Background: The aims of this study were to investigate the prevalence of allergic rhinitis (AR) and the accuracy of the International Study of Asthma and Allergies in Childhood (ISAAC) questions for diagnosis of AR, in Korean children. Methods: Students that participated in an allergic disease prevalence survey in 2010–2017 were evaluated  using questionnaires and a skin prick test (SPT). Age−stratified  prevalence of four rhinitis questions, accuracy of the questions for AR, and proportion of comorbidities in the AR and non-AR (NAR) groups were evaluated. Results: The proportion of students responding to the questionnaire that ever had symptoms of AR since birth, that is, the prevalence of “symptom, ever” was 47.6%. Based on the questionnaire and SPT, overall prevalence of AR and NAR were 21% and 26.5%, respectively. The sensitivity, specificity, and accuracy of “symptom, ever” were 57.5%, 58.4%, and 58.1%, respectively, and those of “diagnosis, ever”, who had ever been diagnosed with AR, were 39.8%, 76.9%, and 63.4%, respectively. Questionnaire−based asthma, atopic dermatitis, and food allergy were significantly associated with the AR group compared to the NAR group. Conclusions: Since the AR accuracy of the questionnaire is about 60%, it should be considered that the questionnaire based survey overestimates the true prevalence of AR in Korean children. The term rhinitis indicates nasal dysfunction that causes nasal itching, sneezing, rhinorrhea, or nasal blockage. Allergic rhinitis (AR) is defined as IgE-mediated nasal inflammation confirmed by sensitization to inhaled allergens, and non−allergic rhinitis (NAR) is defined by rhinitis but an allergic and an infectious etiology are excluded .The prevalence of AR differs from region to region, due to differences in living environments, and the different allergens involved. The International Study of Asthma and Allergies of Childhood (ISAAC) questionnaires are a widely accepted standardized tool for comparing interregional differences in asthma and allergic disease prevalence . The preThus, the aims of this study were to investigate the prevalence of AR and NAR and determine the accuracy of the ISAAC questions on AR in a general population of Korean children and adolescents based on the symptoms and skin prick test (SPT) results.The subjects included were an unselected general population of 18,425 children and adolescents living in various areas, namely metropolitan , middle inland (Chungbuk), islands (Gyeonggi Bay archipelago), southern inland (Gwangju), and seaside (Busan) in the Republic of Korea from 2010 to 2017.The AR was defined as having symptoms of rhinitis with one or more positive results of SPT, whereas NAR was defined as rhinitis symptoms with negative SPT results. From the questionnaire, basic personal demographic information was obtained. Rhinitis symptoms were identified through the Korean version of the ISAAC core questions on rhinitis: “Has your child ever had a problem with sneezing or a runny or a blocked nose when he/she did not have a cold or the flu?” , “In the past 12 months, has your child had a problem with sneezing or a runny or a blocked nose when he/she did not have a cold or the flu?” , “Has your child ever been diagnosed as AR?” , and “In the past 12 months, has your child underwent or been treated for AR?”  . WrittenDermatophagoides pteronyssinus, Dermatophagoides farinae), pollen , mold , and pet . The allergens were manufactured by Allergopharma .All participants were tested with 18 inhalant allergens. The allergens included house-dust mites  was defined as the proportion of children with a positive SPT among those acknowledging that they have symptoms in the questionnaire. Negative predictive value (NPV) was defined as the proportion of children whose questionnaire results were concordant with a negative SPT among those with negative questionnaire responses. The accuracy was calculated as follows:2 test. Association between other allergic diseases and comparisons between the AR and NAR groups were further evaluated by logistic regression models with an adjusted odds ratio (aOR) and a 95% confidence interval (95% CI). All of the statistical data were calculated by using SAS version 9.4 .Symptom prevalence rates were analyzed for all participants and stratified by age−group and sex. Differences between categorical variables were assessed by the Pearson χThe institutional review board of Inha University Hospital approved this study .p < 0.001 with significant difference between two compared groups) with a significant increase with age. The total prevalence of NAR was 26.5%  (p < 0.001).The flowchart of the study participants is illustrated in = 0.045) . Boys haThe diagnostic accuracy of question on rhinitis “symptom, ever” was as follows: sensitivity of 57.5%, specificity of 58.4%, PPV of 44.2%, NPV of 70.6%, and accuracy of 58.1%. The questionnaire validity was compared among the different age groups, the highest sensitivity (63.2%) and NPV (82.0%), and the lowest PPV (30.6%) and accuracy (56.1%) were reported in the 5−7 years age group. The specificity, PPV, and accuracy tended to increase with age. The accuracy of “treatment, current” was the highest (63.8%), followed by “diagnosis, ever” (63.4%), “symptom, current” (59.2%), and “symptom, ever” (58.1%) .The ISAAC core questions for rhinitis were incorporated into our questionnaire as they represent a widely accepted standardized tool for the assessment of the prevalence of rhinitis in children and adolescents . Howeverp < 0.001) and age (p < 0.001). The AR prevalence increased with age from 15.4% among those aged 5−7 years to 23% in the 14−16 years age group. Conversely, the prevalence of NAR in children was highest in those aged 5− years (34.9%), and decreased with age. We found that NAR was twice as common as AR in those aged 5−7 years (NAR 34.9% vs. AR 15.4%), which was different from those aged 14−16 years (NAR 23.4% vs. AR 23.0%).There are a lack of epidemiological studies on the prevalence of NAR, probably because of an inconsistent definition and a lack of allergy skin testing. To our knowledge, this is the first population−based study on the prevalence of AR and NAR in children from various geographical locations in Korea. We found that the prevalence of AR and NAR was 21.0% and 26.5%, respectively, quite different from those in rhinitis patients who visited hospitals (AR 68.3% vs. NAR 14.5%) . The preIn the present study, we were able to evaluate the validity of the ISAAC questionnaire for rhinitis with greater accuracy since it was performed in the general population, comprising many individuals from different regions. The question on the rhinitis “symptom, ever” in the ISAAC questionnaire was found to have moderate sensitivity and specificity  with a high NPV (70.6%), low PPV (44.2%), and moderate accuracy (58.1%). The specificity and PPV were observed to increase with age, indicating that only 30% of 5 to 7−year−old children with “symptom, ever” and 50% of 11 to 16−year−old children are AR. Our results are different from those of a Swiss study on the validation of a rhinitis questionnaire , which showed lower sensitivity and higher specificity compared to those in this study with Korean children . Thus, tOur findings demonstrate that the ISAAC questionnaire is moderately useful in detecting AR in the general population of children and adolescents, and highlights that further diagnostic tools including SPT are needed to confirm diagnoses, especially in younger children. This study also showed that the validity of a questionnaire might vary with age, indicating that the expected validity cannot be simply generalized for the whole pediatric age group.We examined the prevalence of comorbid asthma, atopic dermatitis, food allergy, and sinusitis in the AR and NAR groups. We found that AR was more likely to be associated with asthma, atopic dermatitis, and food allergy, however, sinusitis was equally associated with both groups. In a Swedish study, comorbid allergic diseases associated in the AR versus the NAR groups  were asthma , eczema , and food hypersensitivity  [This study demonstrates the strong association between the AR and atopic dermatitis in various pediatric age groups. The concept of the “atopic march” was developed to describe the progression of allergic disorders from atopic dermatitis to allergic rhinitis and asthma in children. This progression depends on various factors, such as the presence of filaggrin mutations and the time of onset, severity, and control of atopic dermatitis, which were found to be related to a higher risk for the development of AR . Cases oThis study has some limitations. Due to the nature of epidemiological studies, anatomical deformities such as adenoid vegetation, or infectious causes such as chronic rhinosinusitis could not be excluded from the NAR group. Thus, the true prevalence of NAR may be overestimated. In addition, there should be mixed allergic and nonallergic rhinitis, which we were unable to identify in this study.In summary, the prevalence of questionnaire−based AR was 47.6%. Utilizing both questions and SPT results, the prevalence of AR and NAR were 21% and 26.5%, respectively. Considering that the AR accuracy is about 60% based on the ISAAC questionnaire, further diagnostic tools including SPT should be considered to confirm diagnosis, especially in younger children.This nationwide study showed prevalence rate of both AR and NAR among a general population of children aged 5−16 years by combining a modified questionnaire with SPT. Considering that the diagnostic accuracy based on the questionnaire alone was approximately 60%, the questionnaire survey therefore, overestimated the prevalence of AR in Korean children and adolescents. We hope that these findings extend our perspectives on the epidemiologic study in Korea.


