Many health organizations have promoted the importance of the health-related benefits of physical fitness and physical activity. Studies have evaluated effective public health practice aiming to understand the cognition of physical activity among youths and adolescents. However, studies investigating the level of cognition and knowledge of physical fitness among Asian adults are lacking.This study aimed to investigate the self-awareness level of physical fitness and exercise prescription and the demand for physical fitness assessment among Taiwanese adults.In January–July 2019, a cross-sectional anonymous survey was conducted using Research Electronic Data Capture to gather data on demographic data, cognition investigation of physical fitness and exercise prescription, cognitive test of physical fitness and exercise prescription, and demand for physical fitness assessment.The questionnaire was answered by 200 respondents. The rating for cognition investigation of physical fitness was 2.63–3.13 (unclear to mostly clear) and for exercise prescription was 2.05–2.76 (unclear) . Results show that lack of awareness was highest for health-related physical fitness, exercise prescription, and exercise progress planning. 98% of subjects did not know the latest recommended guidelines for physical activity, despite most agreeing that physical fitness and exercise are good for health. Most subjects (72%) indicated a willingness to accept self-pay service for physical fitness assessments.This is the first study to report on the demand for cognition, assessment, and promotion of physical fitness among Taiwanese adults. The study shows that the subjects widely lack knowledge in the cognition of physical fitness and exercise prescription. Furthermore, a self-pay service for the physical fitness assessment and individualized exercise prescription were acceptable to most subjects, especially those undergoing regular health examinations. The findings are encouraging and will aid support for health organizations and professionals in the development and management of promotion strategies on health-related physical fitness in preventive medicine and health promotion. Physical fitness, a state of good health and strength, which is a comprehensive ability, is a multifactorial concept covering five components: muscular strength, muscular endurance, flexibility, body composition, and especially cardiorespiratory fitness , 2, all In this cross-sectional study, an anonymous questionnaire was used for the survey, which was conducted from January 2019 to July 2019 at the Health Management Center of the National Taiwan University Hospital (NTUH) and public websites. We put up flyers with an introduction and a website link for a questionnaire investigation on these public study fields. No inclusion and exclusion criteria were defined for the subject recruitment. When participants learned about the recruitment, they joined voluntarily and were assured confidentiality and that they were free to withdraw from the survey at any time. A double-checked button was used to signify that the participant was done answering the questionnaire. No compensation was provided to the participants for their time. Of 268 responses during the study period, 68 questionnaires with missing data were excluded from the study. A total of 200 individuals voluntarily answered the anonymous questionnaire, along with complete records, which were included in this study for further data analysis. The institutional review board of the NTUH approved the study protocol (201908044RINC).Research Electronic Data Capture (REDCap), a web-based platform hosted at the NTUH, was used to develop, collect, and manage the anonymous questionnaire. REDCap, which is a compatibility, scalability, and security data collection tool, along with built-in analysis tools was developed at Vanderbilt University in 2004 for the purpose of clinical research . REDCap The custom-designed REDCap online survey can be accessed and filled out by participants using a smartphone, tablet, or computer with internet connection. The Hypertext Transfer Protocol Secure (HTTPS) protocol for secure internet communication transmits the feedbacks automatically into the hospital database. NTUH maintains the research database, and to access the management tool, researchers must enter their username and password. The hospital portal system, along with one‐time password from email or Google Authenticator Application, validates the login session. User behavior and manipulation on the platform are tracked as log messages into database. The data format of REDCap fulfills the Clinical Data Interchange Standards Consortium standard. The researchers could use automatic export procedures for seamless data downloads including raw data and variable schema to common data types such as CSV, Excel, SPSS, SAS, R, Stata, and XML.α of 0.912  for cognition investigation of physical fitness and 0.920  for cognition investigation of the exercise prescription, both of which indicate excellent internal consistency. All sensitive information, such as name, identification number, telephone number, email, and Internet Protocol address (IP address) of the electrical devices were not collected in this study. A 5-point Likert scale  was used to rate the cognition items. Higher scores indicate positive cognition. The true-or-false tests were presented as positive and negative statements. Finally, the questionnaire was divided into five pages on the REDCap online survey, with each page having an intuitive interface for the validation of data entry without any missing item. The study team conducted complete checks for content typo and interactive buttons before starting the survey. Although this was an anonymous questionnaire, the participants still had to click a button to accept and continue with the survey at the beginning of the questionnaire.A research team that specialized in exercise physiology, medicine, and health management developed the questionnaire material in a roundtable conference for fulfilling the need of investigation , undergoing regular health examination and frequency . In addition, the following data were gathered to determine the demand for physical fitness assessment: factors that affected the choose of a physical fitness test service, willingness to accept self-funded physical fitness tests and the acceptable price, having undergone a physical fitness test in the past 5 years, and having received guidance related to exercise prescriptions in the past. Furthermore, we evaluated the distribution of the demand for physical fitness assessment stratified by subjects with and without regular health examinations.P < 0.05. Reliability analysis was evaluated by Cronbach’s alpha for the content validity of questionnaire.To calculate numbers and percentages for all question items among respondents, descriptive statistics were initially performed in REDCap. Figures were generated using Microsoft Office Excel 2019. The results of cognition items are presented as means with 95% confidence intervals (CIs). Further statistical analysis was conducted using SPSS software . Chi-square test or Fisher exact test was used for comparisons of willingness between men and women among different age groups, as appropriate. Statistical significance was set at n = 64, 40.5%)  for further analysis. Age distributions, in order, were as follows: 40–49 years , 50–59 years , and 30–39 years . Most subjects were educated at the university  and postgraduate or above  level. Seventy-nine percent of the subjects receive regular health examinations and usually undergo examination once a year , what diseases people with poor cardiopulmonary functions will easily contract , and body mass index . Results of the investigation of cognition of physical fitness in this study are presented in With regard to cognition of physical fitness, subjects’ replies using the 5-point Likert scale ranged from 2.63 to 3.13, indicating that the general cognition of physical fitness is “unclear” to “mostly clear.” Higher scores were found for cognition for flexibility , impact of cardiopulmonary function on life and work , and muscle and muscular endurance . Physical fitness items that had the highest response rates of “very unclear” and “unclear” are health-related physical fitness , exercise prescription , and impacts of different types of activity/exercise on physical fitness . Results of the investigation on cognition of exercise prescription, along with rank ordering according to the distribution of “very unclear” and “unclear” responses, are presented in For cognition of exercise prescription, the results show that participant responses on the 5-point Likert ranged from 2.05 to 2.76, indicating that the cognition of exercise prescription among subjects is, in general, “unclear.” Results show that cognition for exercise intensity , the relationship between duration of exercise and effective exercise , and the relationship between exercise intensity and effective exercise  had higher ratings. The items with the highest rates of “very unclear” and “unclear” ratings are exercise progression planning (a) that understanding one’s own physical fitness is important to self-management of health and (b) that, because the activity content of each person is different, the design of exercise prescription varies from one person to another. However, results show that subjects lack knowledge in the following areas: (1) a single vigorous exercise can make up for the inadequacy of regular exercise if a person has not exercised for a long time ; (2) engaging in vigorous or intensive exercise can greatly improve the body’s immune system ; and (3) having good physical fitness means that the body must be healthy . In addition, although the 333 Plan had already been considered out-of-date, up to 98% of respondents consider it as the latest exercise recommendations. With regard to cognitive test of physical fitness, results indicate that all subjects know (P = 0.017). In the comparison of sexes in each age category, significant differences were found only in subjects between 40 and 49 years . The distribution of willingness to accept a self-pay service for physical fitness assessment by sex, according to different age groups, is displayed in n = 42 (29%); $1,001–$1,500, n = 36 (25%); $1,501–$2,000, n = 24 (17%); $2,001–$2,500, n = 20 (14%); under $500, n = 11 (8%); and greater than $2,500, n = 11 (8%). Overall, 144 (72%) subjects are willing to accept a self-pay service for physical fitness assessments, with more men (79%) than women (63%) agreeing to this , (2) individualized report according to the physical fitness examination results , and (3) reasonable and achievable health-related physical fitness goals . In contrast, the underlying reasons for not choosing a self-pay service for physical fitness assessment are the following: (1) does not want to pay for physical fitness tests , (2) no time , (3) feeling that one is healthy , and (4) does not know any place that provides physical fitness examinations .With regard as to why participants were willing to accept a self-pay service, the main reasons were the provision of (1) individualized exercise prescription according to physical fitness examination results  conducted in the United States, only 36.1% of US adults had ever received messages regarding government physical activity guidelines from any type of media, of which only 1% were aware that for substantial health benefits, the recommended volume of exercise was at least 150 minutes of moderate-intensity exercise per week . SimilarIn Taiwan, life expectancy for the general population is 80.4 years, with rates of 77.3 and 83.7 years among men and women, respectively . With reFor public health organizations such as government agencies and nongovernmental organizations, the main purpose of health dissemination is carried out through multiple channels to raise general public awareness on the importance of the health-related benefits of physical fitness, especially in physical activity and moderate-intensity exercise. In addition, physical inactivity and sedentary lifestyle are global adverse health effects, which have been identified to contribute to the burden of many chronic diseases and premature mortality –53. The P = 0.02; In this study, a self-pay service for physical fitness assessments was acceptable to most subjects (72%). The most acceptable price for a self-pay service for physical fitness assessments was approximately $500−$1,500 New Taiwan Dollars (NTDs) (30 NTDs = 1 US dollar). In Taiwan, the prices of visiting a clinic, undergoing a labor health examination, and undergoing a health examination package generally are approximately $150−$350, $1,000−$1,500, and $20,000−$150,000, respectively. Providing this augmentation service in health examination packages would require the setting of an affordable price. Moreover, compared with subjects who did not go for regular physical examinations, individuals who undergo regular health examinations were more accepting of a self-pay service for physical fitness assessments Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.


