A bilirubin-inducible fluorescent protein from eel muscle.

The fluorescent protein toolbox has revolutionized experimental biology. Despite this advance, no fluorescent proteins have been identified from vertebrates, nor has chromogenic ligand-inducible activation or clinical utility been demonstrated. Here, we report the cloning and characterization of UnaG, a fluorescent protein from Japanese eel. UnaG belongs to the fatty-acid-binding protein (FABP) family, and expression in eel is restricted to small-diameter muscle fibers. On heterologous expression in cell lines or mouse brain, UnaG produces oxygen-independent green fluorescence. Remarkably, UnaG fluorescence is triggered by an endogenous ligand, bilirubin, a membrane-permeable heme metabolite and clinical health biomarker. The holoUnaG structure at 1.2 Å revealed a biplanar coordination of bilirubin by reversible π-conjugation, and we used this high-affinity and high-specificity interaction to establish a fluorescence-based human bilirubin assay with promising clinical utility. UnaG will be the prototype for a versatile class of ligand-activated fluorescent proteins, with applications in research, medicine, and bioengineering.

Treslin collaborates with TopBP1 in triggering the initiation of DNA replication.

TopBP1 has important roles in both DNA replication and checkpoint regulation in vertebrates. We have identified a protein called Treslin that associates with TopBP1 in Xenopus egg extracts. Depletion of Treslin from egg extracts strongly inhibits chromosomal DNA replication. Binding of Treslin to chromatin in egg extracts occurs independently of TopBP1. However, loading of the initiator protein Cdc45 onto chromatin cannot take place in the absence of Treslin. Prior to the initiation of DNA replication, Treslin associates with TopBP1 in a Cdk2-dependent manner. Ablation of Treslin from human cells also strongly inhibits DNA replication. Taken together, these results indicate that Treslin and TopBP1 collaborate in the Cdk2-mediated loading of Cdc45 onto replication origins. Thus, Treslin regulates a pivotal step in the initiation of DNA replication in vertebrates.

Third molar maturity index (I3M) assessment according to different geographical zones: a large multi-ethnic study sample.

Identification of living undocumented individuals highlights the need for accurate, precise, and reproducible age estimation methods, especially in those cases involving minors. However, when their country of origin is unknown, or it can be only roughly estimated, it is extremely difficult to apply assessment policies, procedures, and practices that are accurate and child-sensitive. The main aim of this research is to optimize the correct classification of adults and minors by establishing new cut-off values for four different continents (Africa, America, Asia, and Europe). For this purpose, a vast sample of 10,701 orthopantomographs (OPTs) from four continents was evaluated. For determination and subsequent validation of the new third molar maturity index (I3M) cut-off values by world regions, a cross-validation by holdout method was used and contingency tables (confusion matrices) were generated. The lower third molar maturity indexes, from both left and right side (I3ML and I3MR) and the combination of both sides (I3ML_I3MR) were calculated. The new cut-off values, that aim to differentiate between a minor and an adult, with more than 74.00% accuracy for all populations were as follows (I3ML; I3MR; I3ML_I3MR, respectively): Africa = (0.10; 0.10; 0.10), America = (0.10; 0.09; 0.09), Asia = (0.15; 0.17; 0.14), and Europe = (0.09; 0.09; 0.09). The higher sensitivity (Se) was detected for the I3ML for male African people (91%) and the higher specificity (Sp) of all the parameters (I3ML; I3MR; I3ML_I3MR) for Europeans both male and female (> 91%). The original cut-off value (0.08) is still useful, especially in discriminating individuals younger than 18 years old which is the goal of the forensic methods used for justice.

Interaction of Chk1 with Treslin negatively regulates the initiation of chromosomal DNA replication.

Treslin helps to trigger the initiation of DNA replication by promoting integration of Cdc45 into the replicative helicase. Treslin is a key positive-regulatory target of cell-cycle control mechanisms; activation of Treslin by cyclin-dependent kinase is essential for the initiation of replication. Here we demonstrate that Treslin is also a critical locus for negative regulatory mechanisms that suppress initiation. We found that the checkpoint-regulatory kinase Chk1 associates specifically with a C-terminal domain of Treslin (designated TRCT). Mutations in the TRCT domain abolish binding of Chk1 to Treslin and thereby eliminate Chk1-catalyzed phosphorylation of Treslin. Significantly, abolition of the Treslin-Chk1 interaction results in elevated initiation of chromosomal DNA replication during an unperturbed cell cycle, which reveals a function for Chk1 during a normal S phase. This increase is due to enhanced loading of Cdc45 onto potential replication origins. These studies provide important insights into how vertebrate cells orchestrate proper initiation of replication.

