Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

OsLRR-RLP2 Gene Regulates Immunity to Magnaporthe oryzae in Japonica Rice.

Rice is an important cereal crop worldwide, the growth of which is affected by rice blast disease, caused by the fungal pathogen Magnaporthe oryzae. As climate change increases the diversity of pathogens, the disease resistance genes (R genes) in plants must be identified. The major blast-resistance genes have been identified in indica rice varieties; therefore, japonica rice varieties with R genes now need to be identified. Because leucine-rich repeat (LRR) domain proteins possess R-gene properties, we used bioinformatics analysis to identify the rice candidate LRR domain receptor-like proteins (OsLRR-RLPs). OsLRR-RLP2, which contains six LRR domains, showed differences in the DNA sequence, containing 43 single-nucleotide polymorphisms (SNPs) in indica and japonica subpopulations. The results of the M. oryzae inoculation analysis indicated that indica varieties with partial deletion of OsLRR-RLP2 showed susceptibility, whereas japonica varieties with intact OsLRR-RLP2 showed resistance. The oslrr-rlp2 mutant, generated using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), showed increased pathogen susceptibility, whereas plants overexpressing this gene showed pathogen resistance. These results indicate that OsLRR-RLP2 confers resistance to rice, and OsLRR-RLP2 may be useful for breeding resistant cultivars.

PRX102 Participates in Root Hairs Tip Growth of Rice.

Root hairs are extensions of epidermal cells on the root tips that increase the root contract surface area with the soil. For polar tip growth, newly synthesized proteins and other materials must be incorporated into the tips of root hairs. Here, we report the characterization of PRX102, a root hair preferential endoplasmic reticulum peroxidase. During root hair growth, PRX102 has a polar localization pattern within the tip regions of root hairs but it loses this polarity after growth termination. Moreover, PRX102 participates in root hair outgrowth by regulating dense cytoplasmic streaming toward the tip. This role is distinct from those of other peroxidases playing roles in the root hairs and regulating reactive oxygen species homeostasis. RNA-seq analysis using prx102 root hairs revealed that 87 genes including glutathione S-transferase were downregulated. Our results therefore suggest a new function of peroxidase as a player in the delivery of substances to the tips of growing root hairs.

Beyond Attention-Deficit Hyperactivity Disorder: Exploring Psychiatric Comorbidities and Their Neuropsychological Consequences in Adults.

Objectives: This study aimed to identify the psychiatric comorbidity status of adult patients diagnosed with attention-deficit hyperactivity disorder (ADHD) and determine the impact of comorbidities on neuropsychological outcomes in ADHD. Methods: The study participants were 124 adult patients with ADHD. Clinical psychiatric assessments were performed by two boardcertified psychiatrists in accordance with the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition. All participants were assessed using the Mini-International Neuropsychiatric Interview Plus version 5.0.0 to evaluate comorbidities. After screening, neuropsychological outcomes were assessed using the Comprehensive Attention Test (CAT) and the Korean version of the Wechsler Adult Intelligence Scale, Fourth Edition (K-WAIS-IV). Results: Mood disorders (38.7%) were the most common comorbidity of ADHD, followed by anxiety (18.5%) and substance use disorders (13.7%). The ADHD with comorbidities group showed worse results on the Perceptual Organization Index and Working Memory Index sections of the K-WAIS than the ADHD-alone group (p=0.015 and p=0.024, respectively). In addition, the presence of comorbidities was associated with worse performance on simple visual commission errors in the CAT tests (p=0.024). Conclusion: These findings suggest that psychiatric comorbidities are associated with poor neuropsychological outcomes in adult patients with ADHD, highlighting the need to identify comorbidities in these patients.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

Sucrose preferentially promotes expression of OsWRKY7 and OsPR10a to enhance defense response to blast fungus in rice.

Sucrose controls various developmental and metabolic processes in plants. It also functions as a signaling molecule in the synthesis of carbohydrates, storage proteins, and anthocyanins, as well as in floral induction and defense response. We found that sucrose preferentially induced OsWRKY7, whereas other sugars (such as mannitol, glucose, fructose, galactose, and maltose) did not have the same effect. A hexokinase inhibitor mannoheptulose did not block the effect of sucrose, which is consequently thought to function directly. MG132 inhibited sucrose induction, suggesting that a repressor upstream of OsWRKY7 is degraded by the 26S proteasome pathway. The 3-kb promoter sequence of OsWRKY7 was preferentially induced by sucrose in the luciferase system. Knockout mutants of OsWRKY7 were more sensitive to the rice blast fungus Magnaporthe oryzae, whereas the overexpression of OsWRKY7 enhanced the resistance, indicating that this gene is a positive regulator in the plant defense against this pathogen. The luciferase activity driven by the OsPR10a promoter was induced by OsWRKY7 and this transcription factor bound to the promoter region of OsPR10a, suggesting that OsWRKY7 directly controls the expression of OsPR10a. We conclude that sucrose promotes the transcript level of OsWRKY7, thereby increasing the expression of OsPR10a for the defense response in rice.