In this study, allergic rhinitis (AR) disease targets and Xiang Ju tablet-associated targets were determined through the use of databases for the identification of putative therapeutic targets and then combined. After the production of a putative therapeutic target interaction network for Xiang Ju tablets against AR, topological analysis was used to determine the core targets of Xiang Ju tablets in AR treatment. For all putative therapeutic targets, analyses of biological function and pathway enrichment were performed to optimize the biological processes and key signaling pathways of Xiang Ju tablets in AR treatment. The top 5 therapeutic targets of Xiang Ju tablets in AR treatment were identified and included CXCL8, IL1B, IL6, IL10, and TNF. The biological processes, molecular functions, and cell composition related to the use of Xiang Ju tablets in AR treatment were predominantly associated with cytokine production, regulation of protein secretion, and regulation of peptide secretion; cytokine activity, cytokine receptor binding, and receptor ligand activity; and platelet alpha granule lumen, collagen-containing extracellular matrix, and platelet alpha granule. In addition, the top 64 key signaling pathways were identified. Allergic rhinitis (AR) is a very common disease that is most prevalent in children and adolescents. AR is spreading rapidly worldwide and affects approximately 40% of the population , 2. AR iPlatycarya strobilacea Sieb. et Zucc (with seeds removed), Prunellae Spica, Chrysanthemi Indici Flos, Astragali radix, Magnoliae Flos, Saposhnikoviae Radix, Angelicae dahuricae radix, Glycyrrhizae radix et rhizoma, and Chuanxiong Rhizoma. The Xiang Ju tablets have a Xinsan Qufeng, Qingre Tongqiao effect, on the treatment of acute and chronic sinusitis rhinitis [TCM has been widely used for the prevention and treatment of various diseases for thousands of years, mainly in China and the surrounding areas. TCM has increasingly attracted worldwide attention from clinicians and researchers, owing to its demonstrated efficacy, broad applications, and less side effects . In accorhinitis . Studiesrhinitis , 9. In cNetwork pharmacology, proposed by Hopkins in 2007, is used to elucidate drugs' effects on multiple targets . It can In this study, network pharmacology was used to establish a herb-compound-target-pathway network to explore the mechanism of Xiang Ju tablets in treating AR. The flowchart of the experimental procedures of our study is shown in http://lsp.nwu.edu.cn/tcmsp.php), a unique system pharmacology platform designed for Chinese herbal medicines and containing information on their absorption, distribution, metabolism, and excretion (ADME) characteristics, targets, related diseases, and pathways [To build a database of the compounds in Xiang Ju tablets, we used the Traditional Chinese Medicine Systems Pharmacology Database , 20 , drug likeness (DL), and Caco-2 permeability (Caco-2), the three most important indicators for bioinformatic evaluation of ADME characteristics. Specifically, the ingredients meeting the criteria of OB ≥ 30%, DL ≥ 0.18, and Caco-2 ≥ 0.4 were chosen as candidate compounds for further analysis , 23.OB, the rate and percentage of an oral dose of a drug that is absorbed into the blood circulation and produces pharmacological effects, is one of the most important pharmacokinetic characteristics of oral drugs. It reflects the effectiveness of oral drugs in entering human circulation, which is particularly important in drug discovery of TCM for most oral Chinese herb formulas . In thisIn the early stages of drug development, the evaluation of DL aids in screening excellent compounds . DL is aCaco-2 cell monolayers are widely applied as a standard permeability-screening assay for predicting a compound's intestinal absorption and the fraction of the oral dose absorbed in humans . The Cachttp://www.uniprot.org/) containing the predicted targets of candidate compounds in Xiang Ju tablets.According to TCMSP, the effective compounds in Xiang Ju tablets were matched with potential targets one by one, and the target species was selected as “humans” through the UniProt database (https://db.idrblab.org/ttd/), DrugBank v5.1.2 (https://www.drugbank.ca), and DisGeNET v6.0 [http://www.disgenet.org) database retrieval to obtain targets related to AR and applied the UniProt database (https://www.uniprot.org) to disease targets for standardization. We determined the UniProt accession numbers and gene symbols.We used Therapeutic Targets Database v4.3.2  (https:/NET v6.0  (http://http://bioinfogp.cnb.csic.es/tools/venny/index.html) software to obtain the target intersection. The selected common target proteins of diseases and drug components were used to construct the protein-protein interaction (PPI) network model on the STRING v11.0 [https://string-db.org/) platform. We set the protein type to “Homo sapiens” and left the default settings in place for other parameters. We then exported the “string_interactions.tsv” file and imported it into Cytoscape 3.7.0 [In order to clarify the interaction between AR-related targets and potential targets of Xiang Ju tablets, we intersected the disease targets and drug targets and input the disease target and drug component targets into Venny 2.1  is high, and the number of edges (the number of gene nodes that they are associated with) of each node is quite large . These results demonstrate that these genes are closely related to other genes in the network and consequently may play an important role in AR. IL8 (CXCL8) attracts and activates polymorphonuclear cells in sinonasal mucosa, and it can induce granulocyte recruitment . SeveralThe Merge function of Cytoscape 3.7.0 software was used to combine the candidate compound-candidate target network and the putative therapeutic target interaction network, and 48 compounds that play a role in treating AR in the composite were obtained . The visXanthii fructus contribute to the main anti-inflammatory effects. According to spectral correlation theory, the relationship between the chemical composition of Xanthii fructus and its effect on AR has been described by using the co-correlation coefficient, and apicin has been confirmed to be the main active compound of Magnoliae Flos [In previous studies, quercetin has been found to inhibit mast cell activation, inflammatory cytokine production, and histamine release after immunological stimulation. Quercetin may suppress AR in rats by increasing Cl transport and ciliary beat frequency . Quercetiae Flos .Data analysis of the network topology showed that the maximum degree of freedom and the mean degree of freedom were 25 and 14.75, respectively. The network of the top 16 core therapeutic targets was constructed, and the graphic visualization was performed according to the degree of node freedom and the edge thickness . The degTo further clarify the biological effects involved in the treatment of AR with Xiang Ju tablets, we performed GO analysis of AR-related putative therapeutic targets. GO annotation and enrichment of genes encoding Xiang Ju tablet protein targets were conducted from three aspects: biological process (BP), molecular function (MF), and cell composition (CC). We identified 937 enrichment results in the related items of biological process, which included positive regulation of cytokine production, regulation of protein secretion, regulation of peptide secretion, and other biological processes. Eighteen enrichment results were related to molecular function, including cytokine activity, cytokine receptor binding, receptor ligand activity, and other molecular functions; three enrichment processes were related to cell composition, including platelet alpha granule lumen, collagen-containing extracellular matrix, and platelet alpha granule. We selected the 18 items of molecular function and the first 20 items of BP and then performed GO enrichment analysis in RStudio software Figures  and 7.p value <0.05 were considered significant. A total of 64 KEGG signaling pathways were obtained through pathway enrichment analysis, 57 of which were significantly enriched. In the bubble diagram  database. We input a gene list of major AR-related putative therapeutic targets into RStudio, generating relevant pathways that might have an important influence on the biological process of Xiang Ju tablets in treating AR. Only pathways with a  diagram , the colβ, and NF-κB. AGE-RAGE has been found to promote the occurrence and development of various diseases, owing to its influence on many downstream genes as well as signaling pathways [The inflammation response plays an important role in host survival and also leads to acute and chronic inflammatory diseases such as rheumatoid arthritis, bowel diseases, AR, asthma, atopic dermatitis, and various neurodegenerative diseases . IL17, apathways .In order to more directly understand the mechanism of Xiang Ju tablets in treating AR, we used the Cytoscape 3.7.0 Merge function to combine the “presumed therapeutic active ingredient-assumed therapeutic target” network, the presumed therapeutic target interaction network, and the involved KEGG pathways. The results showed that the interactions among active components, targets, and related pathways of Xiang Ju tablets played a therapeutic role .TCM compounds have multiple Chinese medicinal materials and ingredients and can treat diseases through multiple targets, channels, and links . Fully cTCM has a long history in the treatment of sinusitis and has achieved good results. The Xiang Ju tablets produced by Shaanxi Xiangju Pharmaceutical Group Co., Ltd. are well known in the field of sinusitis treatment and have a considerable market share in China. Dong Qin brand Xiang Ju tablets produced by Shaanxi Xiangju Pharmaceutical Group Co., Ltd. are one of the only protected varieties of TCM. In this study, many methods based on network pharmacology were used to predict the targets, determine putative therapeutic targets, and build the network. This approach, combined with target prediction, was used to clarify the molecular synergistic effects of Xiang Ju tablets on AR. This approach provides clues to explore ethnopharmacology and/or herbal or even multidrug synergies. The potential pharmacological and molecular mechanisms of Xiang Ju tablets in treating AR were revealed through analysis of the putative therapeutic target network and biological functions.In this study, network pharmacology was used to preliminarily screen and analyze the active component targets and mechanisms of Xiang Ju tablets, thus providing a basis for subsequent studies on the pharmacodynamic components and mechanisms of Xiang Ju tablets. However, this study has some shortcomings. First, owing to the limitation of screening conditions, only the main compounds in Xiang Ju tablets could be analyzed. Second, although a large number of targets and pathways can be screened through network pharmacology technology, these results must be verified by pharmacological experiments. Active compound of Xiang Ju tablets and the molecular mechanisms of its action on AR were predicted as a whole, and multiple potential therapeutic targets were explored, thus laying a good theoretical foundation for further experimental verification and providing directions for further investigations of its molecular mechanism.


