such a genotype-phenotype association was mediated through white matter tract integrity in speech-language circuitry. Risk genotype at rs7794745 was associated with decreased benefit from visual speech and lower fractional anisotropy (FA) in several WM tracts (right precentral gyrus, left anterior corona radiata, right retrolenticular internal capsule). These structural connectivity differences were found to mediate the effect of genotype on audiovisual speech perception, shedding light on possible pathogenic pathways in autism and biological sources of inter-individual variation in audiovisual speech processing in neurotypicals.

Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus.

The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC), plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT)-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th) mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat.

A Common CYFIP1 Variant at the 15q11.2 Disease Locus Is Associated with Structural Variation at the Language-Related Left Supramarginal Gyrus.

Copy number variants (CNVs) at the Breakpoint 1 to Breakpoint 2 region at 15q11.2 (BP1-2) are associated with language-related difficulties and increased risk for developmental disorders in which language is compromised. Towards underlying mechanisms, we investigated relationships between single nucleotide polymorphisms (SNPs) across the region and quantitative measures of human brain structure obtained by magnetic resonance imaging of healthy subjects. We report an association between rs4778298, a common variant at CYFIP1, and inter-individual variation in surface area across the left supramarginal gyrus (lh.SMG), a cortical structure implicated in speech and language in independent discovery (n = 100) and validation cohorts (n = 2621). In silico analyses determined that this same variant, and others nearby, is also associated with differences in levels of CYFIP1 mRNA in human brain. One of these nearby polymorphisms is predicted to disrupt a consensus binding site for FOXP2, a transcription factor implicated in speech and language. Consistent with a model where FOXP2 regulates CYFIP1 levels and in turn influences lh.SMG surface area, analysis of publically available expression data identified a relationship between expression of FOXP2 and CYFIP1 mRNA in human brain. We propose that altered CYFIP1 dosage, through aberrant patterning of the lh.SMG, may contribute to language-related difficulties associated with BP1-2 CNVs. More generally, this approach may be useful in clarifying the contribution of individual genes at CNV risk loci.

The effect of consumption of ethanol on subfoveal choroidal thickness in acute phase.

BACKGROUND/AIMS: To investigate the acute effect of ethanol consumption on subfoveal choroidal thickness. METHODS: This prospective interventional study included the right eyes of 30 healthy subjects (30 eyes). Ethanol (1.0 g/kg) was administered orally on the first visit. A matching volume of water was administered orally on the second visit. Oral administration of ethanol and water was performed at 14:00, and choroidal thickness was measured every 30 min until 16:00. Change of choroidal thickness after oral administration of ethanol and water was the main outcome measure. RESULTS: At baseline, choroidal mean subfoveal thickness was 299.0±73.4 µm (range, 186.5-472.5 µm) before ethanol consumption and 297.1±71.1 µm (range, 187.0-470.5 µm) before water consumption. After consumption of ethanol, mean subfoveal choroidal thickness increased during the first 60 min and then decreased during the next 60 min, which was a significant change over time (p<0.001). After consumption of water, there was no significant change in mean subfoveal choroidal thickness over time (p=0.310). Comparison of changes in the mean subfoveal choroidal thickness during 120 min showed significant difference between ethanol and water consumption (p<0.001). CONCLUSIONS: The results of current study show that consumption of ethanol significantly affected the choroidal thickness. Mean subfoveal choroidal thickness increased during the first 60 min and then decreased during the next 120 min after ethanol consumption.

Clinical Association of Thyroid Stimulating Hormone Receptor Antibody Levels with Disease Severity in the Chronic Inactive Stage of Graves' Orbitopathy.