RYR2 and RYR1 genes have been found to harbor three main mutation “hot spots”, where mutations change the channel structure, its interdomain interface properties, its interactions with its binding partners, or its dynamics. In all cases, the result is a defective release of CaCardiac arrhythmias are serious, life-threatening diseases associated with the dysregulation of Ca Ryanodine receptors (RyRs) are the largest ion channels presently known. They are very large 2.2 MDa) Cacoupling ,2,3,4,5..2 MDa Cacoupling ,7. Threecoupling ,10,11,12coupling . RyR2 iscoupling ,14, but coupling ,16. RyR3coupling ,17. The coupling ,19: micecoupling ,21,22 bucoupling ,24. In nand RyRβ ; lower oand RyRβ .Structurally, RyRs are homotetramers, with each subunit having around 5000 amino acids. They are most often found embedded in the sarcoplasmic reticulum (SR) membrane of striated muscle cells at t-tubule triad junctions . At thes subunit ,31,32,33 subunit ,35. EC c subunit ,36. Two ating it ,40,41,42ating it . RyR opetraction ,45,46. Tl levels ,48,49. Il levels ,50. In tl levels .apo and CaRyRs are regulated and influenced by a large number of factors ,51,52. Tns of it . It is g closing ,55. The o the SR . Other rcaffeine . RyRs hacaffeine ,58.Mutations that disrupt the functioning of the RyRs are often associated with myopathies. In the following sections, we will outline the most frequent diseases associated with RyR1 and RyR2 and then review the results of recent structural and computational studies with an emphasis on how individual mutations might disrupt either the structure or the regulation of one of the receptors.RYR1, which are associated with life-threatening myopathies , the central region (roughly 2100–2500) and the C-terminal region (from around 3800 to the C-terminus) ,101,102.RyR3 has recently been linked to Alzheimer’s disease in a mouse model  and famiMalignant hyperthermia is a pharmacogenetic disorder that manifests in a sudden increase in body temperature, muscle stiffness and, in some cases, the breakdown of skeletal muscle fibers (rhabdomyolysis). It is mainly triggered by volatile anesthesia or muscle relaxants; stress can also trigger the disease in some cases . O. Oβ-tref104, α2) , which w104, α2) ,160 as w104, α2) . In the isoforms . Based oisoforms ,12,148.http://www.hgmd.cf.ac.uk/ac/gene.php?gene=RYR1) and CPVT . T. T79]. TThe ryanodine receptor contains four tandem repeats that occur in two sets of two each. These are also called RYR domains, because they have so far only been found in the ryanodine receptors , or someStructurally, the first two RYR repeats (Repeat1–2) are composed of two asymmetrical halves c. Each t repeats . The twotructure . That sttructure ,12,162.The RYR3–4 domain (Repeat3–4), also referred to as the phosphorylation domain, is one of the few domains that has high resolution structures from all three mammalian isoforms ,167. Thereceptor ,167,181.isoforms .Recently, a crystal structure of the mouse RyR2 Repeat3–4 domain bound to the catalytic domain of PKA (PKAc) in complex with the non-hydrolyzable ATP analog adenylyl-imidodiphosphate (AMP-PNP) was determined . In thisFive mutations associated with malignant hyperthermia are found in the RyR1 Repeat1–2 domain. Four of them are located at the domain surface, and are therefore unlikely to cause domain misfolding, but one of them R1043C (R1044C in rabbit RyR1), affects a residue that is involved in multiple hydrogen-bonds, including ones to main-chain atoms from the U-lid; breaking these may therefore affect the U-lid conformation and stability . Two CPVEleven mutation sites associated with malignant hyperthermia, central core disease, multi-minicore disease, King–Denborough syndrome and atypic periodic paralysis have been found in the RyR1 Repeat3–4 domain (summarized in ). Seven E. coli [Before detailed structural studies had been performed, a number of studies sought to partially characterize the structure of RyR with the help of bioinformatics. Bioinformatics studies on RyR2 focused on identifying individual domains based on their amino-acid sequences using the Pfam database ,183. TheE. coli ,157,184.Before the high-resolution cryo-EM structures had been completed, some effort was made to fit the existing high-resolution crystal structures into the low-resolution cryo-EM maps; nearly every structural study featured at least some effort to fit the studied domain into at least one of the available cryo-EM maps ,164,182.Determining the three-dimensional structure of the ryanodine receptor in both its open and closed forms does provide the answer to a number of questions about the architecture of the molecule and its likely conformational changes upon opening and closing, but it says little about the pathway by which the channel opens, what other conformational states are possible, and what the effects might be on the structure of the binding of the various regulatory molecules. Moreover, though the structures can be used to identify the location and possible significance of individual point mutations, by themselves, they still cannot be used to describe in detail their mechanistic consequences. To address these questions, computational studies are needed.The first high-resolution cryo-EM structures of RyR1 were all in the closed form ,11,12. OSeveral computational studies have been carried out on the structure of the ryanodine receptor that combine in vitro or in vivo functional studies with dynamics modeling to assess the functional and structural impact of one or more of the known disease-associated mutations. The part of the receptor most frequently studied in this way is the pore domain, followed by the NTD. The earliest dynamics study examining the channel pore region predated the reporting of the high-resolution cryo-EM structures and used a homology model of the RyR1 pore region that was based on the pore of Kg et al. ,189 usedThree studies in the laboratory of Gerhard Meissner have examined the effect of mutating RyR1 channel residues G4934 and G4941, both of which are in the pore-lining helix S6. The first study  looked aing Eris  to creating Eris  to optiming Eris , they font study  exploredIn a similar study, homology modeling was combined with clinical assessment of a family suffering from CPVT and functional characterization using Camutation . The homPlutella xylostella [Finally, sequence alignment and homology modeling was used to locate and describe the site of a mutation, G4946E, that confers resistance to diamide insecticides to the diamondback moth lostella . The sitde novo A165D mutation.Similar functional-modeling studies have also been carried out on the RyR1 NTD. The earliest was by Zheng and Liu , who simBauer et al.  carried The most comprehensive NTD modeling study to date was by Yamazawa et al. , who comjkq), mouse RyR2 NTD (3im6), mouse RyR2 SPRY1 (5c33), rabbit RyR1 Repeat1–2 (5c30), mouse RyR2 SPRY2 (4P9I), mouse Repeat3–4 (4etv) and rabbit RyR1 (3j8h)  , were usRyanodine receptors play an essential role in excitation-contraction coupling in striated muscle cells. The importance of their role is highlighted by the large number of regulators they have and by the consequences that individual point mutations can have on their resulting activity and the health of those who suffer from them. In addition to the most recent structural research, we have also reviewed here some of the diseases arising most frequently from the disruption of the skeletal and cardiac receptors together with the structural and functional consequences of some of the mutations giving rise to them.Although much has been done, even more still remains unknown about the detailed operation and regulation of these molecules. Ryanodine receptors represent a challenge due to their size, complexity and physiological importance: they are connected with many cellular processes and, as a consequence, interact with many cellular proteins and other ligands, often in a specific cascade or in tandem. High-resolution structures of several RyR domains are still lacking. RyR2, which was shown to be more flexible than RyR1 by EM, in particular requires extra study, both by itself and in complex with some of its regulatory ligands. This would allow more details to be understood regarding both its structure and its physiological role. The differences between the mammalian RyR isoforms need to be better clarified as well as how they are activated in particular tissues. In this respect, RyR3 especially requires more attention. It would also be interesting to elucidate the differences between the RyR isoforms in mammals and their homologs in other animals. Research into how mutant forms of RyRs that are associated with muscular dystrophies presents an ongoing challenge: in most cases it is still unclear how the given mutations influence or initiate a particular disease. To answer such complex questions will require a multidisciplinary approach involving in vitro, in vivo and in silico studies. Structural research and structure-based drug design will certainly be an important part of it. All this knowledge should provide the foundation for establishing new medical treatments, likely individualized, for curing or at least treating the diseases with minimal side-effects.