On-Site Monitoring of Postoperative Bile Leakage Using Bilirubin-Inducible Fluorescent Protein.

BACKGROUND: Bile leakage is the most common postoperative complication associated with hepatobiliary and pancreatic surgery. Until now, however, a rapid, accurate diagnostic method for monitoring intraoperative and postoperative bile leakage had not been established. METHOD: Bilirubin levels in drained abdominal fluids collected from 23 patients who had undergone hepatectomy (n = 22) or liver transplantation (n = 1) were measured using a microplate reader with excitation/emission wavelengths of 497/527 nm after applying 5 µM of UnaG to the samples. UnaG was also sprayed directly on hepatic raw surfaces in swine hepatectomy models to identify bile leaks by fluorescence imaging. RESULTS: The bilirubin levels measured by UnaG fluorescence imaging showed favorable correlations with the results of the conventional light-absorptiometric methods (indirect bilirubin: rs = 0.939, p < 0.001; direct bilirubin: rs = 0.929, p < 0.001). Approximate time required for bilirubin measurements with UnaG was 15 min, whereas it took about 40 min with the conventional method at a hospital laboratory. Following administration of UnaG on hepatic surfaces, the fluorescence imaging identified bile leaks not only on the resected specimens but also in the abdominal cavity of the swine hepatectomy models. CONCLUSION: Fluorescence imaging techniques using UnaG may enable real-time identification of bile leaks during hepatectomy and on-site rapid diagnosis of bile leaks after surgery.

Sex-Related Differences in Cardiac Remodeling and Reverse Remodeling After Transcatheter Aortic Valve Implantation in Patients with Severe Aortic Stenosis in a Japanese Population.

Left ventricular (LV) remodeling with aortic stenosis (AS) appears to differ according to sex, but reverse remodeling after transcatheter aortic valve implantation (TAVI) has not been elucidated in a Japanese population. This study aims to determine whether any sex-related differences in LV or reverse remodeling after TAVI exist in the context of severe AS.Of 208 patients who received TAVI for severe AS in our institution, 100 (men, 42; mean age, 83.0 ± 4.9 years) underwent transthoracic echocardiography before and 3 months after TAVI. Despite similar valvular gradients, women with severe AS had lower indexed LV mass (LVMi) than did men (152.3 ± 35.4 versus 173.2 ± 44.6 g/m2, P = 0.005), with smaller indexed LV end-diastolic (LVEDVi) (50.2 ± 13.3 versus 61.4 ± 20.7 mL/m2, P = 0.001) and end-systolic (LVESVi; 17.9 ± 8.7 versus 24.3 ± 13.8 mL/m2, P = 0.006) volumes. After TAVI, women (-6.0% ± 14.4%) had higher reduction in the rate of change of relative wall thickness (RWT) than did men (4.4% ± 19.0%, P = 0.003). Men (-8.9% ± 3.9%) had higher reduction in the rate of change of LVEDVi than did women (1.5% ± 3.3%, P = 0.045). Incidence of LV reverse remodeling defined as a reduction in LVESV of >15% was significantly higher in men (50%) than in women (26%, P = 0.013).In addition to sex differences in the pattern of LV remodeling with AS, reverse LV remodeling after TAVI also differed between sexes.

Age estimation combining radiographic information of two dental and four skeletal predictors in children and subadults.

Improved age estimates may result from combining different age predictors. This study aimed to validate age estimation performances combining the radiographic development of teeth, cervical vertebrae, hand and wrist bones, skull, and mandible. The sampled consisted of 256 individuals aged between 4 and 20 years. Bayes' rule with a multivariate continuation ratio model was applied for the distribution of the dental scores. The additional age information of the skeletal variables was assessed extending the dental model separately and combining the variables. The performances of all models were quantified and compared using mean error (ME), mean absolute error (MAE), and root mean squared error (RMSE). The best performance resulted combining all variables (ME - 0.04 for F and M; MAE 0.91 for F and 0.95 for M; RMSE 1.14 for F and 1.19 for M). To improve performances and minimize radiation doses, the combination of teeth and hand and wrist bones information is recommended.

3D-computed tomography to compare the dimensions of the left atrial appendage in patients with normal sinus rhythm and those with paroxysmal atrial fibrillation.

