Biology subjects

Choi, M.

Publications and source records attributed to Choi, M..

69 records · Page 4Linked to original sources

bioRxiv2021

Mechanosensory input shapes Drosophila motor behavior through Patterned Spontaneous Network Activity

Neural activity sculpts circuit wiring in many animals. In vertebrates, patterned spontaneous network activity (PaSNA) generates sensory maps and establishes local circuits 1-3. However, it remains unclear how PaSNA might shape neuronal circuits and behavior in invertebrates. Previous work in the developing Drosophila embryo discovered spontaneous muscle activity that did not require synaptic transmission, and hence was myogenic, preceding PaSNA 4-6. These studies, however, monitored muscle movement, not neural activity, and were therefore unable to observe how myogenic activity might relate to subsequent neural network engagement. Here we use calcium imaging to directly record neural activity and characterize the emergence of PaSNA. We demonstrate that the spatiotemporal properties of PaSNA are highly stereotyped across embryos, arguing for genetic programming. Consistent with previous observations, we observe neural activity well before it becomes patterned, initially emerging during the myogenic stage. Remarkably, inhibition of mechanosensory input as well as inhibition of muscle contractions results in premature and excessive PaSNA, demonstrating that muscle movement serves as a brake on this process. Finally, using an optogenetic strategy to selectively disrupt mechanosensory inputs during PaSNA, followed by quantitative modeling of larval behavior, we demonstrate that mechanosensory modulation during development is required for proper larval foraging. This work thus provides a foundation for using the Drosophila embryo to study the role of PaSNA in circuit formation, provides mechanistic insight into how PaSNA is entrained by motor activity, and demonstrates that spontaneous network activity is essential for locomotor behavior. These studies argue that sensory feedback during the earliest stages of circuit formation can sculpt locomotor behaviors through innate motor learning. HighlightsO_LIPaSNA in the Drosophila embryonic CNS is spatiotemporally stereotyped C_LIO_LIMechanosensory neurons negatively modulate PaSNA C_LIO_LIEmbryonic PaSNA is required for larval locomotor behavior C_LI

neuroscience↗

bioRxiv2021

Disease-specific eQTL screening reveals an anti-fibrotic effect of AGXT2 in nonalcoholic fatty liver disease

Background & AimsNonalcoholic fatty liver disease (NAFLD) poses an impending clinical burden. Genome-wide association studies have revealed a limited contribution of genomic variants to the disease, requiring alternative but robust approaches to identify disease-associated variants and genes. We carried out a disease-specific expression quantitative trait loci (eQTL) screen to identify novel genetic factors that specifically act on NAFLD progression on the basis of genotype. MethodsWe recruited 125 Korean biopsy-proven NAFLD patients and healthy individuals and performed eQTL analyses using 21,272 transcripts and 3,234,941 genotyped and imputed SNPs. We then selected eQTLs that were detected only in the NAFLD group, but not in the control group (i.e., NAFLD-eQTLs). An additional cohort of 162 Korean NAFLD individuals was used for replication. The function of the selected eQTL toward NAFLD development was validated using HepG2, primary hepatocytes and NAFLD mouse models. ResultsThe NAFLD-specific eQTL screening yielded 242 loci. Among them, AGXT2, encoding alanine-glyoxylate aminotransferase 2, displayed decreased expression in NAFLD patients homozygous for the non-reference allele of rs2291702, compared to no-NAFLD subjects with the same genotype (P = 4.79 x 10-6). This change was replicated in an additional 162 individuals, yielding a combined P-value of 8.05 x 10-8 from a total of 245 NAFLD patients and 48 controls. Knockdown of AGXT2 induced palmitate-overloaded hepatocyte death by increasing ER stress, and exacerbated NAFLD diet-induced liver fibrosis in mice. However, overexpression of AGXT2 reversely attenuated liver fibrosis and steatosis as well. ConclusionsWe implicate a new molecular role of AGXT2 in NAFLD. Our overall approach will serve as an efficient tool for uncovering novel genetic factors that contribute to liver steatosis and fibrosis in patients with NAFLD. Lay summaryElucidating causal genes for NAFLD has been challenging due to limited tissue availability and the polygenic nature of the disease. Using liver and blood samples from 125 biopsy-proven NAFLD and no-NAFLD Korean individuals and an additional 162 individuals for replication, we devised a new analytic method to identify causal genes. Among the candidates, we found that AGXT2-rs2291702 protects against liver fibrosis in a genotype-dependent manner with the potential for therapeutic interventions. Our approach enables the discovery of NAFLD causal genes that act on the basis of genotype.

