Computational Exploration of Minimum Energy Reaction Pathway of N2O Formation from Intermediate I of P450nor Using an Active Center Model.

P450nor is a heme-containing enzyme that catalyzes the conversion of nitric oxide (NO) to nitrous oxide (N2O). Its catalytic mechanism has attracted attention in chemistry, biology, and environmental engineering. The catalytic cycle of P450nor is proposed to consist of three major steps. The reaction mechanism for the last step, N2O generation, remains unknown. In this study, the reaction pathway of the N2O generation from the intermediate I was explored with the B3LYP calculations using an active center model after the examination of the validity of the model. In the validation, we compared the heme distortions between P450nor and other oxidoreductases, suggesting a small effect of protein environment on the N2O generation reaction in P450nor. We then evaluated the electrostatic environment effect of P450nor on the hydride affinity to the active site with quantum mechanics/molecular mechanics (QM/MM) calculations, confirming that the affinity was unchanged with or without the protein environment. The active center model for P450nor showed that the N2O generation process in the enzymatic reaction undergoes a reasonable barrier height without protein environment. Consequently, our findings strongly suggest that the N2O generation reaction from the intermediate I depends sorely on the intrinsic reactivity of the heme cofactor bound on cysteine residue.

Prediction of Protein Function from Tertiary Structure of the Active Site in Heme Proteins by Convolutional Neural Network.

Structure-function relationships in proteins have been one of the crucial scientific topics in recent research. Heme proteins have diverse and pivotal biological functions. Therefore, clarifying their structure-function correlation is significant to understand their functional mechanism and is informative for various fields of science. In this study, we constructed convolutional neural network models for predicting protein functions from the tertiary structures of heme-binding sites (active sites) of heme proteins to examine the structure-function correlation. As a result, we succeeded in the classification of oxygen-binding protein (OB), oxidoreductase (OR), proteins with both functions (OB-OR), and electron transport protein (ET) with high accuracy. Although the misclassification rate for OR and ET was high, the rates between OB and ET and between OB and OR were almost zero, indicating that the prediction model works well between protein groups with quite different functions. However, predicting the function of proteins modified with amino acid mutation(s) remains a challenge. Our findings indicate a structure-function correlation in the active site of heme proteins. This study is expected to be applied to the prediction of more detailed protein functions such as catalytic reactions.

Performance of a novel computer-aided diagnosis system in the characterization of colorectal polyps, and its role in meeting Preservation and Incorporation of Valuable Endoscopic Innovations standards set by the American Society of Gastrointestinal Endoscopy.

Background and aims: There has been an increasing role of artificial intelligence (AI) in the characterization of colorectal polyps. Recently, a novel AI algorithm for the characterization of polyps was developed by NEC Corporation (Japan). The aim of our study is to perform an external validation of this algorithm. Methods: The study was a video-based evaluation of the computer-aided diagnosis (CADx) system. Patients undergoing colonoscopy were recruited to record videos of colonic polyps. The frozen polyp images extracted from these videos were used for real-time histological prediction by the endoscopists and by the CADx system, and the results were compared. Results: A total of 115 polyp images were extracted from 66 patients. Sensitivity, negative predictive value and accuracy for diminutive polyps on white light imaging (WLI) and image-enhanced endoscopy (IEE) when assessed by CADx was 90.9% [95% confidence interval (CI) 77.3-100] and 95.8% [95% CI 87.5-100], 80% [95% CI 44.4-97.5] and 90.9% [95% CI 58.7-99.8], 84.8% [95% CI 72.7-97] and 84.6% [95%CI 71.8-94.9], respectively, compared to 48.1% [95%CI 37.7-59.1] and 72% [95% CI 62.5-81], 37.5% [95% CI 28.8-46.8] and 55% [95% CI 44.7-65.0], 53.7% [95% CI 44.2-63.2] and 66.7% [95% CI 59.7-73.3] when assessed by endoscopists. Concordance between histology and CADx-based post-polypectomy surveillance intervals was 93.02% on WLI and 96% on IEE. Conclusion: AI-based optical diagnosis is promising and has the potential to be better than the performance of general endoscopists. We believe that AI can help make real-time optical diagnoses of polyps meeting the Preservation and Incorporation of Valuable endoscopic Innovations standards set by the American Society of Gastrointestinal Endoscopy.