Although paroxysmal atrial fibrillation (PAF) is an important cause of cardioembolic stroke, in contrast to chronic AF patients, the anatomical features of the left atrial appendage (LAA) in PAF patients remain unknown. Here, we investigated differences in LAA structures in patients with PAF and those with normal sinus rhythms (NSR) using 3D-computed tomography (3D-CT), which allows us to visualize complicated LAA structures at high spatial resolution. Study subjects were 30 consecutive PAF and 30 NSR patients with complete enhanced cardiac 3D-CT images available. After reconstruction of 3D LAA images, anatomical parameters of the LAA were measured and compared according to three proposed definitions of the LAA orifice plane determined by the following anatomical landmarks: DEF#1, center of warfarin ridge and centerline of proximal left circumflex artery; DEF#2, slope of warfarin ridge and mitral valve annulus; DEF#3, observers' discretion by progressive rotation using the observers' best estimate without the use of landmarks. The LAA volumes of the PAF groups were significantly greater than the NSR group according to all 3 definitions (DEF#1: 1.43 times, DEF#2: 1.44 times, and DEF#3: 1.36 times greater). The LAA orifice area was significantly larger in PAF than in NSR according to DEF#2, but was similar by DEF#1 and DEF#3. Intra-observer and inter-observer variations for any LAA measurements were very low. In conclusion, 3D-CT-based quantitative assessment of the LAA provides highly reproducible and detailed measurements, which can successfully discriminate differences of LAA volume between patients with NSR and those with PAF, suggesting significantly greater volumes in the latter.

Two Distinct Fluorescence States of the Ligand-Induced Green Fluorescent Protein UnaG.

UnaG is a recently discovered ligand-induced fluorescent protein that utilizes bound bilirubin (BR) as its fluorophore. The fluorescence of the UnaG-BR complex (holoUnaG) compares in quantum efficiency to that of enhanced green fluorescent protein, but it is superior in that the fluorophore formation is instantaneous and not dependent on oxygen; hence, much attention has been paid to UnaG as a new fluorescent probe. However, many important molecular properties of fluorescent probes remain unknown, such as the association/dissociation rates of BR, which determine the stability thereof, and the dispersibility of UnaG in aqueous solutions, which influence the functions of labeled proteins. In this study, we found, in the process of investigating the association rate, that the holoUnaG takes two distinct fluorescence states, which we named holoUnaG1 and holoUnaG2. The holoUnaG1 initially forms after binding BR and then changes to the brighter holoUnaG2 by a reversible intra-molecular reaction, thereby finally reaching an equilibrium between the two states. Spectroscopic analysis indicated that the intra-molecular reaction was associated not with a chemical change of BR but with a change in the environmental conditions surrounding BR. We also revealed that the molecular brightness ratio and equilibrium population ratio of the two states (holoUnaG1/holoUnaG2) were 1:3.9 and 6:4, respectively, using photon number counting analysis. From these results, we have suggested a novel schema, to our knowledge, for the formation of the UnaG and BR complex system and have determined the various rate constants associated therein. Additionally, using analytical ultracentrifugation, we established that UnaG in the apo-state (apoUnaG) and the holoUnaG are monomeric in aqueous solution. These findings provide not only key information for the practical use of UnaG as a fluorescent probe, but also the possibility for development of a brighter UnaG mutant by genetic engineering to constitutive holoUnaG2.

Characteristics of Anatomy and Function of the Left Atrial Appendage and Their Relationships in Patients with Cardioembolic Stroke: A 3-Dimensional Transesophageal Echocardiography Study.

BACKGROUND: Increasing attention is being paid to the left atrial appendage (LAA) in the context of risk stratification in cardioembolic stroke (CES) and the requirement for meticulous planning of percutaneous closure device implantation. However, detailed systematic assessment of the LAA remains limited. METHODS: This study evaluated the anatomy and function of LAA using 3-dimensional transesophageal echocardiography (3D-TEE) on 194 consecutive patients older than 50 years old hospitalized exclusively for CES. Patients were stratified into 3 groups on the basis of cardiac rhythm: (1) chronic atrial fibrillation (AF), n = 53; (2) paroxysmal AF, n = 26; and (3) no detected AF, n = 115. RESULTS: Significant differences between the groups were observed for anatomical (orifice area [OA], depth, diastolic volume) and functional parameters (ejection fraction [EF], flow velocity [FV]), as measured by 3D-TEE. The anatomical parameters were consistently the greatest, and functional parameters were the poorest, in the group with chronic AF. There were significant inverse correlations between them (r = -.33, P = .0003 for depth and EF; r = -.27, P = .0020 for depth and FV; r = -.22, P = .016 for OA and EF; and r = -.38, P < .0001 for OA and FV). CONCLUSIONS: LAA morphology and function were strongly affected by cardiac rhythm disturbances. Patients with chronic AF had the greatest LAA dimensions, areas, and volumes as well as the lowest LAA functions. An inverse correlation was observed between LAA size and function.