Most of our medical physicists in training are longing for the day when they receive notification that they have passed the American Board of Radiology Certification examination in one of the subfields of medical physics practice. This Certificate gives the holder the needed qualification to apply for positions requiring such a certification and designates the candidate as a Qualified Medical Physicist (QMP). Given its significant importance in our industry, it is desirable periodically to review the ABR Board Certification structure to assure it is meeting the needs of the medical physics community and the patients served by that community.One drawback of the current structure is that the medical physics trainee is required to select a specialty area very early in their career as a medical physicist. It is difficult enough to obtain and maintain certification in one specialty; maintaining certification in two or three areas is both very onerous and rare. However, if a student wishes to preserve the option of changing their specialty practice or of practicing more than one specialty, there seems little option but to plan for this early in their career.I am noticing that a great many articles submitted to the JACMP are broadly conceived. Many radiation oncology physics papers contain substantial imaging science and clinical practice. Many imaging articles contain radiation oncology imaging and clinical concepts. There are Associate Editors in the JACMP who handle both radiation oncology and imaging manuscripts. Radiation oncology physicists must support the imaging equipment in the department in many if not most institutions, so is the strong division of practice between radiation oncology and imaging at least somewhat artificial?A review of physician ABR certifications and subspecialties within radiology reveals a well‐thought listing of professional certifications with specific additional competencies in subspecialty divisions. Most of these subspecialty competencies require an additional year of training within the subspecialty. There is no counterpart to this respecting medical physics clinical training and certifications.https://www.theabr.org/about/certificate-history):Consider these legacy certifications awarded by the ABR  to be eligible for Maintenance of Certification.Radiologic Physics , 1947–1997X‐Ray and Radium Physics, 1947–1960Roentgen Ray & Gamma Ray Physics, 1961–1975Therapeutic and Diagnostic Medical Physics, 1973–1997Diagnostic and Nuclear Medical Physics, 1976‐1998Therapeutic and Nuclear Medical Physics, 1976–1992Radiologic Physics was certified by the ABR for 50 years, and all of these certificates are still active. Additionally, one could get certified individually in two or three areas, if desired, in order to exhibit certification in each of their practice areas. However, given the difficulty of maintaining multiple certifications, some institutions hire a radiation oncology medical physicist to manage all of their imaging equipment (radiation oncology and radiology) on a full‐time basis, thus effectively having the physicist practicing imaging physics under a radiation oncology physics certificate.All of these factors lead me to make the following observations and offer a suggestion: Many medical physicists practice broadly, and often without specialty certification in some components of their practice. This is often overlooked. It is possible we have made a collective error in judgment when as a community we engaged in a headlong rush into specialization a generation ago? Would we have been better off continuing to examine for primary certification only in Medical Physics? Could we have added subspecialties in radiation oncology physics, imaging physics and nuclear medical physics? Would we have the flexibility to add (and perhaps remove at some point) more subspecialties presently and in the future? Would initial certification as a medical physicist and maintenance of certification be of better service to the community and to our patients if it were conceptually broader and not limited to narrowly conceived specialty questions?Conflated with all of these facts is the overall evolution of our service parameters. New and large subgroups of practice have emerged and are emerging, such as brachytherapy physics, radiosurgery physics, radiation oncology particle physics, mammography physics, MRI physics and others. New technologies such as machine learning, radiomics, artificial intelligence, remote provision of services, and other workforce issues further complicate the medical physics specialty landscape.I further submit that our leadership in the AAPM, CAMPEP and the ABR should seriously think about what is best for the profession and ask ourselves whether we have the structure that best serves the interests of our membership and the patients they serve. (Radiologic) Medical Physics was offered by the ABR as a physics certification as late as 1997. Maybe it should have never gone away.In full disclosure, my own career included ABR certification in all three areas, not as a Radiologic Physicist, but rather I took each of the three certifications individually. I believe this broad practice training and certification experience helped me greatly in my career, and I wish this type of pathway were more accessible for physicists today. While I elected not to participate in Maintenance of Certification with the ABR, I do maintain ABMP certification in Radiation Oncology Physics.


Their properties, such as solubilities, reduced viscosities, and thermal properties, were compared with one another. Similar comparisons were also carried out among the corresponding methoxy group polymers, and the size of the molecules was shown to affect the properties.Poly(ether ether ketone) dendrimers and hyperbranched polymers were prepared from 3,5-dimethoxy-4′-(4-fluorobenzoyl)diphenyl ether and 3,5-dihydroxy-4′-(4-fluorobenzoyl)diphenyl ether through aromatic nucleophilic substitution reactions. 1-( Th. ThHyper solvent . The wattandards . With th2, followed by cleavage of the protective group. Since we could not synthesized one such hydroxyl group protected compound, 3,5-bis(4-fluorobenzoyl)phenol (3) was used for the synthesis of the linear type polymer, which was similar to the linear type poly(ether ether ketone) from 2.Linear poly(ether ketone)s having hydroxyl groups in the side chains can be synthesized by polycondensation of an AB-type monomer derived by protecting only one hydroxyl group in tert-Butyldimethylsilyloxy)-3,5-bis(4-fluorobenzoyl)benzene (4), derived from 3 [4 with the bisphenols was carried out in the presence of K2CO3 at 120–165 °C using a mixture of DMAc and toluene as the solvent [Linear-OH-BisA and Linear-OH-HQ) having hydroxyl groups in the side chains. The formations of Linear-OHs were confirmed by appearance of adsorption bands at around 1150 cm−1 characteristic due to ether group and 3600–3200 cm−1 due to hydroxyl group in the IR spectra.1-anisole (5), the methyl protected compound of 3, for linear poly(ether ketone)s as in the case of the dendrimers. However, while poly(ether ketone) dendrons could be synthesized from 5 [Linear-OH-BisA and Linear-OH-HQ. The yields of Linear-OH-BisA and Linear-OH-HQ were 87% and 85%, respectively. GPC analysis of Linear-OH-BisA gave Mn of 11000 and Mw of 23000, and that of Linear-OH-HQ gave Mn of 8200 and Mw of 18000 (Hyper-OH. Molecular weight distribution of Hyperbranched polymer is thought to be higher than that linear polymer due to distribution of DB as well as molecular weight. Linear poly(ether ketone)s (Linear-OMe-BisA and Linear-OMe-HQ) having methoxyl groups in the side chains were prepared by polycondensation of 5 and the bisphenols.In the  spectra  measuredof 18000 . Their mDenG3-OMe, Hyper-OMe, Linear-OMe-BisA, and Linear-OMe-HQ were also compared to evaluate effect of terminal group.The solubilities, solution viscosities, and thermal properties of poly(ether ether ketone) dendrimers, hyperbranched poly(ether ether ketone)s, and linear poly(ether ketone)s having hydroxyl groups in the side chains were compared, and the effect of the size of the molecules on the properties was evaluated. Those of DenG3-OMe, Hyper-OMe, Linear-OMe-BisA and Linear-OMe-HQ. They were all soluble in NMP, DMAc, and tetrahydrofuran at room temperature. DenG3-OMe and Hyper-OMe were also soluble in pyridine and non-polar solvents, such as chloroform and toluene, showed broad solubility, but Linear-OMe-BisA and Linear-OMe-HQ were insoluble in pyridine and toluene. DenG3-OH, Hyper-OH, Linear-OH-BisA and Linear-OH-HQ. They were soluble in pyridine as well as NMP, DMAc, and tetrahydrofuran, and the functional groups in the termini or side chains were reflected in the solubility.DenG3-OH was soluble in 1 M aqueous NaOH solution, Hyper-OH was soluble upon heating, and Linear-OH-BisA and Linear-OH-HQ were insoluble even with heating. DenG3-OH was also soluble in methanol. The effect of the size of the molecules on the solubility was observed in alkaline aqueous solution. DenG3-OMe, Hyper-OMe, Linear-OMe-BisA, and Linear-OMe-HQ and the concentration in DMAc. The reduced viscosities of DenG3-OMe, Hyper-OMe, Linear-OMe-BisA, and Linear-OMe-HQ at the concentration of 1.0 g/dL were 0.11, 0.15, 0.43, and 0.37, respectively. The reduced viscosities of DenG3-OMe and Hyper-OMe were independent of the concentration, and those of Linear-OMe-BisA and Linear-OMe-HQ increased slightly with concentration. DenG3-OH, Hyper-OH, Linear-OH-bisA and Linear-OH-HQ and the concentration in DMAc. The reduced viscosities of DenG3-OH, Hyper-OH, Linear-OH-BisA, and Linear-OH-HQ at the concentration of 1.0 g/dL were 0.11, 0.17, 0.46, and 0.39, respectively, and the GPC results , and the surface area of the hyperbranched molecule was larger. The interaction between the hyperbranched molecules was thought to be larger due to the larger surface area. Tg of hyperbranched molecules is thought to be higher than linear