PURPOSE: To investigate associations between serum thyroid stimulating hormone (TSH) receptor antibody (TRAb) levels and Graves' orbitopathy (GO) activity/severity in chronic-stage GO and compare the performance of two newly-developed TRAb assays (third-generation TSH-binding inhibition immunoglobulin [TBII] assay versus Mc4 thyroid-stimulating immunoglobulin [TSI] bioassay). METHODS: This study is a retrospective review of medical charts and blood tests from Korean GO patients who first visited the departments of ophthalmology and endocrinology, Yonsei University College of Medicine from January 2008 to December 2011, were diagnosed with GO and Graves' hyperthyroidism, and were followed up for ≥18 months. Third-generation M22-TBII and Mc4-TSI assays were performed in the chronic-inactive GO patients in whom euthyroidism status was restored. Patients' GO activity/severity clinical activity scores (CAS), and modified NOSPECS scores were examined for a correlation with TRAb assays. RESULTS: Fifty patients (mean age, 41.3 years; 41 females) were analyzed. The mean duration of Graves' hyperthyroidism symptom was 63 months (range, 18 to 401 months) and that of GO was 46 months (range, 18 to 240 months). All patients had been treated previously with anti-thyroid drugs for a median period of 52.3 months, and two patients underwent either radioiodine therapy or total thyroidectomy. Mean CAS and NOSPECS scores were 0.5 ± 0.9 (standard deviation) and 4.8 ± 3.1, respectively. Mean M22-TBII and Mc4-TSI values were 7.5 ± 10.2 IL/L and 325.9 ± 210.1 specimen-to-reference control ratio. TSI was significantly correlated with NOSPECS score (R = 0.479, p < 0.001); however, TBII was not associated with NOSPECS score (p = 0.097). Neither TSI nor TBII correlated with CAS (p > 0.05), because GO inflammatory activity subsided in the chronic stages of GO. CONCLUSIONS: In chronic-inactive GO after euthyroid restoration, GO activity score did not associate with serum levels of TRAb or TBII. However, levels of the functional antibody Mc4-TSI did correlate with GO severity. Therefore, the TSI bioassay is a clinically relevant measure of disease severity even in chronic inactive GO.

Reciprocal Relationship between Head Size, an Autism Endophenotype, and Gene Dosage at 19p13.12 Points to AKAP8 and AKAP8L.

Microcephaly and macrocephaly are overrepresented in individuals with autism and are thought to be disease-related risk factors or endophenotypes. Analysis of DNA microarray results from a family with a low functioning autistic child determined that the proband and two additional unaffected family members who carry a rare inherited 760 kb duplication of unknown clinical significance at 19p13.12 are macrocephalic. Consideration alongside overlapping deletion and duplication events in the literature provides support for a strong relationship between gene dosage at this locus and head size, with losses and gains associated with microcephaly (p=1.11x10(-11)) and macrocephaly (p=2.47x10(-11)), respectively. Data support A kinase anchor protein 8 and 8-like (AKAP8 and AKAP8L) as candidate genes involved in regulation of head growth, an interesting finding given previous work implicating the AKAP gene family in autism. Towards determination of which of AKAP8 and AKAP8L may be involved in the modulation of head size and risk for disease, we analyzed exome sequencing data for 693 autism families (2591 individuals) where head circumference data were available. No predicted loss of function variants were observed, precluding insights into relationship to head size, but highlighting strong evolutionary conservation. Taken together, findings support the idea that gene dosage at 19p13.12, and AKAP8 and/or AKAP8L in particular, play an important role in modulation of head size and may contribute to autism risk. Exome sequencing of the family also identified a rare inherited variant predicted to disrupt splicing of TPTE / PTEN2, a PTEN homologue, which may likewise contribute to both macrocephaly and autism risk.

Comparison of the Astigmatic Power of Toric Intraocular Lenses Using Three Toric Calculators.