Structural basis for the recognition of Lewis antigens by genogroup I norovirus.

Noroviruses (NoVs) bind to histo-blood group antigens, namely, ABH antigens and Lewis antigens. We previously showed the NoVs GI/2, GI/3, GI/4, and GI/8 were able to strongly bind to Lewis a (Le(a)) antigen, which is expressed by individuals who are nonsecretors. In this study, to investigate how Lewis antigens interact with GI NoV virion protein 1 (VP1), we determined the crystal structures of the P domain of the VP1 protein from the Funabashi 258 (FUV258) strain (GI/2) in complexes with Le(a), Le(b), H type 1, or A type 1 antigens. The structures were compared with those of the NV/68 strain (GI/1), which does not bind to the Le(a) antigen. The four loop structures, loop P, loop S, loop A, and loop B, continuously deviated by more than 2 Å in length between the Cα atoms of the corresponding residues of the FUV258 and NV/68 P domains. The most pronounced differences between the two VP1 proteins were observed in the structures of loop P. In the FUV258 P domain, loop P protruded toward the next protomer, forming a Le(a) antigen-binding site. The Gln389 residue make a significant contribution to the binding of the Le(a) antigen through the stabilization of loop P as well as through direct interactions with the α4-fucosyl residue (α4Fuc) of the Le(a) antigen. Mutation of the Gln389 residue dramatically affected the degree of binding of the Lewis antigens. Collectively, these results suggest that loop P and the amino acid residue at position 389 affect Lewis antigen binding.

Dental characteristics on panoramic radiographs as parameters for non-invasive age estimation: a pilot study.

The dental characteristics created by acquired dental treatments can be used as age estimators. This pilot study aimed to analyze the correlation between the number of teeth observed for dental characteristics and chronological age and to develop new non-invasive age estimation models. Dental features on panoramic radiographs (420 radiographs of subjects aged 20-89 years) were classified and coded. The correlation between the number of teeth for each selected code (codes V, X, T, F, P, and L) and age was observed, and multiple regression was performed to analyze the relationship between them. Eleven regression models with various combinations of dental sextants were presented. The model with the data from both sides of the posterior teeth on both jaws showed the best performance (root mean square error of 14.78 years and an adjusted R2 of 0.461). The model with all teeth was the second-best. Based on these results, we confirmed statistically significant correlations between certain dental features and chronological age. We also observed that some regression models performed sufficiently well to be used as adjunctive methods in forensic practice. These results provide valuable information for the design and performance of future full-scale studies.

Validation of London Atlas for forensic age estimation in Koreans by comparing with Lee's and Willems' methods.

This cross-sectional study aimed to evaluate the applicability of the London Atlas for age estimation in the Korean population by comparing with Lee's and Willems' methods. Dental ages of 475 orthopantomographs from Korean individuals aged 4-15 years (mean 10.32 ± 3.31 years) were estimated using the London Atlas, Lee's and Willems' methods. Correlation between dental and chronological age was determined using Pearson's correlation coefficient, and the statistical difference between dental and chronological age was analyzed using a paired t-test. The bias (mean differences), mean absolute error, and root mean square error between dental and chronological age, stratified by age groups and estimation methods, were calculated. Differences in bias and absolute error between sexes were scrutinized using an independent-samples t-test. Age estimates of the three tested methods were combined and compared to those of each individual method. The London Atlas and Willems methods resulted in overestimations, whereas the Lee method led to an underestimation on the entire sample. The overall accuracy was observed in the order of Lee's method, the London Atlas, and Willems' method. The London Atlas demonstrated superior consistency of estimation performance across age groups and no significant differences in estimation performance between sexes. The combination of estimates from the London Atlas and Lee's method resulted in an enhancement in bias and accuracy. We conclude that the London Atlas, due to its bias and accuracy comparable to Lee's and Willems' methods, is applicable for forensic practice in the Korean population.

Validation of data mining models by comparing with conventional methods for dental age estimation in Korean juveniles and young adults.