polymers due to both branching and the interaction among molecules.Thermal properties were evaluated by differential scanning calorimetry (DSC). DenG3-OH, Hyper-OH, Linear-OH-BisA, and Linear-OH-HQ. DenG3-OH and Hyper-OH also seemed to be non-crystalline, and Tgs were observed at 144 and 190 °C, respectively. Linear-OH-BisA exhibited Tg at 145 °C followed by exothermic crystallization at 250–305 °C, and Linear-OH-HQ exhibited Tg at 155 °C followed by exothermic crystallization at 280–340 °C. Linear-OH-BisA exhibited an endothermic melting peak at 305–320 °C. Tgs of Hyper-OMe and Hyper-OH were also much higher than the corresponding dendrimers and linear polymers, and in contrast to the polyesters having hydroxyl groups [Tgs of hyperbranched and dendritic polyesters having hydroxyl groups in the chain ends were almost the same as the Tg of the linear polyester having hydroxyl groups in the side chains. DenG3-OH and Hyper-OH did not crystallize, but Linear-OH-BisA and Linear-OH-HQ crystallized. Dendritic polymers could not be crystallized due to their branching structures, and attachment of crystalline molecules to the chain ends as a molecular scaffold was reported to be necessary for the crystallization of hyperbranched polymers [Linear-OH-BisA and Linear-OH-HQ without branching were flexible, the crystallizations were thought to be induced due to hydrogen bonding by the hydroxyl groups.polymers ,24. Sinc1H- and 13C- NMR spectra were recorded on a JNM-GSX400 FT-NMR spectrometer  and IR spectra were recorded on a IR 435 spectrophotometer . GPC was carried out on a PL gel 5μ MIXED-C analytical column  with tetrahydrofuran as the eluent. Reduced viscosity was measured at various concentrations using an Ubbelohde-type capillary viscometer  in DMAc at 30 °C. MALDI-TOF MS were measured on a Shimadzu/Kratos Kompact MALDI II (Shimadzu) equipped with a 337 nm nitrogen laser. DSC was performed with a Shimadzu DSC-60 (Shimadzu), instrument and measurements were made at a heating rate of 10 °C min−1 in nitrogen. The results from first scan were displayed in 1 , DenG0-OH , potassium carbonate , toluene (30 mL), and DMAc (60 mL) was stirred at 130 °C for 1 h. The temperature was raised to 160 °C and water formed during the reaction was removed as an azeotrope with toluene. The reaction mixture was stirred at this temperature for 1.5 h. After the reaction was complete, the mixture was cooled to about 80 °C, and the solvent was evaporated under reduced pressure of 15–20 torr. The residue was washed with water (300 mL) and extracted twice with methylene chloride (200 mL). After the combined extract was dried over anhydrous magnesium sulfate, the solvent was evaporated. Pure DenG1-OMe was obtained by silica gel column chromatography beginning with methylene chloride as the eluent and gradually changing to methylene chloride and ethyl acetate (20:1), yield: 92%.In a flask, a mixture of DenG1-OMe  was heated together with pyridine hydrochloride (50 g) at reflux temperature for about 30 min. After the reaction mixture was homogeneous, it was poured into water (1000 mL), and extracted twice with ethyl acetate (100 mL). The combined extract was dried over anhydrous magnesium sulfate. After evaporation of the solvent, pure DenG1-OH was obtained by silica gel column chromatography by methylene chloride and ethyl acetate (1:1). Yield: 95%.DenG2-OMe was prepared by the same procedure as that for synthesis of DenG1-OMe using DenG1-OH , 1 , potassium carbonate , toluene (23 mL), and DMAc (60 mL). Pure DenG2-OMe was obtained by silica gel column chromatography beginning with methylene chloride and ethyl acetate (25:1) as the eluent and gradually changing to methylene chloride and ethyl acetate (15:1), yield: 83%.DenG2-OH was prepared by the same procedure as that for synthesis of DenG1-OH using DenG2-OMe , and pyridine hydrochloride (60 g). Pure DenG2-OH was obtained by silica gel column by methylene chloride and ethyl acetate (3:7), yield: 91%.DenG3-OMe was prepared by the same procedure as that for synthesis of DenG1-OMe using DenG2-OH , 1 , potassium carbonate , 15 toluene (15 mL), and DMAc (30 mL). Pure DenG3-OMe was obtained by silica gel column chromatography beginning with methylene chloride and ethyl acetate (20:1) as the eluent and gradually changing to methylene chloride and ethyl acetate (13:1). Yield 62%; white powder; Elemental Analysis: C888H630O117; Calculated %C = 75.47, %H = 4.49. Observed. %C = 75.35, %H = 4.41. FT-IR/ATR ν (cm−1): 2940, 1655, 1590, 1225, and 1160 cm−1; TOF-MS: 14133, C888H630O177 (Mw = 14131); 1H-NMR (CDCl3) ppm: 7.83–7.47 , 7.68 , 7.65 , 7.15 , 7.10–7.06 , 7.04 , 6.58 , 6.48 , 6.28 , 6.22 , 3.75 ; 13C-NMR (CDCl3) ppm: 193.9 (C=O), 193.9 (C=O), 193.8 (C=O), 161.9 (Ar), 161.1 (Ar), 160.1 (Ar), 160.1 (Ar), 160.0 (Ar), 159.9 (Ar), 159.9 (Ar), 159.8 (Ar), 158.5 (Ar), 158.5 (Ar), 158.5 (Ar), 158.2 (Ar), 157.5 (Ar), 155.6 (Ar), 141.6 (Ar), 137.2 (Ar), 133.5 (Ar), 133.3 (Ar), 133.2 (Ar), 133.2 (Ar), 133.0 (Ar), 132.3 (Ar), 132.2 (Ar), 132.2 (Ar), 132.1 (Ar), 131.9 (Ar), 128.9 (Ar), 124.7 (Ar), 120.0 (Ar), 118.5 (Ar), 118.2 (Ar), 118.1 (Ar), 117.6 (Ar), 106.3 (Ar), 106.3 (Ar), 106.2 (Ar), 105.5 (Ar), 105.3 (Ar), 98.6 (Ar), 96.8 (Ar), 55.5(-CH3).DenG3-OH was prepared by the same procedure as that used for synthesis of DenG1-OH using DenG3-OMe , and pyridine hydrochloride (80 g). Pure DenG3-OH was obtained by silica gel column by methylene chloride and ethyl acetate (3:7). Yield 87%; white powder; Elemental Analysis: C840H534O117; Calculated %C = 74.96, %H = 4.00. Observed. %C = 75.01, %H = 4.03. FT-IR/ATR ν (cm−1): 3400–3000, 1650, 1590, 1227, and 1162 cm−1; TOF-MS: 13,459, C840H534O117 ; 1H-NMR (DMSO-d6) ppm: 9.02, 7.77–7.65 , 7.20–7.09 , 7.05 , 6.63 , 6.61 , 6.55 , 6.08 , 5.93 ; 13C-NMR (DMSO-d6) ppm: 192.6 (C=O), 192.5 (C=O), 192.5 (C=O), 160.2 (Ar), 159.4 (Ar), 159.1 (Ar), 159.0 (Ar), 159.0 (Ar), 158.9 (Ar), 158.9 (Ar), 158.7 (Ar), 157.7 (Ar), 157.7 (Ar), 157.6 (Ar), 157.5 (Ar), 157.5 (Ar), 155.6 (Ar), 154.9 (Ar), 140.4 (Ar), 135.8 (Ar), 132.7 (Ar), 132.4 (Ar), 132.4 (Ar), 132.0 (Ar), 131.4 (Ar), 131.4 (Ar), 131.2 (Ar), 128.3 (Ar), 124.0 (Ar), 119.2 (Ar), 117.8 (Ar), 117.7 (Ar), 117.2 (Ar), 117.1 (Ar), 105.8 (Ar), 105.7 (Ar), 105.7 (Ar), 105.6 (Ar), 104.7 (Ar), 104.5 (Ar), 98.9 (Ar), 97.7 (Ar).2 , potassium carbonate , toluene (10 mL) and N,N-dimethylacetamide (DMAc) (20 mL) was stirred in a flask at 120 °C. The temperature was then raised from 120 °C to 165 °C, to remove water formed during the reaction as an azeotrope with toluene. The reaction mixture was stirred at this temperature for 6 h. The polymerization proceeded homogeneously. After the reaction was completed, the mixture was cooled to room temperature and poured into 300 mL of methanol. The precipitated polymer was collected by filtration, washed thoroughly with water and methanol, and dried under vacuum. Yield 88%; white powder; Elemental Analysis: C19H12O4; Calculated %C = 74.99, %H = 3.97. Observed. %C = 74.52, %H = 3.64. FT-IR/ATR ν (cm−1): 3400–3000, and 1240 cm−1; 1H-NMR (DMSO-d6) ppm: 7.86–7.58 , 7.30–7.00 , 6.72–6.64 , 6.36–6.31 , 6.31–6.24 , 6.08–6.04 , 5.96–5.90 , 5.93 .A mixture of 2 , potassium carbonate , toluene (10 mL) and DMAc (20 mL) was stirred in a flask at 120 °C. The temperature was then raised from 120 °C to 165 °C to remove water formed during the reaction as an azeotrope with toluene, and the reaction mixture was stirred at this temperature for 6 h. After the reaction mixture was cooled to room temperature, methyl sulfate  and potassium carbonate  were added. The mixture was stirred at 80 °C for another 6 h, cooled to room temperature, and poured into 300 mL of methanol. The precipitated polymer was collected by filtration, washed thoroughly with water and methanol, and dried under vacuum. Yield: 85%; white powder; Elemental Analysis: C20H14O4; Calculated %C = 75.46, %H = 4.43. Observed. %C = 75.05, %H = 4.23. FT-IR/ATR ν (cm−1): 2945, 1650 and 1240 cm−1; 1H-NMR (DMSO-d6) ppm: 7.86–7.58 , 7.30–7.00 , 6.72–6.64 , 6.36-6.31 , 6.31–6.24 , 6.07–6.01 , 6.00–5.93 , 3.81–3.75 .A mixture of tert-butyldimethylchlorosilane  in N,N-dimethylformamide (DMF) (20 mL) was added dropwise to a solution of 3  and imidazole  in DMF (40 mL) at 0 °C. After stirring at 25 °C for 3 h, the precipitated imidazole salt was removed by filtration, and the solvent was evaporated. The residue was distilled under reduced pressure (glass tube oven) to pure 1-(tert-butyldimethylsilyloxy)-3,5-bis(4-fluorobenzoyl)benzene. Yield 72%; transparent oil; Bp: 240 °C (1.0 mmHg); Elemental Analysis: C26H26F2O3Si; Calculated %C = 69.00, %H = 5.79. Observed. %C = 68.72, %H = 5.61. FT-IR/ATR ν (cm−1): 3060, 2930, 1650, 1600, 1230, and 1150 cm−1; TOF-MS: 451, C26H26F2O3Si (Mw = 452.6); 1H-NMR (CDCl3) ppm: 7.88 , 7.65 , 7.54 , 7.17 , 0.96 , 0.09 ; 13C-NMR (CDCl3) ppm: 194.0 (C=O), 160.5  (Ar), 159.5 (Ar), 139.4 (Ar), 133.0  (Ar), 132.5  (Ar), 123.7 (Ar), 118.5 (Ar), 116.0  (Ar), 34.6(-CH3), 32.8(-CH3).A solution of 4 , bisphenol A , potassium carbonate , toluene (10 mL) and DMAc (20 mL) was stirred in a flask at 120 °C. The temperature was then raised from 120 °C to 165 °C, to remove water formed during the reaction as an azeotrope in toluene. The reaction mixture was stirred at this temperature for 6 h. The polymerization proceeded homogeneously. The mixture was cooled to room temperature, and poured into water (300 mL). The precipitated polymer was collected by filtration, and dried under vacuum. Yield: 95%; white powder; FT-IR/ATR ν (cm−1): 3060, 2930, 1650, 1600, 1230, and 1150 cm−1; 1H-NMR (DMSO-d6) ppm: 8.10–7.70 , 7.50–7.30 , 7.30–6.96 , 3.4 , 0.90 , 0.15 . Hydrochloric acid  was added to a solution of the obtained poly(ether ketone) (1.50 g) in DMF (25 mL), and stirred at 80 °C for 3 h. The reaction mixture was poured into methanol (250 mL). The precipitated polymer was collected by filtration and dried under vacuum. Yield 92 %; white powder; Td 360 °C; Elemental Analysis: C35H26O5; Calculated %C = 79.83, %H = 4.98. Observed %C = 79.40, %H = 4.98. FT-IR/ATR ν (cm−1): 3600–3200, 3060, 2930, 1650, 1600, 1230, and 1150; 1H-NMR (DMSO-d6) ppm: 8.20–7.70 , 7.50–7.30 , 7.30–6.90 , 3.35 .A mixture of 5 , bisphenol A , potassium carbonate , toluene (10 mL) and DMAc (20 mL) were stirred in a flask at 120 °C. The temperature was then raised from 120 °C to 165 °C to remove water formed during the reaction as an azeotrope in toluene. The reaction mixture was stirred at this temperature for 6 h. The polymerization proceeded homogeneously. After the reaction was completed, the mixture was cooled to room temperature, and poured into water (300 mL). The precipitated polymer was collected by filtration, and dried under vacuum. Yield 93%; white powder; Td 370 °C; Elemental Analysis: C36H28O5; Calculated %C = 79.98, %H = 5.22. Observed %C = 79.65, %H = 5.05. FT-IR/ATR ν (cm−1): IR(liquid film): 3060, 2930, 1650, 1600, 1230, and 1150 cm−1; 1H-NMR (DMSO-d6) ppm: 8.10–7.70 , 7.50–7.30 , 7.30–6.96 , 3.93 , 3.35 .Compound tert-Butyldimethylsiloxy)-3,5-bis(4-fluorobenzoyl)benzene was polycondensed with bisphenols, followed by cleavage of the protective group to form linear poly(ether ketone)s, and the properties, such as solubilities, reduced viscosities and thermal properties, were compared with those of poly(ether ether ketone) dendrimer having hydroxyl chain ends and hyperbranched poly(ether ether ketone) having hydroxyl chain ends. A similar comparison was carried out with the corresponding methoxy group polymers. The solubilities of the dendrimers and the hyperbranched polymers were higher than the linear polymers. The reduced viscosities in DMAc of the hyperbranched polymers and the linear polymers increased with the concentrations, but those of the linear polymers increased more significantly. The order of increasing Tgs was hyperbranched polymers > linear polymers > dendrimers, and the hydroxyl linear polymers crystallized thermally. The molecular size was shown to affect their properties.1-(


Moreover, capacity remained at 727.3 mAh·g−1 after 100 cycles, showing improved cycling performance. This synthesis strategy can be extended to prepare other Si/C anode materials of LIBs.Silicon/carbon (Si/C) composite has been proven to be an effective method of enhancing the electrochemical performance of Si-based anodes for lithium-ion batteries (LIBs). However, the practical application of Si/C materials in LIBs is difficult because of the weak interaction between Si and C. In this study, we applied two-step diazotization reactions to modify graphite nanosheets (GNs) and Si nanoparticles (Si NPs), yielding a stable Si–Ar–GNs composite. Owing to aryl (Ar) group bonding, Si NPs were dispersed well on the GNs. The as-prepared Si–Ar–GNs composite delivered an initial reversible capacity of 1174.7 mAh·g Lithium-ion batteries (LIBs) are the energy storage devices that drive today’s global portable electronic device market and show great potential for wider applications such as electric vehicles (EVs) because of their high specific energy, long cycle life, lightweight properties, and environmental friendliness ,2,3,4,5.−1, compared with graphite’s 372 mAh·g−1, because it is great insert/de-insert many more lithium ions than the graphite in current LIBs. However, after a few charge-and-discharge cycles, silicon is prone to fracturing and breaking due to its expansion and contraction during de-alloying/alloying processes [Many groups have spent decades working to develop novel anode materials for LIBs ,10,11,12rocesses ,15,16,17rocesses ,19, Si/crocesses ,21, and rocesses ,23, whicsp2 carbon atoms of GNs and Si NPs are weak. It is difficult to deposit Si NPs on the surface of GNs. Diazonium chemistry is an effective method to modify the surface of GNs and improve the binding of GNs and nanoparticles [p-phenylenediamine contains two NH2 groups that can result in two diazotization reactions. Thus, we covalented modified GNs with p-phenylenediamine, forming NH2–Ar–GNs by diazotization reaction. The resulting NH2–Ar–GNs reacted with Si to yield a stable Si–Ar–GNs composite by another step diazotization reaction. Owing to Ar group bonding, the as-prepared Si–Ar–GNs composite anode showed better cycling stability compared with the bare Si anode. Graphite nanosheets (GNs) are a kind of two-dimensional (2D) carbon material. Because of their excellent electrical conductivity, GNs have been widely used as electrical conductive filler ,25. In oarticles . p-phenyThe natural graphite powders (400 mesh) were purchased from Nanjing XFNANO Materials Tech Co., Ltd. . Si NPs (200 nm) were purchased from Sigma-Aldrich . Other chemicals were provided by Sinopharm Chemical Reagent Co., Ltd. .v/v) at 25 °C for one day. The acidized graphites were heated at 1050 °C for 12 s in a muffle furnace to prepare expanded graphite. The expanded graphite was stirred in an 80% aqueous alcohol solution at 25 °C for one day. Then the mixture was sonicated to break expanded graphite into nanoscale sheets. Finally, the resulting dispersion was filtered, washed, and dried in a freezing vacuum dryer to yield GNs. The schematic illustration of the synthesis of GNs is illustrated in The GNs were prepared as follows. The natural graphite powders were added to a mixed acid containing nitric acid and sulfuric acid . The internal morphologies of the Si–Ar–GNs composite and the Si/GNs composite were studied using a transmission electron microscope  and HRTEM.The phase compositions of GNs were analyzed by X-ray powder diffraction  with Cu Kα radiation. Raman spectra were taken by a Reinishaw Raman (RE01) equipped with a 514 nm diode laser. FTIR analyses were conducted using a Bruker Vertex 70 spectrometer from 750 to 4250 cm6 in a mixture of diethyl carbonate (DEC) and ethylene carbonate (EC)  as the electrolyte, and Celgard 2320 membrane as the separator. The galvanostatic charge-discharge performance was tested on a battery test system  from 0.01 to 3.0 V. Cyclic voltammetry (CV) property was tested by an electrochemical station  in the 0.01–2.0 V window at a scan rate of 0.2 mV s−1.Homogeneous slurry was prepared by mixing 80 wt % of Si–Ar–GNs composite, 10 wt % of Super-P carbon black, and 10 wt % of polyacrylic acid (PAA) binder in deionized water. The slurry was then applied to Cu foil as a current collector and dried in a vacuum oven at 100 °C for 24.0 h. The thickness of the electrodes was 70 μm. Coin-type 2025 half-cells were fabricated in an argon-filled glove box  using the coated Cu disc as the working electrode, lithium foil as the counter electrode, 1 M LiPF2SO4–graphite intercalation composite. During the synthesis, huge energy led to H2SO4 decomposition that resulted in a high expansion of graphite. Thus, the expanded graphite formed a loose and porous structure with a high surface area.To investigate the morphology of the expanded graphite, SEM analysis was carried out. 2·g−1, suggesting a high specific surface for Si nanoparticle dispersion. However, the surface carbon atoms of graphite have a weak interaction with particles. It was difficult to deposit Si NPs on the GNs’ surface. −1 and 1575 cm−1 corresponded to the D-band (defect) and G-band, respectively. The G-band originated from graphitic structures (sp2), and the D-band arose from sp3 carbon and was sensitive to carbon impurities. The crystal structure of the GNs was identified using a Raman spectrum as shown in ID/IG), indicated the degree of carbonization [2–Ar–GNs showed peaks at around 3450, 1635, 1489, and 1395 cm−1. The peaks at 3450 and 1635 cm−1 belonged to N–H stretching and bending vibration, while peaks at around 1489 and 1395 cm−1 were assigned to C=C and C–H stretching vibration [2–Ar was grafted onto the GNs’ surface. After reacting with Si NPs, the peaks belonging to phenyl retained while peaks assigned to NH2 disappeared, which revealed that Si NPs were successfully linked to the GNs’ surface via a phenyl group. Moreover, a new weak peak at 3440 cm−1 appeared that was assigned to the stretching mode of the Si–OH bonds. −1 and 930 cm−1 were typical features of the Si vibration mode [The ratio of the integrated area of the D-band to the G-band, R . Moreover, this result was higher than that of other similar Si/C anodes [The electrochemical performance of Si–Ar–GNs is shown in x . The peax . Figure to LixSi . The firC anodes ,40,41. TThe improvement of the capacity retention of the Si–Ar–GNs composite could be ascribed to the following effects. First, owing to Ar group bonding, Si NPs can disperse well on GNs and avoid aggregation during cycling processes. To study this banding effect, the morphologies of the Si–Ar–GNs composite after 100 cycles was examined. SEM images a,b show −1 at a current density of 100 mA·g−1. A capacity of 727.3 mAh·g−1 was obtained at 100 mA·g−1 for 100 cycles, which was higher than that of pure Si (only 45.4 mAh·g−1) and other similar Si/C anodes. The electrochemical performances suggest that the Si–Ar–GNs composite is an ideal anode material for LIBs. Our work provides a simple surface chemistry method to improve the cycling performance of Si/C anode materials.In summary, Si–Ar–GNs composites were successfully prepared via a two-step diazotization reaction. This method made use of the chemical functions of GNs and Si NPs to yield a covalent bond (Ar group) between GNs and Si NPs. Thus, Si NPs were uniformly coated on the GNs’ surface. The covalent bond avoided Si NPs aggregation during cycling processes, and the GNs accommodated the large volume changes of Si NPs, thus improving the cycling stability of the anode. The Si–Ar–GNs composite delivered an initial reversible capacity of 1174.7 mAh·g