PURPOSE: To compare the astigmatic power of toric intraocular lenses (IOLs) obtained from the AcrySof, TECNIS, and iTrace toric calculator in patients with preoperative with-the-rule (WTR) or against-the-rule (ATR) corneal astigmatism. MATERIALS AND METHODS: Fifty eyes with cataract and corneal astigmatism greater than 0.75 diopters were enrolled in each group (WTR and ATR). Keratometric values were measured using autokeratometry, an IOLMaster, and an iTrace, which incorporated corneal topography and ray-tracing aberrometry. Based on measured keratometric values, the astigmatic power of each toric IOL was calculated using three toric calculators. RESULTS: Bland-Altman plots showed good agreement between six pairwise corneal astigmatism values in both groups. The TECNIS calculator tended to suggest a higher astigmatic power of the toric IOL than the AcrySof calculator. With the higher astigmatism and keratometric values from the IOLMaster, in both groups, calculations from the AcrySof and TECNIS calculators resulted in higher calculated astigmatic powers than those from same calculators with autokeratometry-measured values, demonstrating good agreement. With the higher calculated astigmatic power values, the values from the iTrace toric calculator using keratometric values obtained from iTrace ray tracing wavefront aberrometry or iTrace simulated keratometry showed fair to moderate agreement with those from the other calculator-keratometry pairs in both groups. CONCLUSION: To achieve the best refractive outcome after toric IOL implantation, understanding the differences in keratometric values between instruments and in calculated astigmatic power among toric calculator programs is necessary. Moreover, systemic analysis of each toric calculator in conjunction with postoperative data is required.

Comparison of 3 marking techniques in preoperative assessment of toric intraocular lenses using a wavefront aberrometer.

PURPOSE: To assess the accuracy of 3 corneal horizontal meridian marking techniques using the iTrace Surgical Workstation in preoperative assessment for toric intraocular lens (IOL) implantation. SETTING: Department of Ophthalmology, Yonsei University College of Medicine, Seoul, South Korea. DESIGN: Prospective comparative observational study. METHODS: Patients were allocated into 3 groups. Three techniques-marking with a nonpendular marker with a surgeon's direct visualization, slitlamp-assisted marking with a pendulum-attached marker, and slitlamp-assisted marking with a horizontal slit beam-were used to perform the horizontal meridian marking. The marks were immediately documented and aligned with the Zaldivar toric caliper transparent topographic map. Using a horizontal reference meridian determined by the toric caliper, the accuracy of corneal horizontal marking techniques was quantitatively evaluated by determining the rotational deviation and vertical misalignment. RESULTS: For rotational deviation, the pendular marking showed the fewest errors to the horizontal reference meridian (mean error -0.66 degrees; mean absolute error [MAE] 3.77 degrees). There was a significant difference between the pendular marking and the surgeon's direct visual marking. For the MAE, the pendular marking showed less deviation than the surgeon's direct visual marking and the horizontal slit-beam marking. With vertical misalignment, there was no significant difference between the 3 different corneal horizontal meridian marking techniques. CONCLUSION: The slitlamp-assisted technique with the pendulum-attached marker was more precise than marking with a nonpendular marker with a surgeon's direct visualization and marking with a horizontal slit beam in preoperative assessment for toric IOLs. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Postoperative outcomes of anophthalmic socket reconstruction using an autologous buccal mucosa graft.

Socket contracture is one of the most common and difficult problems in anophthalmic patients. This study intended to evaluate postoperative outcomes of anophthalmic socket reconstruction using an autologous buccal mucosa graft in patients with socket contracture. Medical records and photographs of 44 anophthalmic patients who underwent socket reconstruction surgery using an autologous buccal mucosa graft were reviewed retrospectively. The time necessary for the graft surface to be completely vascularized was assessed, and fornix depth was measured before and 6 months after surgery. Postoperative cosmetic and functional outcomes were evaluated, and the factors that influence postoperative outcomes were investigated. The surgery was performed without any significant complications, and the patients only complained of oral discomfort within 1 week. The graft surface was fully vascularized about 1.1 months after surgery. Mean fornix depth after surgery was significantly deeper than that before surgery (9.1 mm, about 68.2% of the vertical size of the implanted graft). Preoperatively, 50.0% of the patient had cosmetic grades 1 and 2; however, 63.6% of the patients achieved grade 4, and 93.2% had higher than grade 3 after surgery. In functional outcomes, 86.4% of the patients presented functional success. Graft recontracture occurred in only 2 patients. Preoperative severe socket contracture was a factor associated with worse cosmetic outcome (P = 0.001). An autologous buccal mucosa can be a safe and effective graft material for the reconstruction of a contracted socket.