Fetal exposure to particulate matter (PM) is associated with infant developmental delay likely via neuroinflammation and prefrontal cortex lesions; however, whether air-purifier usage, which can reduce indoor PM levels, is related to infant developmental delay remains unknown. We therefore examined the prospective relationship between air-purifier usage during pregnancy and infant developmental delay by analyzing 82,441 mother–infant pairs using a simple yes/no questionnaire. Developmental delays at 6 and 12 months were assessed in five areas using the Ages and Stages Questionnaire, Third Edition. A generalized linear mixed model analysis was used to derive adjusted odds ratios (AORs) and 95% confidence intervals (95% CIs) while controlling for 20 covariates. The analysis revealed that air-purifier usage was associated with developmental delays in fine motor  and problem solving  at 6 months and in communication , fine motor , problem solving , and personal–social  at 12 months. In conclusion, a negative association exists between air-purifier usage during pregnancy and infant neurodevelopmental delay that strengthens with time. Our results outline the potential role of air purifiers in inhibiting infant neurodevelopmental delay. Pollution is the leading environmental cause of disabilities and early death . Among tExposure to PM during pregnancy has been shown to exert adverse effects on offspring. For example, Guxens et al.  revealedAlthough the detailed mechanisms underlying the relationship between high fetal exposure to PM and impaired child neurodevelopment remain unknown, one possible pathway possibly involved is neuroinflammation. The basis of this hypothesis is the fact that pregnancy and the first year of life are critical periods of vulnerability for human neural development ,11. ThusOne countermeasure to air pollution may be the use of air purifiers at home. Present-day air purifiers are often equipped with high-efficiency particulate air (HEPA) filters, which can catch PM2.5 with over 99.97% efficacy at the rated airflow. Previous studies ,15 have n > 80,000 records, while controlling for possible confounders such as basic characteristics and dwelling environment. We hypothesized that the use of air purifiers is related to intact neurodevelopment.Therefore, in this study, we examined the prospective relationship between the use of air purifiers during the fetal period and subsequent neurodevelopment. We assessed five major developmental areas across time using a large dataset from the Japan Environment and Children’s Study (JECS), a nationwide birth cohort with The detailed design and baseline characteristics of the JECS have been published elsewhere ,18,19. BThe JECS protocol was approved by the Ministry of the Environment Institutional Review Board for Epidemiological Studies (100910001) and by the Ethics Committees of all participating institutions. This study was also approved by the Ethics Committee, University of Toyama (R2018023) on 12 June 2018. The JECS was conducted in accordance with the Declaration of Helsinki and other nationally valid regulations, and written informed consent was obtained from all participants.Follow-ups were conducted primarily via mailed letters and scheduled in-hospital check-ups until one month postpartum and via mailed letters at 6 and 12 months. Data were acquired using self-administered questionnaires or transcriptions of medical records recorded by physicians, midwives/nurses, and/or research coordinators. The dataset used in this study, jecs-an-20180131, was released in March 2018 and updated in December 2018. The dataset includes measurements for the first and second/third trimesters as well as for 0, 1, 6, and 12 months postpartum.Among the 103,062 participants in this dataset, 5647 were excluded due to multiple registrations, 949 due to multiple births, and 3676 following miscarriages or stillbirths. Among the remaining 92,790 unique pregnancies with singleton live births, 1693 were further excluded due to no response to the questionnaire requesting information on demographics, psycho-social status, residence type, and dwelling environment characteristics, including the usage of air purifiers, during pregnancy. A further 8656 pregnancies were further excluded due to a lack of responses to the posted Ages & Stages Questionnaire, Third Edition (ASQ-3) , at bothThe use of air purifiers during the previous year was assessed using simple yes/no questions. The data were collected during the second/third trimester.Children’s neurodevelopment at 6 and 12 months after birth was assessed using the ASQ-3, an age-specific, structured, parent-completed child monitoring system . The ASQ2), parity (primipara or multipara), smoking status , passive smoking status , alcohol intake , number of hours spent outdoors , physical activity (yes or no), folic acid intake  [n), living-room flooring material , age of house/apartment building , house renovation/interior completion after becoming pregnant (yes or no), and number of years living in the current place of residence . These covariates include standard variables for socioeconomic status and dwelling environment. In addition, we selected several variables in terms of the possibility of impact on exposure and/or outcome. Potential mediators were not used as covariates. The categorization of these variables was conducted according to usual medical practice or common practice in Japan and/or by referring to our previous studies , body mass index (BMI)  , marital≥338 µg) , number s . AllSeparate generalized linear mixed models, with 19 regions set as a random effect and logit as a link function, were used to calculate odds ratios (ORs) and their 95% confidence intervals (CIs). Nonuse of air purifiers was used as the reference. Outcome variables were screen-positive cases in the five developmental areas: (a) communication; (b) general motor; (c) fine motor; (d) problem solving; and (e) social-personal (in the ASQ-3); as well as (f) the total number of screen-positive cases over the five areas at 6 and 12 months. The exposure variable was the yes/no answer to the question regarding the use of air purifiers.The forced entry method was used to include covariates in the multivariate analysis. In the crude model, only the region was set as a random effect. In model 1, the regression models were adjusted for maternal age, parity, smoking status, passive smoking status, folic acid intake, marital status, highest educational level, annual household income, type of residence, number of rooms in the house/apartment, materials covering the floor of the living room, and age of house/apartment building, with the region set as a random effect. In model 2, the regression models were adjusted for the covariates used in model 1: BMI, alcohol intake, number of hours spent outdoors, physical activity, employment status, high-rise living, house renovation/interior completion after becoming pregnant, and number of years living in the current place of residence.SAS 9.4  and R 4.0.0 were used for all statistical analyses.n = 81,669) and 85.60% , respectively, while 0.83% (n = 772) of the mothers did not respond at 6 months but responded at 12 months. Of the 82,441 mother–infant pairs included in the study, the missing-data rate was ≤1% for all covariates except for parity , number of years living in the current place of residence , physical activity , average number of hours spent outdoors , and annual household income . For exposure measures, the item regarding the use of air purifiers had a missing-data rate of <0.37% (n = 298). Each score of the five areas of the ASQ-3 had a <5.99% (max n = 4931) missing-data rate at 6 months and <10.57% (max n = 8712) at 12 months. A total of 18,985 (23.03%) and 21,908 (26.57%) mother–infant pairs had at least one missing value at 6 and 12 months, respectively.The effective response rates at 6 and 12 months postpartum were 88.01%  were compared with those from the multiply imputed datasets  to assess the differences between the strategies for addressing missing values.The resulting OR patterns from the complete case datasets  were analyzed using the same setting except for using a Gaussian distribution.The characteristics of mothers and dwelling environments together with data regarding