Effects of Active-Center Reduction of Plant-Type Ferredoxin on Its Structure and Dynamics: Computational Analysis Using Molecular Dynamics Simulations.

"Plant-type" ferredoxins (Fds) in the thylakoid membranes of plants, algae, and cyanobacteria possess a single [2Fe-2S] cluster in active sites and mediate light-induced electron transfer from Photosystem I reaction centers to various Fd-dependent enzymes. Structural knowledge of plant-type Fds is relatively limited to static structures, and the detailed behavior of oxidized and reduced Fds has not been fully elucidated. It is important that the investigations of the effects of active-center reduction on the structures and dynamics for elucidating electron-transfer mechanisms. In this study, model systems of oxidized and reduced Fds were constructed from the high-resolution crystal structure of Chlamydomonas reinhardtii Fd1, and three 200 ns molecular dynamics simulations were performed for each system. The force field parameters of the oxidized and reduced active centers were independently obtained using quantum chemical calculations. There were no substantial differences in the global conformations of the oxidized and reduced forms. In contrast, active-center reduction affected the hydrogen-bond network and compactness of the surrounding residues, leading to the increased flexibility of the side chain of Phe61, which is essential for the interaction between Fd and the target protein. These computational results will provide insight into the electron-transfer mechanisms in the Fds.

Elucidation of the Correlation between Heme Distortion and Tertiary Structure of the Heme-Binding Pocket Using a Convolutional Neural Network.

Heme proteins serve diverse and pivotal biological functions. Therefore, clarifying the mechanisms of these diverse functions of heme is a crucial scientific topic. Distortion of heme porphyrin is one of the key factors regulating the chemical properties of heme. Here, we constructed convolutional neural network models for predicting heme distortion from the tertiary structure of the heme-binding pocket to examine their correlation. For saddling, ruffling, doming, and waving distortions, the experimental structure and predicted values were closely correlated. Furthermore, we assessed the correlation between the cavity shape and molecular structure of heme and demonstrated that hemes in protein pockets with similar structures exhibit near-identical structures, indicating the regulation of heme distortion through the protein environment. These findings indicate that the tertiary structure of the heme-binding pocket is one of the factors regulating the distortion of heme porphyrin, thereby controlling the chemical properties of heme relevant to the protein function; this implies a structure-function correlation in heme proteins.

Robust automated prediction of the revised Vienna Classification in colonoscopy using deep learning: development and initial external validation.

BACKGROUND: Improved optical diagnostic technology is needed that can be used by also outside expert centers. Hence, we developed an artificial intelligence (AI) system that automatically and robustly predicts the pathological diagnosis based on the revised Vienna Classification using standard colonoscopy images. METHODS: We prepared deep learning algorithms and colonoscopy images containing pathologically proven lesions (56,872 images, 6775 lesions). Four classifications were adopted: revised Vienna Classification category 1, 3, and 4/5 and normal images. The best algorithm-ResNet152-in the independent internal validation (14,048 images, 1718 lesions) was used for external validation (255 images, 128 lesions) based on neoplastic and non-neoplastic classification. Diagnostic performance of endoscopists was compared using a computer-assisted interpreting test. RESULTS: In the internal validation, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for adenoma (category 3) of 84.6% (95% CI 83.5-85.6%), 99.7% (99.5-99.8%), 90.8% (89.9-91.7%), 89.2% (88.5-99.0%), and 89.8% (89.3-90.4%), respectively. In the external validation, ResNet152's sensitivity, specificity, PPV, NPV, and accuracy for neoplastic lesions were 88.3% (82.6-94.1%), 90.3% (83.0-97.7%), 94.6% (90.5-98.8%), 80.0% (70.6-89.4%), and 89.0% (84.5-93.6%), respectively. This diagnostic performance was superior to that of expert endoscopists. Area under the receiver-operating characteristic curve was 0.903 (0.860-0.946). CONCLUSIONS: The developed AI system can help non-expert endoscopists make differential diagnoses of colorectal neoplasia on par with expert endoscopists during colonoscopy. (229/250 words).

Depolarizing Effects in Hydrogen Bond Energy in 310-Helices Revealed by Quantum Chemical Analysis.