Background. Men and women differ in their susceptibility to cardiovascular disease, though the underlying mechanism has remained elusive. Heart disease symptoms, evolution and response to treatment are often sex-specific. This has been studied in animal models of hypertension or myocardial infarction in the past but has received less attention in the context of heart valve regurgitation. The aim of the study was to evaluate the development of cardiac hypertrophy (CH) in response to left ventricle (LV) volume overload (VO) caused by chronic aortic valve regurgitation (AR) in male and female rats treated or not with angiotensin II receptor blocker (ARB), valsartan. We studied eight groups of Wistar rats: male or female, AR or sham-operated (sham) and treated or not with valsartan (30 mg/kg/day) for 9 weeks starting one week before AR surgical induction.Results. As expected, VO from AR resulted for both male and female rats in significant LV dilation  and CH  compared to sham. Sex differences were observed in LV wall thickening in response to VO. In untreated AR males, relative LV wall thickness  was reduced compared to sham, whereas this ratio in females remained unchanged. ARB treatment did not prevent LV dilation in both male and female animals but reversed LV wall thickening in females. Systolic and diastolic functions in AR animals were altered similarly for both sexes. ARB treatment did not improve systolic function but helped normalizing diastolic parameters such as left atrial mass and E wave slope in female AR rats. Increased LV gene expression of Anp and Bnp was normalized by ARB treatment in AR females but not in males. Other hypertrophy gene markers  were not modulated by ARB treatment. The same was true for genes related to LV extracellular matrix remodeling . In summary, ARB treatment of rats with severe AR blocked the female-specific hypertrophic response characterized by LV chamber wall thickening. LV dilation, on the other hand, was not significantly decreased by ARB treatment. This also indicates that activation of the angiotensin II receptor is probably more involved in the early steps of LV remodeling caused by AR in females than in males. Heart diseases are among the leading causes of mortality for both men and women . CardiacLV VO occurs when either the aortic or the mitral valve is regurgitating. Causes for regurgitation are multiple but worldwide, they are most frequently complications of acute rheumatic fever. Rheumatic valve diseases causing aortic regurgitation (AR) are still occurring at an alarming rate in the third-world and low/middle-income countries. In the Western world, they are still prevalent in poor, remote, Native American communities and in immigrants from countries where rheumatic fever is still endemic. The estimated burden worldwide of rheumatic valve diseases is estimated to more than 15 million existing cases with 280k new cases each year and 230k deaths . SecondaLV remodeling in response to significant VO from experimental severe AR in male Wistar rats results in important eccentric hypertrophy (dilation) to accommodate the excess regurgitating aortic blood to pump . We receActivation of the tissue renin-angiotensin-aldosterone system (RAAS) system is a characteristic feature of the myocardial response to a pathological and chronic stress such as a significant valve regurgitation (VO) or a LV pressure overload such as in hypertension or aortic valve stenosis. We previously showed that blocking the RAAS in male AR rats could reduce development of LV hypertrophy (LVH), improve myocardial function and survival . HoweverOur results suggest that angiotensin receptor blockade (ARB) with valsartan during the development of hypertrophy in female rats with severe AR partly abrogates LV wall thickening leading to a more eccentric remodeling similar to the one observed in males.Severe AR was induced in males (300–325 g) and females (200–225 g) Wistar rats (9–10 weeks of age) by retrograde puncture one or two aortic valve leaflets under echocardiographic guidance as previously described . Only anAn echocardiographic exam was performed two weeks after surgery to confirm AR severity (estimation of regurgitation and LV dimensions) and at the end of the protocol 8 week later as previously described . LV massPpia) was the housekeeping gene.Total RNA was extracted using Trizol reagent as described elsewhere . LV RNA t-test was used for comparison between the groups . A Student’s t-test was used when only two groups were compared. A p-value lower than 0.05 was considered significant.Results are presented as the mean and the standard error of the mean (SEM). Statistical analyses were performed on the log of the data. Two-way ANOVA analysis was performed and Holm-Sidak’s pos Treatment with the angiotensin receptor blocker (ARB), valsartan, was initiated a week before surgery and lasted up until the end of the protocol 9 weeks later. Surgery itself, had no effects on body weight gain during the protocol. Valsartan treatment had no significant effects on heart total weight in sham-operated animals, males and females  and 3. TComparing AR rats to sham ones, every parameter measured with the exception of body weight and tibial length were significantly increased as summarized in In As for the animal and heart characteristics described above, most echocardiographic parameters measured were significantly changed by AR  and 5. AIn AR animals, the effects of valsartan were observed on the LV wall thickness  and posterior wall (PW), which were thinner in females compared to untreated AR rats. ARB had no effects in AR males . DiastolAs illustrated in As illustrated in Anp and Bnp mRNA levels were increased in both male and female AR animals , an index of LV remodeling .The classic view of cardiac remodeling induced by a hemodynamic overload is that pressure overload  is associated with initial concentric LV hypertrophy . On the other hand, volume overload  induces chamber dilatation with no or little increase in wall thickness or eccentric remodeling . This isWe observed that LV dilation was similar and relatively unaffected by angiotensin receptor blockade by valsartan both in males and females. Blood regurgitation from the aorta to the LV during diastole is a relatively stable determinant of the disease and cannot be modulated significantly by ARB. On the other hand, LV wall thickening, more important in female AR rats compared to males, was reversed by ARB leading to similar LV morphology between the sexes. Indexed LV mass estimated by echocardiography showed a similar trend where valsartan treatment partly reversed hypertrophy in females but not in males. The method we used makes the geometrical assumption of an elliptical LV. It is possible that the shape of the dilated LV in female AR rats does not obey this assumption.Aortocaval fistula (ACF) is a model of global cardiac volume overload where sex differences have been studied with some details in the past in rats. Although this form of VO is less relevant for a clinical standpoint, it remains the most popular pre-clinical VO model in the literature. In 2002, Gardner and collaborators first reported that female rats with ACV developed less cardiac hypertrophy, evolved less towards heart failure and had better survival than males . OvariecPharmacological interventions for cardiovascular diseases and heart failure are often less prescribed in women. Their absorption, distribution , metabolism and clearance is often different . It is nSex dimorphism in the response to treatment has not been studied before in VO rodent models. The present study design is also different from previous studies we made on male animals using this model . Here, wThe present study also enlightened that development of LV hypertrophy in this model first involves rapid LV dilation to accommodate the increased blood volume during diastole in males. In previous studies in males, we observed either a mild raise in systolic blood pressure or no changes . Then, lAnp and Bnp genes, expression was more elevated in AR rats and valsartan normalized their expression but only in females suggesting that LV wall tension may have been improved by treatment. For three other hypertrophy markers, namely Fos, Trpc6 and Klf15, gene expression was only altered in untreated AR males but not in females as previously reported (fos expression was lowered by ARB both in males and females. As for genes related to extracellular matrix components and metabolism, very few differences between the sexes or by treatment were registered. Volume overload is not associated with important myocardial fibrosis at least in the early steps of the disease (Expression of various LV genes associated with hypertrophy or extracellular matrix remodeling was assessed. In the case of reported . Interes disease . This is disease .Ctgf. In males, benefits of valsartan were only observed for the decrease of Fox and Lox gene expression and an increase for Itgb1. Beta-1 integrin is one subunit of the dimeric fibronectin surface receptor. Its signaling promotes cardiac hypertrophy but also cardiac myocytes survival via Erk and Akt signaling pathways (It is not clear if the effects of angiotensin receptor blockade we observed in AR females provide benefits in the context of a LV VO. On one side, valsartan recreates in AR females an eccentric LV morphology similar to males, which tend to have a worse outcome than untreated AR females after 6 months . On the pathways .We chose to not directly address the influence of sex hormones by castration or ovariectomy in this study. This would have added a level of complexity. We recently observed that loss of testosterone reduced LV hypertrophy in AR males and helped normalize the myocardial transcriptional profile suggesting an important role for this sex hormone in the response of the heart to a pathological stress In conclusion, we showed that female AR rats have a stronger early response to treatment with an angiotensin receptor antagonist, valsartan than males. This response is mainly concentrated on a female-specific feature of the LV remodeling in response to volume overload, LV wall thickening.10.7717/peerj.7461/supp-1Dataset S1Click here for additional data file.