genetics↗

bioRxiv2020

Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression

Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Several genome editing platforms have emerged to evaluate the functionality of TFBS in animals. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice to demonstrate the essentiality of a single TFBS, called a CArG box, in the promoter region of the Tspan2 gene. HDR-mediated substitution of three base pairs in the Tspan2 CArG box resulted in 20/37 (54%) founder mice testing positive for the correct edit. Mice homozygous for this edit showed near loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Using the same protospacer, PE2-mediated editing of a single base in the Tspan2 CArG box yielded 12/47 (26%) founder mice testing positive for the correct edit. This single base substitution resulted in [~]90% loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Targeted sequencing demonstrated all PE2 and HDR founders with some frequency of on-target editing. However, whereas no spurious on-target indels were detected in any of the PE2 founders, many HDR founders showed variable levels of on-target indels. Further, off-target analysis by targeted sequencing revealed mutations in 5/11 (45%) HDR founders but none in PE2 founders. These results demonstrate high fidelity editing of a TFBS with PE2 and suggest a new paradigm for Cre/loxP-free tissue-specific gene inactivation via single base substitution in a TFBS. The PE2 platform of genome editing represents a powerful approach for modeling and correcting relevant noncoding SNVs in the mouse.

genetics↗

bioRxiv2020

Optical topometry and machine learning to rapidly phenotype stomatal patterning traits for QTL mapping in maize

Stomata are adjustable pores on leaf surfaces that regulate the trade-off of CO2 uptake with water vapor loss, thus having critical roles in controlling photosynthetic carbon gain and plant water use. The lack of easy, rapid methods for phenotyping epidermal cell traits have limited the use of quantitative, forward and reverse genetics to discover the genetic basis of stomatal patterning. A new high-throughput epidermal cell phenotyping pipeline is presented here and used for quantitative trait loci (QTL) mapping in field-grown maize. The locations and sizes of stomatal complexes and pavement cells on images acquired by an optical topometer from mature leaves were automatically determined. Computer estimated stomatal complex density (SCD; R2 = 0.97) and stomatal complex area (SCA; R2 = 0.71) were strongly correlated with human measurements. Leaf gas exchange traits correlated with the dimensions and proportion of stomatal complexes but, unexpectedly, did not correlate with SCD. Genetic variation in epidermal traits were consistent across two field seasons. Out of 143 QTLs in total, 36 QTLs were consistently identified for a given trait in both years. 24 hotspots of overlapping QTLs for multiple traits were identified. Orthologs of genes known to regulate stomatal patterning in Arabidopsis were located within some, but not all, of these regions. This study demonstrates how discovery of the genetic basis for stomatal patterning can be accelerated in maize, a model for C4 species where these processes are poorly understood. One sentence summaryOptical topometry and machine learning tools were developed to assess epidermal cell patterning, and applied to analyze its genetic architecture alongside leaf photosynthetic gas exchange in maize.

plant biology↗

bioRxiv2020

Childhood location correlates with epigenetic age and methylation stability in British-Bangladeshi migrants