the use of air purifiers during pregnancy are presented in Multicollinearity was not detected among all covariates; that is, all generalized variance inflation factors were below 2.73.The prevalence, cases, crude ORs, and adjusted ORs (models 1 and 2) for developmental delay in each developmental area at 6 and 12 months according to the use or nonuse of air purifiers are presented in The mean and SD values of the total numbers of screen-positive cases over the five developmental areas and their crude ORs and adjusted ORs (models 1 and 2) according to the use or nonuse of air purifiers are presented in The results of the sensitivity analysis using the complete case dataset  were notThe analysis using continuous scores of each developmental area in the ASQ-3 is summarized in n > 80,000) [To our knowledge, this is the first study to examine the prospective relationships between the use of air purifiers and child neurodevelopment using a nationwide birth cohort study dataset  ,18,19 whAlthough the usage of air purifiers was associated with the areas of communication, fine motor, problem solving, and personal–social, it is not clear which brain areas correspond to each developmental area in the ASQ-3. That said, the prefrontal cortex would be deeply involved in these areas because the prefrontal cortex is known to be responsible for higher intellectual functions, such as goal-driven behavior, including action planning and motor control, executive functioning, working memory, and adequate social response ,29. ThesGiven the underlying mechanism by which the usage of air purifiers prevents impaired neurodevelopment in infants via elimination of airborne PM ,7,8,9,15The crude ORs hardly changed even when 12 covariates were used (model 1) and all 20 covariates were used (model 2). The stability suggests that all covariates served only as very weak confounders. Thus, it would be very difficult to explain the observed association in terms of these variables, although air-purifier usage was clearly related to many variables, such as high education levels, high income, less smoking, being married, multipara, and living in a newer house . InteresSeveral limitations of this study should be addressed. First, we evaluated the usage of air purifiers using simple yes/no questions. Thus, important information regarding the performance of the devices, the types of air filters used, and their usage patterns  was not available. In addition, we did not have data on the actual decrease in indoor PM levels after use of the air purifiers or on the dose–response relationship. Accordingly, the current estimation of ORs was rough. Second, the relationship between maternal PM2.5 exposure and subsequent developmental delay has not yet been reported for this population, so there is a gap in our hypothesis that air-purifier usage might prevent infant neurodevelopmental delay. We cannot deny the possibility that the observed relationship was a spurious association with the true cause existing outside the current framework. Third, although the ASQ-3 is a validated, parent-completed questionnaire, its aim is screening, not diagnosis. Thus, the developmental delay in this study could be overestimated. Fourth, our participants were recruited within the last 10 years from several areas across Japan, including both urban and rural areas, but the extent to which the results are applicable to specific populations cannot be determined. Finally, because our study used a prospective birth cohort, the observed prospective relationship cannot be regarded as a causal relationship.Despite these limitations, we found inverse prospective relationships between the use of air purifiers during pregnancy and infant neurodevelopmental delay at 6 and 12 months postpartum that were salient over time in 82,441 mother–infant pairs while controlling for up to 20 potential confounders. Our results outline the potential role of air purifiers in inhibiting infant neurodevelopmental delay. This study provides a rationale for conducting randomized controlled trials to address the causal relationship between the use of air purifiers and infant neurodevelopmental delay and in-depth studies elucidating what factors are related to reduced risk for developmental delay in relation to air-purifier usage.


This study explores the inner mechanism that helps to strengthen self-care of childcare teachers, which enables teachers to provide quality care to children and promote their own wellbeing. (2) Methods: The survey was conducted from September to October 2018 for childcare teachers in Seoul and Gyeonggi Province using convenience sampling. Out of 550 questionnaires, 491 were returned, with 466 used for the analysis, excluding those with incomplete responses. The collected data were analyzed using descriptive statistics, correlation analysis, and mediation analysis. (3) Results: There were significant correlations between all variables. The mediation analysis showed a complete mediation of resilience. (4) Conclusion: Childcare teachers first have to take good care of themselves in order to perform well as a childcare professional. Educational materials and counseling programs tailored for childcare teachers need to be developed for better self-care and building greater resilience. Materials for directors of daycare centers, as well as teachers stressing the importance of social support for each other, will help childcare teachers’ effective functioning in their professional and personal life. Prevention and intervention programs for self-care will eventually help lower the costs of healthcare in society. Self-care is the personal ability to maintain psychological and emotional balance. It builds one’s holistic health and wellbeing, thus contributing to taking care of others ,2,3. SelCaregivers such as childcare teachers and those who provide mental/physical health services are consistently confronted with a high risk of negative consequences such as psychological distress ,7. In thDue to the recent increase in the number of employed mothers, young children in South Korea stay at daycare centers for an average of 7–11 hours , while aSelf-care allows childcare teachers to recognize their personal and professional identity, maintain a psychological balance, and accept children as they are  since seResearch has stressed the importance of self-care since mental health problems, such as depression, anxiety, and stress, arise when a counselor’s self-care is insufficient . Mental As daycare centers are generally located in smaller spaces, close cooperation with fellow teachers and directors is important as it increases the psychological stability and positive self-awareness of childcare teachers ,36,37. SIt has been reported that self-care is associated with psychological factors ,45,46,47Resilience of teachers is greatly affected by social resources in the workplace provided by directors and colleagues . In regaThe relationships between self-care, social support in the workplace, and resilience have been reviewed in this section, while research examining the relationships among the three has not been conducted. Moreover, studies focusing on self-care for childcare teachers have been scarce, despite it being one of the most stressful occupations . It has Hypothesis 1.Childcare teachers’ self-care, resilience, and social support in the workplace will be significantly and positively correlated with each other.Hypothesis 2.Resilience of childcare teachers will mediate the relationship between social support in the workplace and self-care.Hypothesis 2a.Social support in the workplace will show a significant and direct effect on the resilience of childcare teachers.Hypothesis 2b.The resilience of childcare teachers will show a significant effect on self-care.This study targeted 466 childcare teachers currently working at daycare centers in Seoul and Gyeonggi Province, South Korea. The survey was conducted from 24 September to 19 October 2018, using convenience sampling after the pilot survey was conducted in early August (IRB File No. 1808-045-02). Researchers visited or called the directors of daycare centers in Seoul and Gyeonggi Province to explain the purpose of the research and recruit participants. With the approval of the directors, the questionnaire was distributed to childcare teachers along with a survey consent form and a gift  was provided as compensation for participants. A total of 550 questionnaires were distributed to childcare teachers, with 491 questionnaires returned (89.3% response rate). Questionnaires with incomplete responses were excluded and a total of 466 were used for the analysis.The demographic characteristics of the subjects are as follows. Of the 446 subjects, 259 teachers (55.6%) were in their 40s or older, 214 (45.9%) had graduated from a vocational college, 209 (44.8%) had less than 5 years of work experience, and 261 (56.0%) had a monthly income of less than 2 million Korean won (KRW), approximately US$1,800. The average amount of monthly income was 2.3 million KRW, while 331 (71.0%) worked 9 h per day and 375 (80.5%) were married. Details of the characteristics of subjects are presented in Health Promoting Life Profile II (HPLPII) developed by Walker, Sechrist, and Pender ,66 was uThe scale comprised of 52 questions in 6 categories regarding health promoting behaviors: health responsibility (9 questions), physical activity (8 questions), nutrition (9 questions), spiritual growth (9 questions), interpersonal relations (9 questions), and stress management (8 questions). Each area included the following statement: “Report any unusual signs or symptoms to a physician or other health professional” for health responsibility; “Take part in light to moderate physical activity . Cronbach’s α coefficient was 0.94 for self-care, 0.82 for health responsibility, 0.90 for physical activity, 0.75 for nutrition, 0.82 for spiritual growth, 0.82 for interpersonal relations, and 0.77 for stress management, respectively.Myung’s  scale, wResilience was measured with Park’s  scale usCollected data was analyzed using SPSS 23 and the PROCESS 3.5 macro program developed by Hayes . First, First, results from the descriptive statistics are presented in Second, Pearson’s correlation analysis was conducted to examine the relationships among childcare teachers’ social support in the workplace, resilience, and self-care . The res2 (63) = 215.55, GFI = 0.94, TLI = 0.96, CFI = 0.97, RMSEA = 0.07).Next, confirmatory factor analysis (CFA) was performed to see whether the three variables in the model distinctively differentiated from one another . The resTo examine the mediating role of resilience in the path between social support in the workplace and self-care of childcare teachers, the PROCESS 3.5 macro program (Model 4) developed by Hayes  was usedp < 0.001; supporting Hypothesis 2a) in the regression model including resilience (M) and social support (X) only. Results from the regression model with all three variables revealed that resilience had a significant effect on self-care . Meanwhile, the direct effect of social support was not significant , which indicated that it was a complete mediation. The bootstrapping result showed that the confidence interval was between 0.14 and 0.27 and confirmed that the mediating role of resilience was significant.Social support for childcare teachers in the workplace had a significant effect on resilience  was lower than those reported in previous studies ,78, withIn terms of Hypothesis 1, it was supported by the results showing significant and positive correlations among self-care of childcare teachers, resilience, and social support in the workplace. The findings are in accordance with previous studies of childcare teachers ,63 as waResilience showed a complete mediation, while the direct effect of social support in the workplace on self-care was not significant. This may explain the question that previous research addressed as to why colleague assistance programs were not so effective ,86. StudThe self-care behavior of professionals who are engaged in a caregiving role was perceived as being selfish as well as a luxury, despite the need for self-care to be considered vital to performing the professional role well and sustaining a state of well-functioning . The resSince social support can be helpful in building and strengthening the resilience of childcare teachers, as was identified in this study, education programs regarding the importance of support from peer groups and directors are urgent in regard to changing the culture of daycare centers (“culture of silence” —not offeThe result of the study, namely discovering the mediating role of resilience in the path between social support and self-care, places greater importance on the resilience of childcare teachers, which was rather recently given attention in Korea compared to other personal traits and characteristics. Teachers with low self-resilience are likely to view their lives negatively, lack self-confidence, be unable to manage or control emotions, and give up easily upon confronting a problem . ChildcaThe limitations of the study and suggestions for future studies are as follows. First, since this study was conducted on daycare teachers located in Seoul and Gyeonggi Province, there is limited generalizability of the results to other cities and thus, generalization of the results across countries should be done with caution. Second, the measure for self-care used in this study was not the one developed specifically for childcare teachers. Generating and developing a scale for childcare teachers would give greater insight since self-care encompasses various domains of life . Hence, This study discovered that resilience of childcare teachers was a pathway by which social support in workplace influenced teachers’ self-care. Little has been known regarding the relationship between self-care, resilience, and social support in the workplace, in particular for a childcare teacher, which is one of the most stressful occupations ,79. This


The histologic types differed in RNA expression of PRAME, KIT, SOX17, NANOG, KLF4, POU5F1, RB1, DNMT3B, and LIN28A (p < 0.01). The histologic types had concordant differences in RNA expression of the genes in the three datasets. Eight genes had overlap with a high RNA expression in at least two histologic types. In contrast, only seminoma (SE) had a high RNA expression of KLF4 and only embryonal carcinoma (EC) had a high RNA expression of DNMT3B. In conclusion, the meta-analysis showed that the development of the histologic types of TGCT was driven by changes in RNA expression of candidate genes. According to the RNA expressions of the ten genes, TGCT develops from NT over GCNIS, SE, EC, to the differentiated types of TGCT.There is no consensus as to how a precursor lesion, germ cell neoplasia in situ (GCNIS), develops into the histologic types of testicular germ cell tumor type II (TGCT). The present meta-analysis examined RNA expressions of 24 candidate genes in three datasets. They included 203 samples of normal testis (NT) and histologic types of TGCT. The Fisher’s test for combined  Testicular germ cell tumor type II (TGCT) is the most frequent malignancy in young adult men. Worldwide, oncologists follow the TNM  classification that separates TGCT into seminoma and nonseminomatous germ cell tumors (NSGCT) .The WHO classification of tumors of the urogenital system and male genital organs 2016 .In 1950, Dixon and Moore described that TGCT developed along two lines, i.e., one line for seminoma and the other line for NSCGT . With reIn 2004, in a meta-analysis of genes in TGCT, von Eyben pointed out that undifferentiated types of TGCT had high levels of CCND2 and low levels of RB1 which combined, programed the malignant germ cells for a high proliferation . A meta-After the WHO classification in 2016, an immunohistochemical study, in 2017, showed that precursor lesions for TGCT had a seminomatous immunophenotype . The GenIn general, solid tumors develop as a result of an accumulation of mutations or epigenetic changes in oncogenes or tumor suppressor gene. As TGCT does not have mandatory mutations, the development of the histologic types should be caused by regulation of gene expression of candidate genes. To further study how TGCT develops, we aimed to investigate whether the histologic types of TGCT differed significantly in RNA expression of candidate genes.Our meta-analysis examined RNA expression of 24 candidate genes in three datasets of TGCT. The three datasets are publicly available ,13,14. PThe three datasets were from studies that differed with respect to time, country, and methodology for the RNA measurements. Nevertheless, the histologic types had concordant RNA expressions of eight significant genes. Therefore, our meta-analysis combined the evaluation of the three datasets.p values for overlap of RNA expressions of concordant genes in the histologic types from the p values of the individual three RNA datasets, as shown in We combined PRAME, as shown in PRAME than EC, YST, and TER combined .Our meta-analysis found the histologic types had overlaps and differences in RNA expressions of the candidate genes. Normal testis (NT), GCNIS, and SE had overlap with high RNA expression of KIT, SOX17, and NANOG. In the second microarray, GCNIS and SE combined had a four times higher RNA expression of KIT than NT , as shown in GCNIS had a higher RNA expression than NT of three genes, i.e., SOX17 than EC, YST, and TER combined , as shown in GCNIS and SE combined