Factors Affecting Depressive Symptoms in Children and Adolescents With Epilepsy.

Objectives: We aimed to evaluate the clinical and psychological factors influencing depressive symptoms in children and adolescents with epilepsy. Methods: We administered self-reported questionnaires assessing children's depressive symptoms (Children's Depression Inventory, CDI) and anxiety (Revised Children's Manifest Anxiety Scale, RCMAS) to children and adolescents with epilepsy (n=87, age range=6-17 years). We asked their parents to complete questionnaires on epilepsy-related variables, parental stress (Questionnaire on Resources and Stress, QRS), parental anxiety (State-Trait Anxiety Inventory, STAI), family functioning (Family Adaptability and Cohesion Evaluation Scale, FACES), children's attention problems (Abbreviated Conners Parent Rating Scale Revised, CPRS), and children's behavioral problems (Korean Child Behavior Checklist, K-CBCL). Stepwise multiple regression analysis was performed to identify predictive variables affecting depressive symptoms. Results: Family adaptability (r=-0.240, p=0.026), family cohesion (r=-0.381, p<0.001), children's attention problems (r=0.290, p=0.006), children's anxiety (r=0.714, p<0.001), children's behavioral problems (r=0.371, p<0.001), parental anxiety (r=0.320, p=0.003), and parental stress (r=0.335, p=0.002) were significantly correlated with children's depressive symptoms. Children's anxiety (ß=0.655, p<0.001) and parental stress (ß=0.198, p=0.013) were significantly related to their depressive symptoms (adjusted R2=0.539). Conclusion: Clinicians should detect and manage children's anxiety and parental stress, which may affect depressive symptoms in children and adolescents with epilepsy.

OsSNDP3 Functions for the Polar Tip Growth in Rice Pollen Together with OsSNDP2, a Paralog of OsSNDP3.

Understanding pollen tube growth is critical for crop yield maintenance. The pollen tube provides a path for sperm cells for fertilization with egg cells. Cells must be subdivided into functionally and structurally distinct compartments for polar tip growth, and phosphoinositides are thought to be one of the facilitators for polarization during pollen tube growth. OsSNDP3 encodes Sec14-nodulin domain-containing protein and localizes in the nucleus and the microdomains of the plasma membrane in tobacco leaf epidermis cells. OsSNDP3 is thought to bind with phosphatidylinositol 4,5-bisphosphate based on the data including the information of basic amino acids in the C-terminal and colocalization with 2X Pleckstrin homology domain of Phospholipase C delta-1. OsSNDP3 interacts with a protein that contains a class I nodulin domain. We discovered that OsSNDP3 plays a significant role in pollen tube germination using CRISPR/Cas9 systems, whereas another pollen-preferential Sec14-nodulin domain-containing protein, OsSNDP2, additively functions with OsSNDP3 during pollen tube germination. Gene Ontology analysis using downregulated genes in ossndp3 indicated that the expression of genes involved in the phosphatidylinositol metabolic process and tip growth was significantly altered in ossndp3. OsSNDP3 aids pollen polar tip growth by binding with phosphatidylinositol 4,5-bisphosphate. We can better understand the roles of phosphoinositides during pollen tube growth by studying the functions of OsSNDP3 and OsSNDP2. And downregulated genes in ossndp3 might be useful targets for future research on polar tip growth.

A myosin XI adaptor, TAPE, is essential for pollen tube elongation in rice.