BackgroundMigration from one environment to another often causes marked changes in developmental conditions. Here we compare epigenetic ageing and stability of the epigenetic maintenance system among British-Bangladeshi women who grew up in Bangladesh (adult migrants), where there are higher pathogen loads and poorer health care, to second-generation Bangladeshis who grew up in the UK. In our previous studies of these migrants, those who spent their childhoods in Bangladesh also had lower levels of reproductive hormones and a shorter reproductive lifespan compared to those who grew up in the UK, suggesting life history trade-offs during development. In the present study, we hypothesised that women who grew up in Bangladesh would have i) an older epigenetic/biological age compared to the women with a childhood in the UK and ii) that differences in the pace of epigenetic ageing might also be reflected by altered stability of DNA methylation marks. ResultsIllumina EPIC array methylation data from buccal tissue was used to establish epigenetic age estimates from 15 adult migrants and 11 second-generation migrants, aged 18-35 years. Using residuals from linear regression of DNA methylation-based biological age (DNAm age) on the chronological age, the results showed significant differences (p=0.016) in epigenetic age estimates: women whose childhood was in Bangladesh are on average 6.02 ({+/-} 2.34) years older, than those who grew up in London. We further investigated the efficiency of the epigenetic maintenance system which purportedly is reflected by epigenetic clocks. Methylation states of CpGs at the LHCGR/LHR locus, which contributes to Horvaths multi tissue epigenetic clock were evaluated. Based on the Ratio of Concordance Preference (RCP) approach that uses double-stranded methylation data, we find that maintenance of epigenetic information is more stable in women who grew up in Bangladesh. ConclusionsThe work supports earlier findings that adverse childhood environments lead to phenotypic life history trade-offs. The data indicate that childhood environments can induce subtle changes to the epigenetic maintenance system that are detectable long after exposure occurred. The implication of such a finding warrants further investigation as it implies that a less flexible epigenetic memory system established early in life could reduce the capacity to respond to different environmental conditions in adult life.

genetics↗

bioRxiv2020

Proteomics of autophagy deficient macrophages reveals enhanced antimicrobial immunity via the oxidative stress response

Defective autophagy is associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohns disease by enhancing innate immunity in macrophages. However, autophagy also mediates clearance of intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in antimicrobial immunity. In this study, we found that loss of Atg16l1 in macrophages enhanced the killing of virulent Shigella flexneri (S.flexneri), an enteric bacterium that resides within the cytosol by escaping all membrane-bound compartments. Quantitative multiplexed proteomics revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which also promoted S.flexneri killing. Consistently, myeloid cell-specific deletion of Atg16l1 accelerated bacterial clearance in vivo. Finally, pharmacological modulation of oxidative stress by suppression of cysteine import conferred enhanced microbicidal properties to wild type macrophages. These findings demonstrate that control of oxidative stress by ATG16L1 regulates antimicrobial immunity against intracellular pathogens. Impact statementMaculins et al utilize multiplexed mass spectrometry to show that loss of the autophagy gene Atg16l1 in macrophages enhances antimicrobial immunity against intracellular pathogens via the oxidative stress response.

cell biology↗

bioRxiv2020

MitoROS due to loss of Slc4a11 in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy

Recent studies from Slc4a11 KO mice have identified mitochondrial dysfunction as a major contributor toward oxidative stress and cell death in Congenital Hereditary Endothelial Dystrophy. Here we asked if this stress activated autophagy in the Slc4a11 KO cell line and in KO mouse endothelial tissue. Early indicators of autophagy, phospho-mTOR and LC3-II indicated activation, however P62 was elevated suggesting an impairment of autophagy flux. The activity and the number of lysosomes, the organelle responsible for the final degradation of autophagy substrates, were found to be reduced in the KO. In addition, the expression of the master regulator of lysosomal function and biogenesis, TFEB, was significantly reduced in the KO corneal endothelia. Also, we observed increased Unfolded Protein Response, as well as elevated expression of ER stress markers, BIP and CHOP. To test if lysosomal and ER stress stems from elevated mitochondrial ROS, we treated Slc4a11 KO corneal endothelial cells with the mitochondrial ROS quencher, MitoQ. MitoQ restored lysosomal enzymes as well as TFEB, reduced ER stress, and increased autophagy flux. MitoQ injections of Slc4a11 KO mice decreased corneal edema, the major phenotype associated with CHED. We conclude that mitochondrial ROS causes ER stress and lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction and ER stress in an animal model of CHED, and to characterize inter-organelle relationship in a corneal cell type.