Teeth are known to be the most accurate age indicators of human body and are frequently applied in forensic age estimation. We aimed to validate data mining-based dental age estimation, by comparing the accuracy of the estimation and classification performance of 18-year thresholds with conventional methods and with data mining-based age estimation. A total of 2657 panoramic radiographs were collected from Koreans and Japanese populations aged 15 to 23 years. They were subdivided into a training and internal test set of 900 radiographs each from Koreans, and an external test set of 857 radiographs from Japanese. We compared the accuracy and classification performance of the test sets from conventional methods with those from the data mining models. The accuracy of the conventional method with the internal test set was slightly higher than that of the data mining models, with a slight difference (mean absolute error < 0.21 years, root mean square error < 0.24 years). The classification performance of the 18-year threshold was also similar between the conventional method and the data mining models. Thus, conventional methods can be replaced by data mining models in forensic age estimation using second and third molar maturity of Korean juveniles and young adults.

Rif1 restrains the rate of replication origin firing in Xenopus laevis.

Metazoan genomes are duplicated by the coordinated activation of clusters of replication origins at different times during S phase, but the underlying mechanisms of this temporal program remain unclear during early development. Rif1, a key replication timing factor, inhibits origin firing by recruiting protein phosphatase 1 (PP1) to chromatin counteracting S phase kinases. We have previously described that Rif1 depletion accelerates early Xenopus laevis embryonic cell cycles. Here, we find that in the absence of Rif1, patterns of replication foci change along with the acceleration of replication cluster activation. However, initiations increase only moderately inside active clusters. Our numerical simulations suggest that the absence of Rif1 compresses the temporal program towards more homogeneity and increases the availability of limiting initiation factors. We experimentally demonstrate that Rif1 depletion increases the chromatin-binding of the S phase kinase Cdc7/Drf1, the firing factors Treslin, MTBP, Cdc45, RecQL4, and the phosphorylation of both Treslin and MTBP. We show that Rif1 globally, but not locally, restrains the replication program in early embryos, possibly by inhibiting or excluding replication factors from chromatin.

Urinary lumirubin excretion in jaundiced preterm neonates during phototherapy with blue light-emitting diode vs. green fluorescent lamp.

Phototherapy converts lipophilic unconjugated bilirubin to hydrophilic bilirubin photoisomers, such as lumirubin. We comparatively used a blue light-emitting diode (LED) and a green fluorescent lamp (FL) as light sources for phototherapy of hyperbilirubinemic preterm neonates with the aim of examining potential differences in urinary lumirubin excretion between these two wavelengths. Urinary lumirubin levels were measured using a fluorescence assay with blue light exposure in the presence of the unconjugated bilirubin-inducible fluorescent protein UnaG, and denoted as urinary UnaG-bound bilirubin (UUB)/creatinine (Cr) (µg/mg Cr). Preterm neonates born at ≤ 33 weeks gestational age and treated with phototherapy were subjected to this study. The maximum UUB/Cr level during phototherapy per device intensity was compared between neonates treated with the blue LED and the green FL. A total of 61 neonates were examined to determine the maximum UUB/Cr levels. The median of maximum UUB/Cr excretion per light intensity of each device (µg/mg Cr/µW/cm2/nm) was 0.83 for the blue LED and 1.29 for the green FL (p = 0.01). Green light was found to be more effective than blue one for bilirubin excretion via urinary lumirubin excretion. This is the first spectroscopic study to compare the efficacy of phototherapy at different wavelengths using fluorescence assay.

Cardiac Reversibility and Survival After Transcatheter Aortic Valve Implantation in Patients With Low-Gradient Aortic Stenosis.