Visual perception of ADHD children with sensory processing disorder.

OBJECTIVE: The aim of the present study was to investigate the visual perception difference between ADHD children with and without sensory processing disorder, and the relationship between sensory processing and visual perception of the children with ADHD. METHODS: Participants were 47 outpatients, aged 6-8 years, diagnosed with ADHD. After excluding those who met exclusion criteria, 38 subjects were clustered into two groups, ADHD children with and without sensory processing disorder (SPD), using SSP reported by their parents, then subjects completed K-DTVP-2. Spearman correlation analysis was run to determine the relationship between sensory processing and visual perception, and Mann-Whitney-U test was conducted to compare the K-DTVP-2 score of two groups respectively. RESULTS: The ADHD children with SPD performed inferiorly to ADHD children without SPD in the on 3 quotients of K-DTVP-2. The GVP of K-DTVP-2 score was related to Movement Sensitivity section (r=0.368(*)) and Low Energy/Weak section of SSP (r=0.369*). CONCLUSION: The result of the present study suggests that among children with ADHD, the visual perception is lower in those children with co-morbid SPD. Also, visual perception may be related to sensory processing, especially in the reactions of vestibular and proprioceptive senses. Regarding academic performance, it is necessary to consider how sensory processing issues affect visual perception in children with ADHD.

Methylphenidate-osmotic-controlled release oral delivery system treatment reduces parenting stress in parents of children and adolescents with attention-deficit/hyperactivity disorder.

OBJECTIVE: The aim of the current study was to investigate the effect of methylphenidate-osmotic release oral delivery system (MPH-OROS) treatment on parenting stress in parents of children and adolescents with attention-deficit/hyperactivity disorder (ADHD). METHODS: Four hundred and ninety-five children and adolescents (391 boys and 104 girls), aged 7 to 18 years who met the Diagnostic and Statistical Manual of Mental Disorders, fourth edition criteria for ADHD, were recruited at 48 psychiatric outpatient clinics across South Korea. Children's symptoms, parenting stress, and parental depression were assessed at baseline, week 4, and week 8 of MPH-OROS treatment using the Korean version of the DuPaul's ADHD Rating Scale (ARS), the Beck's Depression Inventory (BDI), and the Parenting Stress Index, Short Form (PSI-SF). RESULTS: We found significantly decreased scores of ARS, parental BDI, and PSI-SF from baseline to week 4 and from week 4 to week 8. Also, there were positive correlations among baseline PSI-SF, ARS, and BDI scores. The changes in BDI and ARS scores were significantly associated with the PSI score changes, accounting for 20.1% and 10.0%, respectively. CONCLUSIONS: We suggest that the increased parenting stress and depression in parents of children and adolescents with ADHD can be improved following the treatment with MPH-OROS.

Satb1 ablation alters temporal expression of immediate early genes and reduces dendritic spine density during postnatal brain development.

Complex behaviors, such as learning and memory, are associated with rapid changes in gene expression of neurons and subsequent formation of new synaptic connections. However, how external signals are processed to drive specific changes in gene expression is largely unknown. We found that the genome organizer protein Satb1 is highly expressed in mature neurons, primarily in the cerebral cortex, dentate hilus, and amygdala. In Satb1-null mice, cortical layer morphology was normal. However, in postnatal Satb1-null cortical pyramidal neurons, we found a substantial decrease in the density of dendritic spines, which play critical roles in synaptic transmission and plasticity. Further, we found that in the cerebral cortex, Satb1 binds to genomic loci of multiple immediate early genes (IEGs) (Fos, Fosb, Egr1, Egr2, Arc, and Bdnf) and other key neuronal genes, many of which have been implicated in synaptic plasticity. Loss of Satb1 resulted in greatly alters timing and expression levels of these IEGs during early postnatal cerebral cortical development and also upon stimulation in cortical organotypic cultures. These data indicate that Satb1 is required for proper temporal dynamics of IEG expression. Based on these findings, we propose that Satb1 plays a critical role in cortical neurons to facilitate neuronal plasticity.