cell biology↗

bioRxiv2020

Single adeno-associated virus-based multiplexed CRISPR-Cas9 system to nullify core components of the mammalian molecular clock

ABSTRACTThe mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) containing Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1). TTFL robustness is endowed by genetic complementation between these components; therefore, multiple genes must be knocked out to physiologically investigate the molecular clock, which requires extensive research resources. To facilitate molecular clock disruption, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for Pers, Crys, or Bmal1. First, we designed single guide RNAs (sgRNAs) targeting individual clock genes using an in silico approach and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and expressed in viral vectors. CSAC efficiency was demonstrated by decreased protein expression in vitro and ablated molecular oscillation ex vivo. We also measured locomotor activity and body temperature in Cas9-expressing mice injected with CSAC at the suprachiasmatic nucleus. Circadian rhythm disruption was observed under free-running conditions, indicating that CSAC can efficiently and robustly disrupt molecular circadian clock. Thus, CSAC is a simple and powerful tool for investigating the physiological role of the molecular clock in vivo.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Representation Learning of Resting State fMRI with Variational Autoencoder

Resting state functional magnetic resonance imaging (rsfMRI) data exhibits complex but structured patterns. However, the underlying origins are unclear and entangled in rsfMRI data. Here we establish a variational auto-encoder, as a generative model trainable with unsupervised learning, to disentangle the unknown sources of rsfMRI activity. After being trained with large data from the Human Connectome Project, the model has learned to represent and generate patterns of cortical activity and connectivity using latent variables. The latent representation and its trajectory represent the spatiotemporal characteristics of rsfMRI activity. The latent variables reflect the principal gradients of the latent trajectory and drive activity changes in cortical networks. Latent representations are clustered by both individuals and brain states. Representational geometry captured as covariance or correlation between latent variables, rather than cortical connectivity, can be used as a more reliable feature to accurately identify subjects from a large group, even if only a short period of data is available per subjects.

neuroscience↗

bioRxiv2020

Machine learning approach reveals heterogeneous responses to FAK and Rho GTPases inhibition on smooth muscle spheroid formation

Atherosclerosis and vascular injury are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMCs would advance the effort to treat vascular disease. However, the response to treatments aimed at VSMCs is often different among patients with the same disease condition, suggesting patient-specific heterogeneity in VSMCs. Here, we present an experimental and computational method called HETEROID (Heterogeneous Spheroid), which examines the heterogeneity of the responses to drug treatments at the single-spheroid level by combining a VSMC spheroid model and machine learning (ML) analysis. First, we established a VSMC spheroid model that mimics neointima formation induced by atherosclerosis and vascular injury. We found that FAK-Rac/Rho, but not Cdc42, pathways regulate the VSMC spheroid formation through N-cadherin. Then, to identify the morphological subpopulations of drug-perturbed spheroids, we used an ML framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our ML approach reveals that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect the spheroid morphology, suggesting there exist multiple distinct pathways governing VSMC spheroid formation. Overall, our HETEROID pipeline enables detailed quantitative characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis of various drug treatments.

cell biology↗

bioRxiv2019

Genomic profiling of 553 uncharacterized neurodevelopment patients reveals a high proportion of recessive pathogenic variant carriers in an outbred population