Offspring of mothers with hyperglycemia during pregnancy have a higher incidence of long-term neuropsychiatric disorders than offspring from a normal pregnancy, indicating that neocortical neurogenesis might be affected by maternal hyperglycemia. A paucity of study evaluating the effects of hyperglycemia on neocortical neurogenetic differentiation of neural stem cells, and the mechanism remains unclear. We sought to investigate the the roles and possible molecular mechanism of maternal hyperglycemia on neocortical neurogenetic differentiation of neural stem cells. We established a mouse model of a hyperglycemic pregnancy to study effects of intrauterine exposure to maternal hyperglycemia on neocortical neurogenesis. We observed morphological changes in the neocortex and detected the neurogenetic differentiation of neural stem cells in offspring affected by high glucose levels. We investigated the regulatory network between epigenetic modification and transcription factors in differentiated neural stem cells under hyperglycemic conditions. Maternal hyperglycemia disturbs neocortical lamination in some non-malformed offspring. Our results suggested that hyperglycemia altered the early-born neuron fate and the distribution of newborn neurons in deep layers by promoting the earlier differentiation of neural stem cells. Altered histone acetylation and its regulation on the transcription of proneural genes might be correlated to the disrupted differentiation of neural stem cells and altered distribution of newborn projection neurons in the neocortex. Our data raised the possibility that maternal hyperglycemia in pregnancy disturbs the laminar distribution of neocortical projection neurons in some non-malformed offspring via epigenetic regulation on neural stem cell differentiation and the birthdate of neocortical neurons. The severity of harmful effects on the neurodevelopment depends on the degree of hyperglycemia6. During development, the embryonic nervous system is susceptible to elevated blood glucose levels. Neurulation and corticogenesis are two critical stages during the development of the central nervous system8. Severe hyperglycemia in early pregnancy affects the closure of the embryonic neural tube and subsequently increases the risk of neural tube defects (NTDs)11. Moderate hyperglycemia does not substantially increase the risk of NTDs, and more offspring with a relatively normal phenotype are produced12. Considering that offspring from hyperglycemic pregnancy have higher incidences of long-term neuropsychological disorders and cognitive dysfunction than the offspring from a normal pregnancy14, we speculated that the process of neocortical development might be affected by hyperglycemia in some offspring with a normal phenotype from mothers with moderate hyperglycemia during pregnancy. Our collaborators and we previously focused on the effects of maternal hyperglycemia on embryonic neurodevelopment, primarily the effects of severe hyperglycemia on the incidence of NTDs and the mechanism by which high glucose levels cause NTDs23. However, few studies have examined the effects of moderate hyperglycemia on neurodevelopment in offspring without obvious NTDs. The incidence of gestational diabetes continues to increase, which has an enormous influence on embryonic neurodevelopment24. Fortunately, the incidence of NTDs has decreased to a certain extent, which has benefitted from modified glucose control and the widespread use of folic acid25. However, the long-term neuropsychiatric disorders in the non-malformed offspring affected by maternal hyperglycemia require further study. In this paper, our intent is to discover the mechanism of embryonic neurodevelopment disorders in non-malformed offspring from mothers with hyperglycemia in pregnancy.Hyperglycemia in pregnancy (HIP) has been demonstrated to increases risk of neurodevelopmental disorders in the offspring27. Normal development of the embryonic cerebral cortex plays a decisive role in the future physiological function of the central nervous system29. The dorsal pallium of the telencephalon gives rise to the neocortex30. Neocortical neurogenesis is a well-ordered process of the proliferation and differentiation of neural stem cells (NSCs) from the ventricular zone (VZ) and migration of newborn neurons outward31. Early-born neurons start to migrate radially in an orderly manner at embryonic day 12.5 (E12.5) in mice, forming the deep cortex . Later born neurons begin to move to the superficial layers (layers 2–4) at E14.532. The entire migration process is completed after birth. According to previous studies by Yuan et al. from our group, hyperglycemia in pregnancy increases the expression of the neuronal maker Tuj1 in the E13.5 dorsal telencephalon21. However, no relevant studies have examined how high glucose affects the neurogenetic differentiation and the subsequent birth and laminar distribution of neocortical neurons.The normal laminar structure of the cerebral cortex and the formation of complex neuronal circuits are essential for advanced central nervous system functions37. Several studies on NTDs induced by maternal hyperglycemia have identified variations in epigenetic modifications13, particularly changes in histone acetylation. Our collaborators, Dheen et al., observed decreased acetylation of histone 3 on lysine 9 in proliferating NSCs exposed to severe hyperglycemia38. Yang et al. reported increased acetylation of histone 3 at several sites in embryos from diabetic mice39. However, the influence of moderately high glucose levels on the epigenetic mechanism controlling NSC differentiation during the development of the neocortical layers has not been identified. Among the epigenetic modifications that affect the expression of transcription factors (TFs), histone modifications are particularly complicated. Complex and dynamic regulatory networks between histone modifications and TFs regulate the proliferation and differentiation of NSCs during neocortical neurogenesis33. The orderly transcription of proneural genes guides the differentiation of new neurons in the neocortex45. Histone acetylation plays an important role in the transcription activation of proneural bHLH genes during the differentiation of NSCs into neurons46. In the present study, we detected changes in histone acetylation and their regulatory effects on the transcription of proneural genes under hyperglycemic conditions.Epigenetic regulation plays an important role in the development of neocortex, including histone modification, DNA methylation, and non-coding RNAsAdverse effects of hyperglycemia on embryonic neurodevelopment have far-reaching influences on the health of the offspring, which poses a serious threat to public health. Glycemic control during pregnancy does not completely avoid the risk of neurodevelopment disorders in offspring. Our study investigated the mechanism underlying the anomalous neocortical neurogenesis in non-malformed offspring of mothers with hyperglycemia in pregnancy. For the first time, moderate hyperglycemia was shown to disturb the laminar distribution of newborn neurons in the neocortex. Moreover, high glucose influenced the differentiation of NSCs, leading to changes in the birthdate and laminar distribution of neocortical neurons. We explored the mechanism regulating histone modification during the differentiation of NSCs affected by hyperglycemia. These findings improve our understanding of the relationship and network between epigenetic modifications and the transcription of TFs during the differentiation of NSCs under hyperglycemic conditions. We hope to provide targets for the diagnosis, intervention and reversal of embryonic neural dysplasia caused by hyperglycemia. We also describe methods for studying the influence of other maternal and environmental factors on the development of offspring, and provide possible candidate markers for the prevention and treatment of adverse outcomes.21. Exposure to maternal hyperglycemia is confirmed as an independent risk factor for long-term neuropsychiatric morbidity in the offspring4. Thus, it is considerable to study the effects of maternal hyperglycemia on aberrant neurodevelopment. So we further detected the effects of maternal hyperglycemia on embryonic corticogenesis.In the present study, moderate hyperglycemic female C57BL/6J mice were mated with normal males, and the phenotypes of the fetuses were investigated. At E11.5, the fasting blood glucose level (FBG) of the HIP group (9.307 ± 0.796 mmol/l) was higher than the control group (5.719 ± 0.834 mmol/l)  and EdU to determine the effect of the high glucose treatment on the mechanism regulating the cell cycle exit of NSCs. The number of PH3-positive cells was decreased in high glucose-treated differentiated NSCs Fig. , meanwhi49. Therefore, we examined the effect of moderately high glucose levels on the transcription of these bHLH factors in the cerebral cortex and differentiated NSCs, respectively. As expected, the transcription of Ngn1 and NeuroD2 was significantly increased in the cerebral cortex of HIP mice  and decreased repressor bHLH factors ice Fig. . The traice Fig. . The traice Fig.  and highice Fig. . Based o37. In addition, high glucose has been shown to increase histone acetylation during neural development. Therefore, we detected changes in level of histone acetylation in telencephalon of HIP and differentiated NSCs treated with high glucose levels. We detected changes in level of histone H3 acetylated at lysine 9 (H3K9ac), histone H3 at lysine 14 (H3K14ac), histone H4 acetylated at lysine 8 (H4K8ac), and histone H4 acetylated at lysine 16 (H4K16ac)  Fig. , which houp Fig. . We did oup Fig. .Fig. 5Th52. We detected the expression of these enzymes in non-malformed offspring from a moderately hyperglycemic pregnancy and normal pregnancy by quantitative RT-PCR and Western blot analyses. As shown in Fig. Several HATs and HDACs participate in modifying H3K14ac in the developing nervous system. CBP, P300, and GCN5 are HATs, and SIRT1 is an HDACMaternal hyperglycemia alters neurodevelopment, but few reports have examined abnormal cerebral development in the offspring without obvious NTDs. We discovered that maternal hyperglycemia disturbed neocortical lamination in non-malformed offspring via facilitating the cell cycle exit of NSCs and altered the fate of early-born neocortical neurons during neocortical neurogenesis. We further demonstrated that increased accumulation of H3K14 acetylation at the Ngn1 and NeuroD2 promoters activated their transcription; then, Ngn1 and NeuroD2 initiated the premature differentiation of NSCs and sustained the commitment to neuronal fates under high glucose condition.21, so they produced more offspring exposed to maternal hyperglycemia while had normal phenotype without NTDs compared with pregnant mice with severe hyperglycemia. The data from epidemiological survey demonstrated that offspring affected by maternal hyperglycemia had high risks of long-term neuropsychiatric disorders than the offspring of normal pregnancy3, which suggested the effects of maternal hyperglycemia on aberrant neurodevelopment. So it is considerable to further explore the effects of maternal hyperglycemia on embryonic corticogenesis.In the present study, we selected the pregnant mice with moderate hyperglycemia in consideration of that the incidence of NTDs in the HIP group was less than the severe diabetes group (with fasting blood glucose levels exceeding 16.7 mmol/l) in our previous experiments29. Alterations in neocortical lamination contribute to neuropsychological disorders. The position where we acquired image of laminar structure is located in the boundary area of frontal cortex and parietal cortex, which is usually used to observe laminar structure47. Our results revealed that maternal hyperglycemia altered the fates of deep layer (early-born) neocortical neurons of this position during neocortical neurogenesis, which suggested that maternal hyperglycemia might influence embryonic brain function via disturbing corticogenesis. We further postulated that maternal hyperglycemia affected the birthdates of neocortical projection neurons by altering NSC differentiation, resulting in the abnormal laminar distribution of neocortical neurons in the offspring from a moderately hyperglycemic pregnancy.The birthdate of cortical projection (pyramid) neurons is closely associated with laminar identityDuring the neocortical development, multipotent NPCs maintain the balance between self-renewal and neurogenesis. In the present study, a high glucose treatment promoted the early exit of NSCs from the cell cycle and significantly accelerated the progression of neurogenesis. A significant increase in the number of early-born neuron was observed, while no significant change in number of late-born neuron was identified in