Pollen tube (PT) elongation is important for double fertilization in angiosperms and affects the seed-setting rate and, therefore, crop productivity. Compared to Arabidopsis (Arabidopsis thaliana L.), information on PT elongation in rice (Oryza sativa L.) is limited by the difficulty in obtaining homozygous mutants. In a screen of T-DNA insertional mutants, we identified a mutant in the Tethering protein of actomyosin transport in pollen tube elongation (TAPE) gene with an unusual segregation ratio by genotyping analysis. A CRISPR/Cas9 knockout mutant of TAPE that produced a short PT was sterile, and TAPE was expressed specifically in pollen grains. TAPE is a homolog of a myosin XI adaptor in Arabidopsis with three tetratricopeptide repeat and Phox and Bem1 protein domains. TAPE showed latrunculin B-sensitive, actin-dependent localization to the endoplasmic reticulum. Yeast two-hybrid screening and transcriptome analysis revealed that TAPE interacted with pollen-specific LIM protein 2b and elongation factor 1-alpha. Loss of TAPE affected transcription of 1,259 genes, especially genes related to cell organization, which were downregulated. In summary, TAPE encodes a myosin XI adaptor essential for rice PT elongation.

Comparative transcriptome analysis of pollen and anther wall reveals novel insights into the regulatory mechanisms underlying anther wall development and its dehiscence in rice.

To further understand the regulatory mechanism for anther dehiscence in rice, we carried out transcriptome analysis for the following two tissues: the anther wall and pollen at the anthesis stage. With the anatomical meta-expression data, in addition to these tissues, the differentially expressed genes (DEGs) between the two tissues were further refined to identify 1,717 pollen-preferred genes and 534 anther wall-preferred genes. A GUS transgenic line and RT-qPCR analysis for anther wall-preferred genes supported the fidelity of our gene candidates for further analysis. The refined DEGs were functionally classified through Gene Ontology (GO) enrichment and MapMan analyses. Through the analysis of cis-acting elements and alternative splicing variants, we also suggest the feature of regulatory sequences in promoter regions for anther wall-preferred expression and provide information of the unique splicing variants in anther wall. Subsequently, it was found that hormone signaling and the resulting transcriptional regulation pathways may play an important role in anther dehiscence and anther wall development. Our results could provide useful insights into future research to broaden the molecular mechanism of anther dehiscence or anther wall development in rice.

Cognitive Developmental Trajectories in Adult ADHD Patients and Controls: A Comparative Study.

OBJECTIVE: There is an updated conceptualization of whole-lifespan attention-deficit hyperactivity disorder (ADHD), promoted by awareness of probable persistence of impairment into adulthood. We investigated cognition trajectories from adolescence to mid-adulthood in ADHD. METHOD: Data of 240 patients with ADHD and 244 healthy controls (HCs) were obtained; clinical symptoms and neuropsychological functions were assessed using the various tests. RESULTS: Compared to HCs, patients with ADHD except 35 to 44 age interval showed lower full scale intelligence quotient. They showed decreased verbal comprehensive scores except in the 35 to 44 age interval and working memory scores in all intervals. In the Comprehensive Attention Test, patients with ADHD showed increased working memory error frequencies except in the 15 to 17 age interval and divided attention omission error in all intervals. CONCLUSION: Adults with ADHD showed deficits not in simple attention but in complex attention, including divided attention and working memory.

Pre- and postnatal exposure to multiple ambient air pollutants and child behavioral problems at five years of age.

Ambient air pollution is emerging as a risk factor for adverse neurological symptoms and early childhood diseases. This study aimed to evaluate the association between pre- and postnatal exposure to air pollutants and childhood behavior by using MOCEH prospective birth cohort data. In total, 353 mother-child pairs at birth, who completed child behavioral assessments using the Korean version of the Child Behavior Checklist at five years of age, were included in the study. Multivariate linear regression (MLR) for single pollutant and Bayesian kernel machine regression (BKMR) for multiple pollutants were conducted. MLR analysis showed that air pollutant exposures during the first trimester were significantly associated with the internalizing problems score after adjusting for covariates. The estimates were 0.19 (0.05-0.32) per 1 µg/m3 increase in PM2.5, 0.13 (0.04-0.22) per 1 µg/m3 increase in PM10, and 0.20 (0.02-0.37) per 1 ppb increase in NO2. The BKMR model analysis revealed that the overall effects of multiple air pollutants during the first trimester of pregnancy and 0-6 months of the infantile period were significantly associated with behavioral problems. Boys showed a stronger associations than girls. Taken together, these results showed that the first trimester of pregnancy and 0-6 months of the infantile period were important for air pollutant exposure because exposure at these periods was associated with behavioral problems in 5-year-old children. Future efforts are required to control air pollution levels and reduce the health burden of vulnerable populations, including pregnant women and children.

Interaction of OsRopGEF3 Protein With OsRac3 to Regulate Root Hair Elongation and Reactive Oxygen Species Formation in Rice (Oryza sativa).