BackgroundA substantial portion of Mendelian disease patients suffers from genetic variants that are inherited in a recessive manner. A precise understanding of pathogenic recessive variants in a population would assist in pre-screening births of such patients. However, a systematic understanding of the contribution of recessive variants to Mendelian diseases is still lacking.\n\nMethodsGenetic diagnosis and variant discovery of 553 undiagnosed Korean patients with complex neurodevelopmental problems (KND for Korean NeuroDevelopmental cohort) were performed using whole exome sequencing of patients and their parents. Pathogenic variants were selected and evaluated based on a comparison to patient symptoms and genetic properties of the variants were analyzed.\n\nResultsDisease-causing variants, including newly discovered variants, were identified in in 57.5% of the probands of the KND cohort. Of the 553 patients, 47.4% harbored variants that were previously reported as being pathogenic, and 35.1% of the previous reported pathogenic variants were inherited in a recessive manner. Genes that cause recessive disorders tend to be less constrained by loss-of-function variants and enriched in metabolic and mitochondrial pathways. This observation was applied to an estimation that approximately 1 in 17 healthy Korean individuals carry at least one of these pathogenic variants that develop severe neurodevelopmental problems in a recessive manner. Furthermore, the feasibility of these genes for carrier screening was evaluated.\n\nConclusionsWe suggest that the odds are high for healthy individuals carrying a potentially pathogenic variant, and its genetic properties. Our results will serve as a foundation for recessive variant screening to reduce occurrences of rare Mendelian disease patients. Additionally, our results highlight the utility and necessity of whole exome sequencing-based diagnostics for improving patient care in a country with a centralized medical system.

genetics↗

bioRxiv2018

Somatic uniparental disomy with a rare EFL1 variant causes Shwachman-Diamond syndrome through dysregulating ribosomal protein synthesis

Shwachman-Diamond syndrome (SDS; OMIM: #260400) is caused by variants in SBDS (Shwachman-Bodian-Diamond syndrome gene), which encodes a protein that plays an important role in ribosome assembly. Recent reports suggest that recessive variants in EFL1 are also responsible for SDS. However, the precise genetic mechanism that leads to EFL1-induced SDS remains incompletely understood. Here we present three unrelated Korean SDS patients that carry biallelic pathogenic variants in EFL1 with biased allele frequencies, resulting from a bone marrow-specific somatic uniparental disomy (UPD) in chromosome 15. The recombination events generated cells that were homozygous for the relatively milder variant, allowing for the evasion of catastrophic physiological consequences. Still, the milder EFL1 variant was solely able to impair 80S ribosome assembly and induce SDS features in cell line, zebrafish, and mouse models. The loss of EFL1 resulted in a pronounced inhibition of terminal oligo-pyrimidine element-containing ribosomal protein transcript 80S assembly. Therefore, we propose a more accurate pathogenesis mechanism of EFL1 dysfunction that eventually leads to aberrant translational control and ribosomopathy.

genetics↗

bioRxiv2018

Comprehensive genetic analysis by whole exome sequencing in 352 Korean pediatric patients with unknown neurodevelopmental disorders