had a four times higher RNA expression of NANOG than NT , as shown in NANOG differed stepwise among histologic types. SE and EC combined had a three times higher RNA expression of NANOG than NT and GCNIS combined .GCNIS and SE had a ten times higher RNA expression of CCND2, KLF4, and POU5F1 was high in SE but low in GCNIS. Furthermore, SE had a downregulated tumor suppressor gene, RB1, not downregulated in GCNIS. In contrast, both GCNIS and SE had high RNA expression of KIT, SOX17, and NANOG.SE had a set of overexpressed genes that differed from that in GCNIS. The RNA expression of CCND2 than NT , as shown in In the second microarray, SE had a twelve times higher RNA expression of POU5F1 than NT and GCNIS combined , as shown in SE and EC combined had a four times higher RNA expression of KLF4 than all other histologic types combined , as indicated in SE and EC had a seven times higher RNA expression of RB1 that was a quarter of that of NT and GCNIS combined , as shown in SE and EC combined had an RNA expression of DMT3B and LIN28A, but the RNA expressions were low in SE.EC had a set of genes with overexpressed RNA that differed from that in SE. EC had high RNA expressions of DNMT3B than all other histologic types combined , as shown in In the second microarray, EC had an 18 times higher RNA expression of LIN28A than SE , as shown in LIN28A differed stepwise among the histologic types. SE and EC combined had a six times higher RNA expression of LIN28A than NT and GCNIS combined .EC had a three times higher RNA expression of RB1 than EC , as shown in AFP than EC .Differentiated NSGCT, TER, YST, and CC, had genes that were overexpressed, whereas EC did not overexpress the genes. TER had a nine times higher RNA expression of CGB5 than EC .CC had an eleven times higher RNA expression of CDKN1A, CDKN2C. CCNE1, FOXD0, MYC, MYCN, MDM2, and PTTG1.In the second microarray, the histologic types did not differ significantly in RNA expression of The histologic types of TGCT had considerable overlaps of high RNA expressions of many genes, as shown in KLF4 for SE and DNMT3B for EC. The significant differences for RNA expressions of genes among histologic types indicate that the histologic types had different roles for the genes.The histologic types also differed in RNA expression of significant genes, as shown in In undifferentiated histologic types of TGCT, SE, and EC, high RNA expressions of significant genes are associated with the main clinical features such as self-renewal, hyperproliferation, and sensitivity to platin-based chemotherapy, as indicated in PRAME, KIT, SOX17, NANOG, CCND2, KLF4, POU5F1, RB1, DNMT3B, and LIN28A.Our meta-analysis of cancer genomics of 24 candidate genes of TGCT fulfilled the major goal to identify driver genes for initiation of GCNIS, the precursor lesion, and progression to the histologic types of TGCT. The histologic types differed significantly in RNA expression of Eight of the ten significant candidate genes differed concordantly in the three datasets regarding RNA expressions among the histologic types. Thus, our gene analyses indisputably support that NT develops over GCNIS to SE, EC, and the differentiated types of NSGCT.p values of the three datasets by the Fisher’s test [p findings were highly significant.Our meta-analysis evaluated the individual tissue samples in the three datasets for the RNA expressions. Combined in a meta-analysis of the r’s test , the sumCCND2 and NANOG and both genes have their gene locus on chromosome 12p . Thus, the RNA expressions of the two genes in part explains how the high copy numbers of chromosome 12p in TGCT contribute to the pathogenesis of TGCT.Undifferentiated types of TGCT had a high RNA expression of Although the recent WHO classification 2016 gave the precursor lesion an ambiguous name, GCNIS, the precursor lesion in reality is a localized malignancy. Half the GCNIS progress to microinvasive germ cell tumors within five years [CCND2, NANOG, and POU5F1 and, furthermore, both SE and EC had reduced RNA expression of RB1. The stepwise increase among GCNIS and the undifferentiated types of TGCT in levels of RNA expression of the three genes supports that the genes are driver genes in the pathogenesis of TGCT.SE can progress to EC. Both SE and EC, had high RNA expression of PRAME, KIT, and SOX17, not found in EC. EC had a high RNA expression of LIN28A not found in SE. SE had KLF4 as a genetic signatures and EC had DMNT3B as a genetic signature. However, the differences between SE and EC do not prove that SE and EC develop according to separate lines.SE and EC also differed. SE had high RNA expressions of The genes have two roles in the pathogenesis of TGCT. Overlap of high RNA expression of many genes in the histologic types bind the histologic types together. Differences in genes among the histologic types can be primary or secondary. Two genes had a high RNA expression only in one histologic type as a genetic signature. The two genes contribute to the specific characteristics of SE and EC.KLF4 is a genetic signature. KLF4 could block SE for differentiation into differentiated types of NSGCT, TER, YST, and CC. RNA expression of KLF4 had a longer half-life when the cells expressed NANOG and POU5F1 [For SE, d POU5F1 .DNMT3B is a genetic signature. The high RNA expression of DNMT3B causes EC to have more methylation of DNA than SE. Similarly, a proteomic study found that EC had high protein expression of DNMT3B [POU5F1 regulated expression of POU5F1 [DNMT3B is part of the difference in gene expressions as EC differentiates into TER, YST, and CC.DNMT3B methylates DNA. For EC, f DNMT3B . Methylaf POU5F1 . DownregOur results are in accordance with previous research. Undifferentiated and differentiated histologic types of TGCT differed in RNA expression of genes for pluripotency and, similarly, the two histologic types differed in protein levels of the genes in previous publications. Thus, TGCT mainly regulates the proteins at the step of transcription of the genes, like eukaryotic cells, in general, regulate the protein levels.DNMT3B and LIN28A [NANOG and POU5F1.Our findings are consistent with an experimental study that xenografted a seminoma cell line, TCam2, to the flanks of nude mice, and therefore the TCam2 thereby developed into EC ,20,21. DDNMT3B, LIN28A, NANOG, and POU5F1 than SE, similar to the mechanisms reported in the experimental study.The mechanisms caused EC, in our meta-analysis, to have similarly higher RNA expression of CCND2 and downregulated RB1. Our findings were also consistent with the observations that GCNIS overexpressed genes for pluripotency [In addition, a previous systematic review  showed tipotency , that MYipotency , and thaipotency .NANOG and POU5F1 than GCNIS, YST, and TER [NANOG, POU5F1, and SOX17 are like the findings in another study as EC differentiates into TER. Methylations of CpG dinucleotides in NANOG regulatory regions downregulated gene expression of NANOG [Further previous studies also showed that SE and NSGCT had higher expression of  and TER ,26. The of NANOG .Importantly, our meta-analysis showed that genes for pluripotency were upregulated in SE and EC and not only in GCNIS. The genes had a stepwise increase in RNA expression from NT over GCNIS, and SE to EC and a reduction in TER. The changes indicate that epigenetics regulates the RNA expression of the genes.Epigenetics for GCT can involve that methylations and acetylations of specific lysines on histone 3  in promoters and enhancers of the significant candidate genes regulate the RNA expression of the genes ,29,30.NANOG in cells with a bivalent promoter for NANOG.NANOG can act together with SOX2 and POU5F1 and the complex of transcription factors can bind to a motif in DNA . The comKLF4, LIN28A, MYC, NANOG, and POU5F1 can reprogram somatic cells into induced pluripotent stem cells, iPSC [A panel of four genes for pluripotency including ls, iPSC ,33. The As proof of the impact in patients with TGCT of genes for pluripotency, a set of genes for pluripotency could reprogram fibroblasts into iPSC in a report of a seminoma patient .Both EC and embryonal stem cells (ESC) have a high expression of genes for pluripotency , but iPSPRAME showed that PRAME inhibited that SE differentiated into TER [LIN28A showed that LIN28A stimulates expression of LIN28A, NANOG, and POU5F1 [Knockdown studies support that our candidate genes are important. Knockdown of into TER . Knockdod POU5F1 .Nettersheim argued that TGCT developed based on a bidirectional plasticity ,10. The The Genomic Cancer Atlas (TGCA) extensively reported RNA findings in relation to the development of TGCT . TGCA suThe Vogelstein group indicates that a total of three mutations of oncogenes and tumor suppressor genes are sufficient to drive the malignant