Root hairs are tip-growing cells that emerge from the root epidermis and play a role in water and nutrient uptake. One of the key signaling steps for polar cell elongation is the formation of Rho-GTP by accelerating the intrinsic exchange activity of the Rho-of-plant (ROP) or the Rac GTPase protein; this step is activated through the interaction with the plant Rho guanine nucleotide exchange factor (RopGEFs). The molecular players involved in root hair growth in rice are largely unknown. Here, we performed the functional analysis of OsRopGEF3, which is highly expressed in the root hair tissues among the OsRopGEF family genes in rice. To reveal the role of OsRopGEF3, we analyzed the phenotype of loss-of-function mutants of OsRopGEF3, which were generated using the CRISPR-Cas9 system. The mutants had reduced root hair length and increased root hair width. In addition, we confirmed that reactive oxygen species (ROS) were highly reduced in the root hairs of the osropgef3 mutant. The pairwise yeast two-hybrid experiments between OsRopGEF3 and OsROP/Rac proteins in rice revealed that the OsRopGEF3 protein interacts with OsRac3. This interaction and colocalization at the same subcellular organelles were again verified in tobacco leaf cells and rice root protoplasts via bimolecular functional complementation (BiFC) assay. Furthermore, among the three respiratory burst oxidase homolog (OsRBOH) genes that are highly expressed in rice root hair cells, we found that OsRBOH5 can interact with OsRac3. Our results demonstrate an interaction network model wherein OsRopGEF3 converts the GDP of OsRac3 into GTP, and OsRac3-GTP then interacts with the N-terminal of OsRBOH5 to produce ROS, thereby suggesting OsRopGEF3 as a key regulating factor in rice root hair growth.

Global Identification of ANTH Genes Involved in Rice Pollen Germination and Functional Characterization of a Key Member, OsANTH3.

Pollen in angiosperms plays a critical role in double fertilization by germinating and elongating pollen tubes rapidly in one direction to deliver sperm. In this process, the secretory vesicles deliver cell wall and plasma membrane materials, and excessive materials are sequestered via endocytosis. However, endocytosis in plants is poorly understood. AP180 N-terminal homology (ANTH) domain-containing proteins function as adaptive regulators for clathrin-mediated endocytosis in eukaryotic systems. Here, we identified 17 ANTH domain-containing proteins from rice based on a genome-wide investigation. Motif and phylogenomic analyses revealed seven asparagine-proline-phenylalanine (NPF)-rich and 10 NPF-less subgroups of these proteins, as well as various clathrin-mediated endocytosis-related motifs in their C-terminals. To investigate their roles in pollen germination, we performed meta-expression analysis of all genes encoding ANTH domain-containing proteins in Oryza sativa (OsANTH genes) in anatomical samples, including pollen, and identified five mature pollen-preferred OsANTH genes. The subcellular localization of four OsANTH proteins that were preferentially expressed in mature pollen can be consistent with their role in endocytosis in the plasma membrane. Of them, OsANTH3 represented the highest expression in mature pollen. Functional characterization of OsANTH3 using T-DNA insertional knockout and gene-edited mutants revealed that a mutation in OsANTH3 decreased seed fertility by reducing the pollen germination percentage in rice. Thus, our study suggests OsANTH3-mediated endocytosis is important for rice pollen germination.

Cohort profile: the Ewha Birth and Growth Study.

With the introduction of life-course epidemiology, researchers realized the importance of identifying risk factors in early life to prevent chronic diseases. This led to the establishment of the Ewha Birth and Growth Study in 2001; the study is a prospective birth cohort designed to provide evidence of early life risk factors for a child's growth and health. Participants were recruited from those who visited Ewha Womans University Mokdong Hospital (a tertiary hospital in southwest Seoul, Korea) for prenatal care at 24-28 weeks of gestation. In total, 891 mothers enrolled in this study between 2001 and 2006 and their offspring (n=940) were followed-up. Regular check-up examinations of offspring were conducted at 3 years, 5 years, and 7 years of age and every year thereafter. To consider age-related health issues, extensive data were collected using questionnaires and measurements. In 2021, the study subjects will reach 19 years of age, and we are planning a check-up examination for early adulthood. About 20 years have passed since the cohort data were collected, and we have published results on childhood health outcomes associated with prenatal and birth characteristics, genetic and epigenetic characteristics related to childhood metabolism, the effects of exposure to endocrine disruptors, and dietary patterns in childhood. Recently, we started reporting on topics related to adolescent health. The findings will facilitate identification of early life risk factors for chronic diseases and the development of interventions for diseases later in life.