our experiments. Overall, high glucose levels markedly enhanced the premature differentiation of NSCs to produce more early-born deep layer neocortical neurons in an earlier period of neurogenesis. However, more complex and in-depth studies are needed to confirm the effects of maternal hyperglycemia on the late-born neuron fates.43. During the neocortical development, Ngn1 and Ngn2 are transiently expressed and are critical for the commitment of NPCs in the ventricular zone, followed by the expression of NeuroD1 and NeuroD2 during terminal differentiation53. Ngn1 initiates neuronal commitment-related gene expression, while NeuroD2 further sustains neuronal fates55. As shown in our previous studies, the expression of bHLH factors is altered in proliferating and differentiated NSCs exposed to high glucose concentrations23. Maternal hyperglycemia also increases the transcription of bHLH genes in the embryonic telencephalon23. In the present study, we detected significantly increased the expression of the Ngn1 and NeuroD2 mRNAs in differentiated NSCs under high glucose condition during the formation of the laminar structure of the neocortex in fetuses from the HIP group that did not exhibit significant malformations. Therefore, it is an innovation to study the effects of high glucose levels on neurogenetic differentiation by investigating the transcription of bHLH genes in non-malformed offspring from moderately hyperglycemic pregnant mice.The bHLH family of transcription factors plays a key role in cell fate decisions in NPCs and the maturation of terminally differentiated cells13. Moreover, epigenetic regulation plays an important role in the development of the nervous system34. According to numerous studies, the modifications of histones that affect the structure and density of chromatin dynamically regulate gene expression associated with the cell fate decisions in NSCs38. Among the many modifications, histone acetylation relaxes the chromatin architecture and allows transcriptional factors to easily access the genomic DNA; acetylation mainly occurs on histone H3 and histone H4. Numerous studies on NTDs induced by a hyperglycemic pregnancy have identified variations in epigenetic modifications, particularly changes in histone acetylation. Lower acetylation of lysine 9 on H3 (H3K9) was observed in proliferating NSCs exposed to excessively high glucose concentrations (40 mmol/l) and in NSCs isolated from embryos from a diabetic pregnancy38. However, no report has examined the effect of moderate maternal hyperglycemia on histone acetylation in determining the fate of NSCs during neocortical neurogenesis. Interestingly, moderate maternal hyperglycemia and a high glucose (25 mmol/l) treatment both preferentially increased the acetylation of H3K14, but had no significant influence on the acetylation of H3K9, H4K8, or H4K16, which had been shown to participate in transcriptional activation50. Moreover, high glucose levels increased the acetylation of H3K14 at the Ngn1 and NeuroD2 promoters, which then predictably activated their transcription. These findings are the first to directly show that high glucose levels alter the expression of pivotal genes participating in neurogenetic differentiation by affecting histone acetylation.Transcriptional profiles are altered in embryos exposed to maternal hyperglycemia, implicating epigenetic regulation of gene expression58. Dheen et al. observed significantly decreased levels of the SIRT1 mRNA and protein in NSCs from embryos from pregnant diabetic mice59. Moreover, SIRT1 represses the activity of P300 by deacetylation to maintain the balance between histone acetylation and deacetylation during cellular differentiation60. Meanwhile, the imbalance between histone acetylation (upregulated P300) and deacetylation (downregulated SIRT1) under hyperglycemic conditions increases the expression of genes activated by H3K14 acetylation61. The results from the present study improve our understanding of the balance between P300- and SIRT1-based regulation of NSC differentiation.Histone acetylation is reversible and is regulated by two groups of enzymes, HATs and HDACs. P300 and SIRT1 play important roles in the differentiation and fate determination of NSCs during brain developmentIn conclusion, our data suggested the association between the effects of maternal hyperglycemia on neocortex development and epigenetic regulation on neurogenesis. High glucose levels promoted the early exit of NSCs from the cell cycle and the early-born neuron fate commitment. High glucose levels increased H3K14 acetylation levels at the Ngn1 and NeuroD2 promoters by disturbing the balance between P300 and SIRT1, and promoted transcription of Ngn1 and NeuroD2 Fig. . EnhanceWe abide by the Guide for the care and use of laboratory animals, Eighth edition (2011) in animal care and treatment. Our experimental protocol was authorized by the Ethics Committee on Animal Experiments of the Medical School of Shandong University (No. KYLL-2017(KS)-357).23. Pregnancy was designated as E0.5 when a copulation plug was observed after mating. At designated times , embryos were collected from pregnant mice by caesarean section. The forebrains of embryos and neonates were fixed with 4% paraformaldehyde (PFA) in PBS overnight at 4 °C and cryoprotected with 20% sucrose in PBS. Serial sections of the brain were cut at a thickness of 20 μm using a cryostat .Hyperglycemia was induced in 8-week-old female C57BL/6J mice  by an intraperitoneal (ip) injection of streptozotocin  dissolved in citrate buffer  for three consecutive days. Fasting blood glucose (FBG) levels were examined 7 days after the STZ injection using an FAD-GDH System  and were monitored every 3 days and immediately before euthanasia. Mice with FBG levels ranging from 8.3 mmol/l (150 mg/dl) to 11.1 mmol/l (200 mg/dl) were regarded as the HIP group. Control group mice were injected with an equal volume of citrate buffer, and their FBG levels were <6.7 mmol/l (120 mg/dl)The implantations and alive fetuses were counted at E11.5, and the percentage of alive fetuses among implantations was calculated. NTDs were screened in alive fetuses with a stereomicroscope  in a blinded manner.62.At E13.5, pregnant mice were ip injected with EdU , and embryos were collected 24 h later. Frozen sections of the embryonic forebrain were incubated with HCl (2N), blocked with 10% goat serum and incubated with a mouse anti-EdU monoclonal antibody  and a rabbit anti-Ki67 monoclonal antibody . Sections were then incubated with FITC-conjugated goat anti-mouse IgG and TRITC-conjugated goat anti-rabbit IgG , and counterstained with DAPI ; sections were mounted with fluorescent mounting medium . Finally, images of these sections were captured using a fluorescence microscope . For the analysis of proliferating (cell cycle re-entry) and quiescent (cell cycle exit) cells, the percentage of EdU+/Ki67+ and EdU+/Ki67– cells among the total number of EdU+ cells in the dorsal telencephalon were counted and was calculatedDNA fragmentation was detected using TUNEL staining . Briefly, frozen sections from E13.5 embryos were incubated with the TUNEL reaction mixture and then counterstained with DAPI. Images were captured with a fluorescence microscope . The numbers of TUNEL-positive and DAPI-positive cells in the dorsal telencephalon were counted and percentages were calculated.5 cells/ml in DMEM/F12 (1:1) supplemented with 2% B27 , 20 ng/ml basic fibroblast growth factor , 20 ng/ml EGF , 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded into 75 cm2 T-flasks and incubated in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C. After 3 days of incubation, primary neurospheres were dissociated into single cells and cultured for 3 days to form passage 1 neurospheres as shown in Fig. NSCs were isolated from the telencephalon of C57BL/6J mice at embryonic day 12.5. Cells were separated mechanically in DMEM/F12 (1:1) . After centrifugation and resuspension, cells were counted. Cell were seeded at final density of 2 × 10We also isolated NSCs from the telencephalon of non-malformed embryos of HIP and cultured them in differentiation medium containing a physiological glucose concentration to examine epigenetic reprogramming in utero caused by maternal hyperglycemia.63.Frozen sections of the telencephalon from E17.5 embryos or P1 neonates were permeabilized with 0.1% TritonX-100, blocked with 10% goat serum and incubated with the following primary antibodies: rabbit TBR1 monoclonal antibody , rat CTIP2 monoclonal antibody , and rabbit SATB2 polyclonal antibody . FITC-conjugated goat anti-rabbit IgG and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rat IgG were used as secondary antibodies. Cell nuclei were counterstained with DAPI, and sections were mounted with fluorescent mounting medium . Images were captured using a fluorescence microscope . Cells on 300 µm wide sections of the mediolateral axis of cortical sections expressing indicated markers were counted. We analyzed the density of cells by calculating the number of cells per 100 µm mediolateral axis wideCells on the coverslips were fixed with 4% PFA. The cell slides were handled as described above. We used the following primary antibodies for staining: anti-MAP2 antibody  and anti-phosphohistone H3 antibody . Images were captured with a fluorescence microscope .−ΔΔCt method25, with β-actin serving as the normalization control.Total RNA was extracted from dorsal telencephalon tissues of E13.5 embryos and cultured differentiated NSCs using Trizol reagent  and reverse transcriptions were primed with oligo dT and performed on equal amounts of total RNA (2 μg) using ReverAid First Strand cDNA Synthesis Kit . Real-time PCR was performed with SYBR Green Realtime PCR Master Mix  and performed with CFX connect . Primer sequences are listed in Table Mouse dorsal telecephalon tissues from E13.5 embryos and cultured NSCs were lysed in ice-cold RIPA buffer  for 20 min at 4 °C. Supernatants were collected and total protein concentrations were measured by the BCA method. Equivalent amounts of protein in each sample were separated on 8–15% SDS–PAGE gel electrophoresis and then electrotransferred onto 0.22 or 0.45 μm PVDF membranes . The membranes were then blocked with 5% milk in TBST buffer and probed with the following primary antibodies at 4 °C overnight: anti-TUJ1 antibody , anti-histone H3 antibody , anti-acetyl-histone H3 (lys9) antibody , anti-acetyl-histone H3 (lys14) antibody , anti-histone H4 antibody , anti-acetylhistone H4 (lys8) antibody , anti-P300 antibody , anti-CBP antibody , anti-SIRT1 , anti-GCN5 , and anti-β-actin antibody . Secondary antibodies were HRP conjugated to either goat anti-mouse IgG or anti-rabbit IgG. The bands were finally visualized by the enhanced chemiluminescence ECL detection kit . The intensity of bands was determined by the Image-Pro Plus 6.0 software.3 and fixed with 3% glutaraldehyde for 2 h at 4 °C. The fixed tissues were washed no less than three times with PBS. Then, the tissues were post-fixed in 1% OsO4 for 1 h at 4 °C. The tissues were dehydrated through an ascending ethanol series, and infiltrated with 100% epoxy resin:acetone  1 h, 4 h, no less than 12 h, respectively. Finally, the tissues were embedded in fresh epoxy resin and polymerized at 37 °C for 12 h, 45 °C for 12 h, 60 °C for 1–2 h. Ultrathin sections were supported on grids (150 nm) and stained with uranyl acetate for 20 min and lead citrate for 20 min at room temperature. Images were captured with JEM-1200EX transmission electron microscope .Dorsal telecephalon tissues from E13.5 embryos were cutted into 1 mm64 are listed in Table ChIP was performed with an EZ-ChIP kit  according to the manufacturer’s instructions. Mouse dorsal telecephalon from E13.5 embryos were fixed with 1% formaldehyde to cross-link histones to the DNA for 10 min at room temperature and then harvested. Each pellet was resuspended in SDS Lysis Buffer containing protease inhibitors. Cells were lysed and sonicated to shear the chromatin into fragments with an average size of ~150–900 bp. The supernatant was immunoprecipitated with an anti-acetyl-histone H3 (lys14) antibody  or a control rabbit anti-IgG antibody overnight at 4 °C. After washes, elution, and reversing the cross-links, the DNA was purified from each sample and the corresponding input sample and analyzed using qRT-PCR. The primers used for PCRΧ2 test. Other variables were analyzed using Student’s t test or one-way analysis of variance followed by Tukey’s test. Data are reported as the mean ± SD, and differences were considered statistically significant when p values were <0.05.The quantitative data are reported as absolute values, rates, mean ± SD, unless specified otherwise. The percentages of alive fetuses and NTDs were analyzed using the Figure S1Figure S2Figure S3table S1Supplemental figure legends