ImportanceAccurate diagnosis of pediatric patients with complicated neurological problems demands a well-coordinated combination of robust genetic analytic capability and delicate clinical evaluation. It should be tested whether this challenge can be augmented by whole exome sequencing (WES).\n\nObjectiveTo evaluate the utility of WES-based diagnosis and discovery of novel variants of undiagnosed patients with complex neurodevelopmental problems in a country with a centralized medical system.\n\nDesign, setting, and participantsA cohort of 352 Korean patients, believed to cover a major portion of the entire country from July 2014 to April 2017, with a broad spectrum of neurodevelopmental disorders without any pathogenic variants revealed by conventional methods were evaluated by trio-based WES at Seoul National University Childrens Hospital.\n\nExposuresWES of patients and parents and subsequent evaluation of genetic variants.\n\nMain outcomes and measuresGenetic variants from each patient were evaluated for known disease association and novel variants were assessed for possible involvement with neurodevelopment process.\n\nResultsWe identified disease-causing variants, including newly discovered variants, in 57.4% of the probands, who had underwent a mean of 5.6 years of undiagnosed periods and visited mean of 2.3 tertiary hospitals. The cohort included 112 patients with variants that were previously reported as pathogenic (31.8%), 16 patients with copy number variants (4.5%) and 27 patients with variants that were associated with different clinical symptoms (7.7%). We also discovered potentially pathogenic variants from 47 patients that required further functional assessments (13.4%) and demonstrated potential implications in neurodevelopmental disorders. Following the genetic analysis, we provided more precise treatments to selected patients. A few clinical vignettes are presented that illuminate the potential diagnostic pitfalls that one could have encountered without this approach.\n\nConclusions and relevanceOur results highlight the utility of WES-based diagnosis for improved patient care in a country with a centralized medical system and discovery of novel pathophysiology mechanisms.\n\nKey pointsO_ST_ABSQuestionC_ST_ABSWhat is the advantage of whole exome sequencing based diagnosis of pediatric neurology patients with unknown rare symptoms in a large tertiary clinic in a country with a centralized medical system?\n\nFindingsWhole exome sequencing of 352 Korean patients, with a mean of 5.7 years of undiagnosed period, yielded 44.0% of conservative diagnostic yield. A number of cases were directly benefitted by trio-based WES via termination of diagnostic odyssey, genetic counseling for next offspring, or suggestion of more effective and customized treatment options.\n\nMeaningWe report on the establishment of a national-level whole exome-based diagnosis system, with emphasis on deliberate integration of clinical interpretation and genetic analysis. Whole exome sequencing should be a choice of diagnostic tools for pediatric neurologic patients with ambiguous symptoms.

genetics↗

bioRxiv2018

Oncogenic effects of germline mutations in lysosomal storage disease genes

Clinical observations have indicated that patients with Gaucher disease or Fabry disease are at increased risk of cancer. However, a systematic evaluation of the oncogenic effects of causal mutations of lysosomal storage diseases (LSDs) has been lacking. Here we report a comprehensive association analysis between potentially pathogenic germline mutations in LSD genes and cancer interrogating genomic (or exomic) variant datasets derived from the Pan-Cancer Analysis of Whole Genomes project (case cohort), the 1000 Genomes project (primary control cohort), and the Exome Aggregation Consortium that does not include The Cancer Genome Atlas subset (validation control cohort). We show that potentially pathogenic variants (PPVs) in 42 LSD genes are significantly enriched in cancer patients in a histology-dependent manner, cancer risk is higher in individuals with a greater number of PPVs, and cancer develops earlier in PPV carriers. Analysis of tumor genomic and transcriptomic data from the pancreatic adenocarcinoma cohort revealed potential mechanisms that might be involved in the oncogenic contribution of PPVs. Our findings extend the mechanistic understanding of inherited cancer susceptibility and highlight the promise of harnessing available therapeutic strategies to restore lysosomal function for personalized cancer prevention.

genetics↗

bioRxiv2018

Comprehensive functional screening of taste sensation in vivo

The initial event in taste sensation is mediated by taste cells on the tongue that translate ingested chemicals into cellular signals. Current understanding on this cellular level taste encoding process has relied on ex vivo model systems that cannot fully recapitulate natural cellular microenvironment in vivo. To resolve this methodological limitation, we invented a microfluidics-on-a-tongue imaging chamber that has integrated multichannel microfluidics for auto-controlled tastant delivery. Using this system, we screened over 100 fungiform taste cells to the five basic taste qualities, and obtained comprehensive functional maps. We revealed that taste cells are composed of 70% of single-tuned and 30% of dual-tuned cells, and also discovered a novel population of dual-tuned taste cells encoding a positive valence. We believe that our novel screening platform will pave a way for the deeper understanding of taste coding logic.

neuroscience↗