Transcriptome Analysis of Triple Mutant for OsMADS62, OsMADS63, and OsMADS68 Reveals the Downstream Regulatory Mechanism for Pollen Germination in Rice (Oryza sativa).

The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.

GORI, encoding the WD40 domain protein, is required for pollen tube germination and elongation in rice.

Successful delivery of sperm cells to the embryo sac in higher plants is mediated by pollen tube growth. The molecular mechanisms underlying pollen germination and tube growth in crop plants remain rather unclear, although these mechanisms are crucial to plant reproduction and seed formation. By screening pollen-specific gene mutants in rice (Oryza sativa), we identified a T-DNA insertional mutant of Germinating modulator of rice pollen (GORI) that showed a one-to-one segregation ratio for wild type (WT) to heterozygous. GORI encodes a seven-WD40-motif protein that is homologous to JINGUBANG/REN4 in Arabidopsis. GORI is specifically expressed in rice pollen, and its protein is localized in the nucleus, cytosol and plasma membrane. Furthermore, a homozygous mutant, gori-2, created through CRISPR-Cas9 clearly exhibited male sterility with disruption of pollen tube germination and elongation. The germinated pollen tube of gori-2 exhibited decreased actin filaments and altered pectin distribution. Transcriptome analysis revealed that 852 pollen-specific genes were downregulated in gori-2 compared with the WT, and Gene Ontology enrichment analysis indicated that these genes are strongly associated with cell wall modification and clathrin coat assembly. Based on the molecular features of GORI, phenotypical observation of the gori mutant and its interaction with endocytic proteins and Rac GTPase, we propose that GORI plays key roles in forming endo-/exocytosis complexes that could mediate pollen tube growth in rice.

Validity of the Korean Version of DIVA-5: A Semi-Structured Diagnostic Interview for Adult ADHD.

BACKGROUND: This study was to assess the validity of the Korean version of the semi-structured Diagnostic Interview for Adult ADHD, third edition (DIVA-5). The secondary aim was to compare sociodemographic and psychiatric comorbidities in adult patients with and without a diagnosis of ADHD. METHODS: A total of 279 participants were recruited from nine psychiatric outpatient clinics in Korea. All participants were administered the Mini-International Neuropsychiatric Interview (MINI) Plus v.5.0.0, the Adult ADHD Self-Report Scale v1.1 (ASRS-v1.1) Symptom Checklist, and DIVA-5. Diagnosis concordance between two board-certified psychiatrists and DIVA-5 were analysed. RESULTS: The DIVA-5 showed a diagnostic accuracy of 92%, a sensitivity of 91.30%, and a specificity of 93.62%. Significant clinical and demographic differences between ADHD and control groups were found. CONCLUSION: The Korean version of DIVA-5 is a reliable tool for assessing and diagnosing ADHD in adult Korean populations.

Longitudinal association between environmental tobacco smoke exposure and behavioral problems in children from ages 5 to 9.

Few studies have assessed the cumulative effects of exposure to environmental tobacco smoke (ETS1) in relation to children's neurobehavioral problems over time. We assessed the longitudinal associations between ETS exposure at age 5 and behavioral problems at ages 5, 7, and 9 using the Ewha Birth and Growth Cohort, in Seoul, Korea. Children with available urinary cotinine levels at age 5 and one or more behavioral problem scores measured with the Korean Version of Child Behavior Checklist (CBCL2) at age 5, 7, and 9 were included in the study. Those whose mothers smoked during pregnancy were excluded, and a total of 179 children were included in the analysis. A linear mixed-model analysis using a REPEATED statement was conducted to assess whether ETS exposure was associated with the total, internalizing, and externalizing behavioral problem scores of the CBCL. The group with higher levels of cotinine showed continuously higher total and external behavioral problem scores from ages 5 to 9, which was seen after adjusting for preterm birth, father's education level, and television watching time. In addition, the difference in the total and external behavioral problem scores between the higher and lower cotinine groups at age 5 was statistically significant after a Bonferroni correction (p = 0.02 and 0.04, respectively), even though the fixed effect of cotinine level was almost but not statistically significant (p = 0.07 and 0.08, respectively). The results of this longitudinal cohort study provide evidence regarding the negative effects of ETS exposure in early childhood and their behavioral problems over time. This study supports the strengthening of anti-smoking policies used in educational interventions for parents, in order to reduce early ETS exposure in children.