Background Prognostic implications of transcatheter aortic valve implantation (TAVI) in low-gradient (LG) aortic stenosis (AS) remain controversial. The authors hypothesized that differences in cardiac functional recovery may solve this ongoing controversy. The aim was to evaluate clinical outcomes and the response of left ventricular (LV) function following TAVI in patients with LG AS. Methods and Results This multicenter retrospective study included 1742 patients with severe AS undergoing TAVI between January 2015 and March 2019. Patients were subdivided into low-flow (LF) LG, normal-flow (NF) LG, LF high-gradient, and NF high-gradient AS groups according to the mean gradient of the aortic valve (LG <40 mm Hg) and LV stroke volume index (LF <35 mL/m2). Outcomes and changes in echocardiographic parameters after TAVI were compared between the groups. A total of 227 patients (13%) had reduced ejection fraction, and 486 patients (28%) had LG AS (LF-LG 143 [8%]; NF-LG 343 [20%]). During a median follow-up period of 747 days, 301 patients experienced a composite end point of cardiovascular death and rehospitalization for cardiovascular events, which was higher in the LF-LG and NF-LG groups than in the high-gradient groups. LG AS was independently associated with the primary outcome (hazard ratio, 1.69; P<0.001). Among 1239 patients with follow-up echocardiography, LG AS showed less improvement in the LV mass index and LV end-diastolic volume compared with high-gradient AS after 1 year, while LV recovery was similar between the LF AS and NF AS groups. Conclusions LG AS was associated with poorer outcomes and LV recovery, regardless of flow status after TAVI. Careful evaluation of AS severity may be required in LG AS to provide TAVI within the appropriate time and advanced care afterward.

The applicability of Willems' and Lee's dental age estimation methods for Japanese children and the comparison with the Korean population.

This study aimed to examine the applicability of Willems' and Lee's methods in a Japanese population and to find the differences in teeth development between Japanese and Korean children by comparing the accuracy of their estimated age using the two methods. The developmental status of the lower left permanent teeth in 1,475 orthopantomograms of Japanese children were evaluated based on Demirjian's criteria, and the ages were estimated using Willems' and Lee's methods. The errors in estimated ages were calculated and compared with equivalent Korean population data. The accuracy of both methods for estimating the age of the Japanese population was similar to that in the Korean population except for that of Lee's method used in Japanese males. The results showed that there might be differences in teeth development between the Japanese and Korean boys. To increase the accuracy of the dental age estimation, the method that is derived from their own population data should be applied in forensic practice.

Percutaneous repair of systemic atrioventricular valve regurgitation with the MitraClip-NT system in congenitally corrected transposition of great arteries.

In a patient with congenitally corrected transposition of the great arteries, dilatation of the atrioventricular valve annulus related to worsening of systemic ventricular function, which worsened systemic atrioventricular valve (SAVV) functional regurgitation. In this article, we report a case of successful transcatheter treatment with MitraClip (Abbott Vascular, Santa Clara, CA, USA) in a 68-year-old female patient with congenitally corrected transposition of the great arteries and severe SAVV failure using imaging modalities. The patient had been hospitalized four times within 8 months, receiving optimal medical therapy for severe SAVV regurgitation and systemic ventricular failure. In this patient, the risk associated with surgery was considered extremely high owing to severe heart failure and liver cirrhosis. We positioned two clips appropriately, side by side, in between the anterior and septal leaflets, guided by computed tomography and three-dimensional echocardiography. The procedure resulted in optimal post-procedural reduction of regurgitation without stenosis, with a good clinical outcome noted at 2-year follow-up. Learning objective: Since surgery for adult congenital heart disease is linked to high risk, we seek less invasive treatment for such patients. In patients with congenital heart disease and severe valve regurgitation, the use of MitraClip (Abbott Vascular, Santa Clara, CA, USA) could be a feasible option for select cases.

Accuracy of age estimation and assessment of the 18-year threshold based on second and third molar maturity in Koreans and Japanese.

This study aimed to validate Lee's age estimation method and assess the 18-year threshold in Korean and Japanese populations. We evaluated the maxillary and mandibular second (M2) and third molars (M3) in 2657 orthopantomograms of the Korean and Japanese populations aged 15-23 years (19.47±2.62 years for Koreans, 19.31±2.60 years for Japanese), using Demirjian's criteria. Dental age was estimated, and correlations between chronological and dental ages were analyzed. Classification performance was calculated based on the 18-year threshold. The relationship between developmental stage and chronologic age was analyzed using multiple linear regression. Our results revealed that Lee's method was appropriate for estimation in the Korean population. When the Lee's method was applied to the Japanese population, a lower value of correlation coefficients between estimated and chronological age, and lower specificity were observed. Population differences were observed predominantly in the stages of root development (stages F and G) of M2s and M3s in both jaws and more frequently in females than in males. In the multiple linear regression between developmental stage and chronological age, lower values of adjusted r2 were observed in the Japanese population than in the Koreans. In conclusion, the Lee's method derived from the Korean population data might be unsuitable for Japanese juveniles and adolescents. To support the findings of this study, future studies with samples from multiple institutions should be conducted. Future studies with larger sample sizes are also warranted to improve the accuracy of dental age estimation and confirm the developmental pattern of teeth in the Japanese population.