Hydrogen-bond (H-bond) energies in 310-helices of short alanine peptides were systematically examined by precise DFT calculations with the negative fragmentation approach (NFA), a modified method based on the molecular tailoring approach. The contribution of each H-bond was evaluated in detail from the 310-helical conformation of total energies (whole helical model, WH3-10 model), and the results were compared with the property of H-bond in α-helix from our previous study. The H-bond energies of the WH3-10 model exhibited tendencies different from those exhibited by the α-helix in that they depended on the helical position of the relevant H-bond pair. H-bond pairs adjacent to the terminal H-bond pairs were observed to be strongly destabilized. The analysis of electronic structures indicated that structural characteristics cause the destabilization of the H-bond in 310-helices. We also found that the longer the helix length, the more stable the H-bond in the terminal pairs of the WH3-10 model, suggesting the action of H-bond cooperativity.

Development of a short Japanese version of the Self-Stigma of Stuttering Scale (4S-J-16): Translation and evaluation of validity and reliability.

PURPOSE: This study aims to develop a short Japanese version of the Self-Stigma of Stuttering Scale (4S), which assesses the self-stigma of adults who stutter (AWS) in a self-completed form, and evaluate its psychometric properties and reliability and validity. METHODS: After translating the original 4S scale into Japanese (4S-J) through a forward-backward translation process, it was administered to 123 Japanese adults who stutter. A short version of the 4S-J was developed through factor analysis and eliminating items with low loadings to original factors. Reliability was verified by calculating internal consistency and test-retest reliability. Participants also completed the Japanese-translated version of the Rosenberg Self Esteem Scale, General Self-Efficacy Scale, and Subjective Happiness Scale to verify construct validity. As a secondary analysis, our results regarding psychological properties of the short version of the 4S were then compared to those of studies from other countries. RESULTS: Sixteen items were selected for the short version of the scale (4S-J-16), and confirmatory factor analysis verified the original structure of the 4S. We obtained good internal consistency and test-retest reliability. Regarding construct validity, our results showed similar correlation with the other selected scales. The value of all scores on the stigma scale in the Japanese cohort was significantly higher than that in the foreign cohort. CONCLUSIONS: Overall, the 4S-J-16 has good internal consistency, test-retest reliability, and construct validity with a three-factor structure of self-stigma in Japanese AWS. The findings suggest that Japanese cultural background increases the stigma scores, thereby enabling us to briefly assess the psychosocial issues of AWS.

Analysis of Fluctuation in the Heme-Binding Pocket and Heme Distortion in Hemoglobin and Myoglobin.

Heme is located in the active site of proteins and has diverse and important biological functions, such as electron transfer and oxygen transport and/or storage. The distortion of heme porphyrin is considered an important factor for the diverse functions of heme because it correlates with the physical properties of heme, such as oxygen affinity and redox potential. Therefore, clarification of the relationship between heme distortion and the protein environment is crucial in protein science. Here, we analyzed the fluctuation in heme distortion in the protein environment for hemoglobin and myoglobin using molecular dynamics (MD) simulations and quantum mechanical (QM) calculations as well as statistical analysis of the protein structures of hemoglobin and myoglobin stored in Protein Data Bank. Our computation and statistical analysis showed that the protein environment for hemoglobin and myoglobin prominently affects the doming distortion of heme porphyrin, which correlates with its oxygen affinity, and that the magnitude of distortion is different between hemoglobin and myoglobin. These results suggest that heme distortion is affected by its protein environment and fluctuates around its fitted conformation, leading to physical properties that are appropriate for protein functions.

Global Analysis of Heme Proteins Elucidates the Correlation between Heme Distortion and the Heme-Binding Pocket.

Heme proteins play diverse and important biological roles, from electron transfer and chemical catalysis to oxygen transport and/or storage. Although the distortion of heme porphyrin correlates with the physical properties of heme, such as the redox potential and oxygen affinity, the relationship between heme distortion and the heme protein environment is unclear. Here, we tested the hypothesis that the protein environment of the heme-binding pocket determines heme distortion (conformation). We analyzed the correlations between the amino acid composition of the heme-binding pocket and the magnitude of heme distortion along 12 vibrational modes using machine learning. A correlation was detected in the three lowest vibrational modes. Analysis of heme distortions in nearly the same environments of the heme-binding pocket supported this notion. Our analyses indicate that the heme-binding pocket environment is a major factor impacting the distortion of heme porphyrin along the three lowest vibrational modes. In addition, statistical analysis of the distortion of heme porphyrin revealed that the peaks of distributions of the ruffling and breathing distortions are shifted from 0 (the equilibrium structure). Both the ruffling and breathing distortions are correlated with the redox potential of heme, so that heme molecules with these distortions have a lower redox potential than planar molecules. These findings explain the structure-function relationship of heme.