transformation , and thaIn TGCT, NSGCT often includes a combination of the histologic types . HoweverFuture studies could further investigate how epigenetics regulates our candidate genes. It would be interesting to analyze whether RNA expression of the candidate genes among the histologic types of TGCT are associated with methylations and acetylations of lysines on histone 3 of promoters and enhancers for the candidate genes.Our meta-analysis has limitations. It is not a systematic review. Pathologists accept that genes for pluripotency are important in elder patients for the development of GCT ,42,43,44PRAME, KIT, SOX17, CCND2, KLF4, NANOG, POU5F1, DNMT3B, RB1, and LIN28A are important in the pathogenesis of TGCT.In conclusion, http:www.ncbi.gov.gen/query/ace.cgi?acc.GSE 8607). The first microarray included three samples of NT and 40 samples of SE.The meta-analysis evaluated two microarray datasets and an RNA sequencing (seq) dataset. The first microarray  used Affhttp:ww.ncbi.gov.gen/query/ace.cgi?acc.GSE 1818) and included three samples of NT, three samples of GCNIS, three samples of SE, five samples of EC, four samples of YST, four samples of YST, and one sample of CC.The second microarray  used Agi2 scale relative to a reference standard.The microarray summarized gene expression on loghttp://portal.gdc.cancer.gov/legacy-archieve/).The TCGA Research Network made the RNAseq database  availablThe RNAseq dataset included 72 samples of SE, 27 samples of EC, 13 samples of YST, and 16 samples of TER.Regarding the second microarray, we carried out inverse logarithmic transformation before we analyzed and reported the RNA expressions.The original studies of the three datasets followed the Declaration of Helsinki.The meta-analysis selected genes that previous publications had indicated were significant for the development of TGCT. Overall, our meta-analysis included six genes for pluripotency, five proto-oncogenes, four tumor suppressor genes, five genes for the three serum tumor markers, and four genes for biomarkers and a testis cancer antigen.KLF4, LIN28, MYC, NANOG, POU5F1, and SOX17 were among the genes with the highest gene expression in GCNIS [SOX2.The six genes for pluripotency, in GCNIS . Of thesCCND2, MDM2, MYC, NMYC, PTTG1, and CCNE1 [CDK1N1A, CDKN2C, PTEN, and RB1 [TGCT can include upregulated proto-oncogenes such as nd CCNE1 ,46,47,48 and RB1 ,55,56,57AFP, CGB5, LDHA, LDHB, and LDHC for the genes for the serum tumor markers, alpha fetoprotein (AFP), human chorionic gonadotropin (hCG), and lactate dehydrogenase , because the tumor markers are important for the treatment of TGCT. AFP and CGB5 were secondary to the histologic types, and therefore the present meta-analysis included only limited analysis of the tumor marker genes.Our list of candidate genes included KLF4, LIN28A, NANOG, and POU5F1 as genes for pluripotency. We took a low combined p value from more than one dataset to indicate a role of the gene for the development of TGCT. We defined a gene as a driver gene if RNA expression of the gene increased stepwise from NT to SE/EC. We defined other significant genes as passenger genes.The meta-analysis defined We defined RNA expression of a gene as insignificant if the RNA expression of the gene did not differ statistically significantly among NT and the histologic types of TGCT.A high RNA expression in two or more histologic types indicated an overlap of gene expression as a link as a histologic type progresses to the next. A significant change in RNA expression among histologic types indicated that the gene contributed to the development among the histologic types. We defined a significantly high RNA expression in only one histologic type as a genetic signature.p values based on the p values for comparisons of RNA expression of histologic types of the individual datasets using the meta-analytic method of the Fisher’s test [The meta-analysis gave priority to comparisons among gene expressions in histologic types of TGCT that could be based on more than one dataset. We showed the combined r’s test .p value < 0.05 as statistically significant.The meta-analysis did not substitute missing data in the datasets of RNA expression in a tissue sample. The RNA expressions of the candidate genes had non-parametric distributions. Therefore, the meta-analysis used the median values for the RNA expressions of the genes and the Kruskal–Wallis test as we compared two groups of histologic types. All statistical tests were two sided. We considered a We carried out the statistical analyses with Stata 14.2 .


In terms of biological behavior, gene regulation, or signaling pathways, there is a certain similarity between tumorigenesis and embryonic development of humans. Three germ layer structure exhibits the distinct ability to form specific tissues and organs.The present study set out to investigate the genetic mutation characteristics of germ layer differentiation-related genes using the tumor cases of the cancer genome atlas (TCGA) database.GATA6, FOXA2, GATA4, AFP) in the endoderm group, compared with the groups of ectoderm and mesoderm. Additionally, four members  within the Hedgehog signaling pathway genes showed a relatively higher mutation rate in the endoderm group than the other two groups.These tumor samples were divided into three groups, including the ectoderm, mesoderm, and endoderm. Children cases less than 9 years old accounted for a larger proportion for the cases in the ectoderm and mesoderm groups; whereas the middle-aged and elderly individuals (from 50 to 89 years old) were more susceptible to tumors of endoderm. There was a better prognosis for the cases of mesoderm, especially the male with the race of White, compared with the other groups. A missense mutation was frequently detected for the cases of ectoderm and endoderm, while deletion mutation was common for that of mesoderm. We could not identify the ectoderm, mesoderm, or endoderm-specific mutated genes or variants with high mutation frequency. However, there was a relatively higher mutation incidence of endoderm markers (TCGA tumors of ectoderm, mesoderm, and endoderm groups exhibit the distinct subject distribution, survival status, and genomic alteration characteristics. The synergistic mutation effect of specific genes closely related to embryonic development may contribute to the tumorigenesis of tissues or organs derived from the specific germ layers. This study provides a novel reference for exploring the functional connection between embryogenesis and tumorigenesis. It has commonly been assumed that the embryonic period occurred in the first 3 months of human intrauterine development . After tA germ layer is a primary group of cells involved in the preliminary formation of external and internal body shape , 4. In bInterestingly, a series of issues  during embryogenesis show a certain similarity with that of tumorigenesis –16. The https://cancergenome.nih.gov/) were divided into three groups, including the ectoderm, mesoderm, and endoderm. We presented the brief classification information of TCGA cases in Additional file According to the main process of three germ layer differentiation  within the TCGA database, we visualized the information of the top 50 mutated genes for the top 500 tumor cases, including mutation type, mutation frequency, gender, race, ethnicity, age at diagnosis, vital status, and days to death, in the ectoderm, mesoderm and endoderm groups, respectively. Also, we provided the combined Oncogrid results of these groups.Based on the “Oncogrid” module (http://bioinformatics.psb.ugent.be/webtools/Venn/) were applied for the above Venn diagram analysis.Based on the factors of clinical characteristics and sample size, we further classified each germ layer group into three subgroups. Then, these subgroups in each group were subjected to a Venn diagram analysis to obtain the commonly mutated genes or mutation sites, which then overlapped with the other two germ layer groups. Thus, the potential specific mutant genes/sites widely presented in the respective germ layer group were obtained. The “Launch Analysis” module of TCGA and on-line Venn tool , mesoderm markers , and endoderm markers  were selected. The data was visualized by an interactive heatmap. Besides, we utilized the “Survival Analysis” module to perform the prognostic analyses of overall survival (OS) and disease-free survival (DFS). Furthermore, we used the approach of the cBio Cancer Genomics Portal [We utilized the “Multiple Gene Comparison” module of gene expression profiling interactive analysis approach (version 2)  to profis Portal , 27 to pPTCH1, SMO, SHH, GLI1, GLI2, GLI3); Notch pathway ; TGFβ pathway ; Wnt pathway .Also, we performed the genetic mutation profile analysis of the embryogenesis-associated signaling pathways, including Hedgehog, Notch, TGFβ, and WNT pathways , 28, 29,We first divided the tumor cases of TCGA database into three groups, including ectoderm, mesoderm, and endoderm. Then, the distribution characteristics of gender, ethnicity, race, and age at diagnosis of tumor cases were analyzed, respectively. As shown in Fig. P < 0.00001). Nevertheless, there was no significant difference between the ectodermal and endoderm group in the clinical prognosis of cases (P = 0.097).Next, we performed the survival curve and log-rank analyses in the groups of ectoderm, mesoderm, and endoderm, respectively. As shown in Fig. P = 0.472). Nevertheless, we observed a better prognosis for the tumor cases with the race of White  and Black or African American  in the mesoderm group, compared with that in the endoderm or ectoderm group. Additionally, similar results were observed for the all male cases  or specific male cases with the white race , but not the female cases .Based on the two factors (race and gender), we further performed a series of subgroup analyses of survival status. There was no significant difference among the three groups in the clinical prognosis of Asian cases ; XIST, TSIX, RP11-85G18.6 (lincRNAs); and MIR1270, AC215219.2, AC090825. 