Quantum chemical studies on hydrogen bonds in helical secondary structures.

We present a brief review of our recent computational studies of hydrogen bonds (H-bonds) in helical secondary structures of proteins, α-helix and 310-helix, using a Negative Fragmentation Approach with density functional theory. We found that the depolarized electronic structures of the carbonyl oxygen of the ith residue and the amide hydrogen of the (i + 4)th residue cause weaker H-bond in an α-helix than in an isolated H-bond. Our calculations showed that the H-bond energies in the 310-helix were also weaker than those of the isolated H-bonds. In the 310-helices, the adjacent N-H group at the (i + 1)th residue was closer to the C=O group of the H-bond pair than the adjacent C=O group in the 310-helices, whereas the adjacent C=O group at the (i + 1)th residue was close to the H-bond acceptor in α-helices. Therefore, the destabilization of the H-bond is attributed to the depolarization caused by the adjacent residue of the helical backbone connecting the H-bond donor and acceptor. The differences in the change in electron density revealed that such depolarizations were caused by the local electronic interactions in their neighborhood inside the helical structure and redistributed the electron density. We also present the improvements in the force field of classical molecular simulation, based on our findings. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-022-01034-5.

Detection of flat colorectal neoplasia by artificial intelligence: A systematic review.

OBJECTIVES: This study review focuses on a deep learning method for the detection of colorectal lesions in colonoscopy and AI support for detecting colorectal neoplasia, especially in flat lesions. DATA SOURCES: We performed a systematic electric search with PubMed by using "colonoscopy", "artificial intelligence", and "detection". Finally, nine articles about development and validation study and eight clinical trials met the review criteria. RESULTS: Development and validation studies showed that trained AI models had high accuracy-approximately 90% or more for detecting lesions. Performance was better in elevated lesions than in superficial lesions in the two studies. Among the eight clinical trials, all but one trial showed a significantly high adenoma detection rate in the CADe group than in the control group. Interestingly, the CADe group detected significantly high flat lesions than the control group in the seven studies. CONCLUSION: Flat colorectal neoplasia can be detected by endoscopists who use AI.

PyDISH: database and analysis tools for heme porphyrin distortion in heme proteins.

Heme participates in a wide range of biological functions such as oxygen transport, electron transport, oxygen reduction, transcriptional regulation and so on. While the mechanism of each function has been investigated for many heme proteins, the origin of the diversity of the heme functions is still unclear and a crucial scientific issue. We have constructed a database of heme proteins, named Python-based database and analyzer for DIStortion of Heme porphyrin (PyDISH), which also contains some analysis tools. The aim of PyDISH is to integrate the information on the structures of hemes and heme proteins and the functions of heme proteins. This database will provide the structure-function relationships focusing on heme porphyrin distortion and lead to the elucidation of the origin of the functional diversity of heme proteins. In addition, the insights obtained from the database can be used for the design of protein function. PyDISH contains the structural data of more than 13 000 hemes extracted from the Protein Data Bank, including heme porphyrin distortion, axial ligands coordinating to the heme and the orientation of the propionate sidechains of heme. PyDISH also has information about the protein domains, including Uniprot ID, protein fold by CATH ID, organism, coordination distance and so on. The analytical tools implemented in PyDISH allow users to not only browse and download the data but also analyze the structures of heme porphyrin by using the analytical tools implemented in PyDISH. PyDISH users will be able to utilize the obtained results for the design of protein function. Database URL: http://pydish.bio.info.hiroshima-cu.ac.jp/.

Effects of a remote mutation from the contact paratope on the structure of CDR-H3 in the anti-HIV neutralizing antibody PG16.

PG16 is a broadly neutralizing antibody to the human immunodeficiency virus (HIV). A crystal structure of PG16 revealed that the unusually long 28-residue complementarity determining region (CDR) H3 forms a unique subdomain, referred to as a "hammerhead", that directly contacts the antigen. The hammerhead apparently governs the function of PG16 while a previous experimental assay showed that the mutation of TyrH100Q to Ala, which does not directly contact the antigen, decreased the neutralization ability of PG16. However, the molecular mechanism by which a remote mutation from the hammerhead or contact paratope affects the neutralization potency has remained unclear. Here, we performed molecular dynamics simulations of the wild-type and variants (TyrH100Q to Ala, and TyrH100Q to Phe) of PG16, to clarify the effects of these mutations on the dynamics of CDR-H3. Our simulations revealed that the structural rigidity of the CDR-H3 in PG16 is attributable to the hydrogen bond interaction between TyrH100Q and ProH99, as well as the steric support by TyrH100Q. The loss of both interactions increases the intrinsic fluctuations of the CDR-H3 in PG16, leading to a conformational transition of CDR-H3 toward an inactive state.

Development of a real-time endoscopic image diagnosis support system using deep learning technology in colonoscopy.

Gaps in colonoscopy skills among endoscopists, primarily due to experience, have been identified, and solutions are critically needed. Hence, the development of a real-time robust detection system for colorectal neoplasms is considered to significantly reduce the risk of missed lesions during colonoscopy. Here, we develop an artificial intelligence (AI) system that automatically detects early signs of colorectal cancer during colonoscopy; the AI system shows the sensitivity and specificity are 97.3% (95% confidence interval [CI] = 95.9%-98.4%) and 99.0% (95% CI = 98.6%-99.2%), respectively, and the area under the curve is 0.975 (95% CI = 0.964-0.986) in the validation set. Moreover, the sensitivities are 98.0% (95% CI = 96.6%-98.8%) in the polypoid subgroup and 93.7% (95% CI = 87.6%-96.9%) in the non-polypoid subgroup; To accelerate the detection, tensor metrics in the trained model was decomposed, and the system can predict cancerous regions 21.9 ms/image on average. These findings suggest that the system is sufficient to support endoscopists in the high detection against non-polypoid lesions, which are frequently missed by optical colonoscopy. This AI system can alert endoscopists in real-time to avoid missing abnormalities such as non-polypoid polyps during colonoscopy, improving the early detection of this disease.

Hydrogen bond donors and acceptors are generally depolarized in α-helices as revealed by a molecular tailoring approach.

Hydrogen-bond (H-bond) interaction energies in α-helices of short alanine peptides were systematically examined by precise density functional theory calculations, followed by a molecular tailoring approach. The contribution of each H-bond interaction in α-helices was estimated in detail from the entire conformation energies, and the results were compared with those in the minimal H-bond models, in which only H-bond donors and acceptors exist with the capping methyl groups. The former interaction energies were always significantly weaker than the latter energies, when the same geometries of the H-bond donors and acceptors were applied. The chemical origin of this phenomenon was investigated by analyzing the differences among the electronic structures of the local peptide backbones of the α-helices and those of the minimal H-bond models. Consequently, we found that the reduced H-bond energy originated from the depolarizations of both the H-bond donor and acceptor groups, due to the repulsive interactions with the neighboring polar peptide groups in the α-helix backbone. The classical force fields provide similar H-bond energies to those in the minimal H-bond models, which ignore the current depolarization effect, and thus they overestimate the actual H-bond energies in α-helices. © 2019 The Authors. Journal of Computational Chemistry published by Wiley Periodicals, Inc.

Molecular Mechanism of Depolarization-Dependent Inactivation in W366F Mutant of Kv1.2.

Voltage-gated potassium channels play crucial roles in regulating membrane potential. They are activated by membrane depolarization, allowing the selective permeation of K+ ions across the plasma membrane, and enter a nonconducting state after lasting depolarization, a process known as inactivation. Inactivation in voltage-activated potassium channels occurs through two distinct mechanisms, N-type and C-type inactivation. C-type inactivation is caused by conformational changes in the extracellular mouth of the channel, whereas N-type inactivation is elicited by changes in the cytoplasmic mouth of the protein. The W434F-mutated Shaker channel is known as a nonconducting mutant and is in a C-type inactivation state at a depolarizing membrane potential. To clarify the structural properties of C-type inactivated protein, we performed molecular dynamics simulations of the wild-type and W366F (corresponding to W434F in Shaker) mutant of the Kv1.2-2.1 chimera channel. The W366F mutant was in a nearly nonconducting state with a depolarizing voltage and recovered from inactivation with a reverse voltage. Our simulations and three-dimensional reference interaction site model analysis suggested that structural changes in the selectivity filter upon membrane depolarization trap K+ ions around the inner mouth of the selectivity filter and prevent ion permeation. This pore restriction is involved in the molecular mechanism of C-type inactivation.

Zn2+-Binding to the Voltage-Gated Proton Channel Hv1/VSOP.

The voltage-gated proton channel (Hv1/VSOP) is inhibited by Zn2+, of which the binding site is located in the extracellular region. We utilized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the coordination structure by monitoring protein structural changes induced by Zn2+-binding. The Zn2+-induced difference ATR-FTIR spectra of Hv1 showed IR features that can be assigned to the histidine C5-N1 and carboxylate-COO- stretches as well as amide I changes likely in α-helical peptide bonds. Analysis of vibrational frequencies indicated that the Zn2+ is coordinated by the anionic carboxylate with monodentate mode and by the histidine at N1 (Nτ) position of the neutral imidazole form. Combined with quantum chemical calculations, the most probable coordination structure was proposed as a tetrahedral geometry with ligands of carboxylate and imidazole groups in addition to a water molecule.

The evolutionary process of mammalian sex determination genes focusing on marsupial SRYs.

BACKGROUND: Maleness in mammals is genetically determined by the Y chromosome. On the Y chromosome SRY is known as the mammalian male-determining gene. Both placental mammals (Eutheria) and marsupial mammals (Metatheria) have SRY genes. However, only eutherian SRY genes have been empirically examined by functional analyses, and the involvement of marsupial SRY in male gonad development remains speculative. RESULTS: In order to demonstrate that the marsupial SRY gene is similar to the eutherian SRY gene in function, we first examined the sequence differences between marsupial and eutherian SRY genes. Then, using a parsimony method, we identify 7 marsupial-specific ancestral substitutions, 13 eutherian-specific ancestral substitutions, and 4 substitutions that occurred at the stem lineage of therian SRY genes. A literature search and molecular dynamics computational simulations support that the lineage-specific ancestral substitutions might be involved with the functional differentiation between marsupial and eutherian SRY genes. To address the function of the marsupial SRY gene in male determination, we performed luciferase assays on the testis enhancer of Sox9 core (TESCO) using the marsupial SRY. The functional assay shows that marsupial SRY gene can weakly up-regulate the luciferase expression via TESCO. CONCLUSIONS: Despite the sequence differences between the marsupial and eutherian SRY genes, our functional assay indicates that the marsupial SRY gene regulates SOX9 as a transcription factor in a similar way to the eutherian SRY gene. Our results suggest that SRY genes obtained the function of male determination in the common ancestor of Theria (placental mammals and marsupials). This suggests that the marsupial SRY gene has a function in male determination, but additional experiments are needed to be conclusive.

Maternal Active Mastication during Prenatal Stress Ameliorates Prenatal Stress-Induced Lower Bone Mass in Adult Mouse Offspring.

Chronic psychological stress is a risk factor for osteoporosis. Maternal active mastication during prenatal stress attenuates stress response. The aim of this study is to test the hypothesis that maternal active mastication influences the effect of prenatal stress on bone mass and bone microstructure in adult offspring. Pregnant ddY mice were randomly divided into control, stress, and stress/chewing groups. Mice in the stress and stress/chewing groups were placed in a ventilated restraint tube for 45 minutes, 3 times a day, and was initiated on day 12 of gestation and continued until delivery. Mice in the stress/chewing group were allowed to chew a wooden stick during the restraint stress period. The bone response of 5-month-old male offspring was evaluated using quantitative micro-CT, bone histomorphometry, and biochemical markers. Prenatal stress resulted in significant decrease of trabecular bone mass in both vertebra and distal femur of the offspring. Maternal active mastication during prenatal stress attenuated the reduced bone formation and increased bone resorption, improved the lower trabecular bone volume and bone microstructural deterioration induced by prenatal stress in the offspring. These findings indicate that maternal active mastication during prenatal stress can ameliorate prenatal stress-induced lower bone mass of the vertebra and femur in adult offspring. Active mastication during prenatal stress in dams could be an effective coping strategy to prevent lower bone mass in their offspring.