1 (miRNAs), among these three groups. Then, we performed an intersection analysis of three sets of mutant genes by a Venn diagram. Additional file RP11-65L19.4 gene mutation in the ectoderm group occurred only in four breast cancer cases and two skin cancer cases; PPP1R2P9 AP00345.1 and C16orf95 genes in the mesoderm group mainly existed in the cases with uterus tumors. Similarly, only seven cases, including three bladder cancer cases, three lung cancer cases, and one esophagus cancer case, comprised the SNORA71E gene mutation in the ectodermal group  and 23 cases of glioblastoma multiforme (GBM) . Besides, the MEIS1 deletion mutation with the highest incidence in the mesoderm group occurred mainly in 81 patients with uterus corpus endometrial carcinoma. The followed survival analysis data also showed a better prognosis for the patients in the MEIS1 deletion mutation group, compared with the MEIS1 deletion non-mutation group . In the ectoderm group, The BRAF V600E missense mutation with the highest incidence was mainly presented in 288 cases with thyroid carcinoma and 206 cases with skin cutaneous melanoma . However, a similar result was detected for the skin cutaneous melanoma , but not thyroid carcinoma (P = 0.273).We should note that the above-stated mutant gene refers to a gene with one or more mutation sites. To focus on the characteristics of a specific genetic mutation site, we determined the mutation sites with high frequency in the ectoderm, mesoderm, and endoderm groups, respectively. Figure NRAS Q61K missense mutation in the ectodermal group mainly occurs in the patients with skin cancer, while splice acceptor GATA3 X309_splice mutations only present in the breast cancer cases derived from ectoderm  mainly occur in the cases with uterine tumors  in the endoderm group, compared with the groups of ectoderm and mesoderm. Nevertheless, we did not observe similar results for the markers of mesoderm  or the endoderm markers  in the ectoderm or endoderm group were more likely to be higher than the other groups. Moreover, the high expression levels of ectoderm or endoderm markers were correlated with the poor prognosis of overall survival and disease-free survival .We further analyzed the mutation profile of the markers of the ectoderm, mesoderm, and endoderm among the three germ layer-derived tumors, respectively. As shown in Fig. PTCH1 and SHH, four members  within the Hedgehog signaling pathway genes showed a relatively higher mutation rate in the endoderm group than the other two groups. Similarly, we observed the distinct mutation rates for the selected members within the Notch  than other groups. Additionally, there exists the distinct mutation prevalence of the three mutation sites of the TP53 gene, including the R175H, R273C, and R248Q, in the ectoderm, mesoderm, or endoderm-derived tumors.We found that several highly frequent gene variants exhibit the different distribution characteristics and genetic mutation profiles in the ectoderm, mesoderm, and endoderm groups. For instance, the male Caucasian cases in the mesoderm group with common deletion mutations showed a better prognosis than that in the ectoderm and endoderm groups with the missense mutations. The GATA6, FOXA2, GATA4, AFP) in the endoderm group showed a relatively higher mutation rate than that in the other groups. However, similar results were not detected for the ectoderm markers  and mesoderm markers . The subsequent expression and prognosis analysis data showed a high expression of the four ectoderm markers in the tumor tissues in the ectoderm group, which is also correlated with the poor clinical prognosis of cases. Thus, besides the mutation mechanism, the reactivation or functional enhancement of more embryonic development-related driver genes through expression regulation is worthy of further investigation.The determination of three germ layers is essential for the diversification of vertebrate cells , 11. GerOur results suggested that a high proportion of children patients aged from 0 to 9 years old in the groups of ectoderm and mesoderm, but a high percentage of tumor cases aged 50–89 years old in the endoderm group. How to understand the difference in the age distribution among the three germ layer groups? In the ectoderm group, tumors were mostly in the breast, nervous system, or epidermis. Our mesoderm group includes tumors of the kidney, ovary, blood, uterus, blood, prostate, and so on. And our endoderm group mainly comprises the tumors of the bronchus, lung, colon, pancreas, thyroid, esophagus, and liver. The susceptibility of children to the ectoderm and mesoderm-derived tumors may be associated with the factor of genetic development. And the gained environmental stimuli, such as air pollution, irregular diet, smoking, or drinking, may partly contribute to the propensity of middle-aged and elderly individuals to the endoderm-derived tumors.SMO, GLI1, GLI2, GLI3) and four markers of endoderm  showed a relatively high mutation rate in the endoderm-derived tumors, suggesting the potential role of these driver genes or specific signaling pathways in the occurrence and development of tumors in the tissues or organs derived from endoderm. But, it should be noted that the germ layer differentiation involves the synergy of multiple driving genes or signaling pathways. We only selected a few specific genes for the preliminary analysis, based on the published evidence. The expression, mutation, and oncogenic role analysis of more driver genes involved in the development of the germ layers or the specific organs are required for a more scientific investigation. In addition, it is meaningful to integrate the clinical-pathological indicators , disease risk factors , clinical treatment , and other factors to establish an efficient analytical model for the investigation of the crosstalk between the embryogenesis and tumorigenesis. During this process, the influence of promiscuous genes or non-critical mutation sites should be excluded.Although we have observed the differences in the distribution feature, mutation types, and clinical prognosis, there were no specific mutation sites with a high incidence that can effectively distinguish the tumors of different germ layers. Four members within the Hedgehog signaling pathway (In summary, we took advantage of TCGA datasets to explore the genetic mutation spectrum of different germ layer-derived tumors in terms of embryonic development. We failed to obtain the mutated genes or mutation sites with high frequency, which are relatively restricted to the ectoderm, mesoderm, or endoderm-derived tumors. There was a difference among the three germ layers in the issues of distribution characteristics, survival status, and mutation profile. The synergistic mutation effect of driver genes involved in the germ layer development may contribute to the functional connection between embryogenesis and tumorigenesis. Additional work is still required to further investigate the role of gene regulatory networks of germ layer specification and organogenesis in the development of specific cancers.Additional file 1: Fig. S1. Schematic diagram of early embryonic development.Additional file 2: Fig. S2. Classification of TCGA tumor cases.Additional file 3: Fig. S3. Overall survival curve analysis by race and gender. a Asian race; b White race; c Black or African American; d male; e males in the white race; f Female.Additional file 4: Fig. S4. Analysis of most mutated genes. a Top ten most mutated mRNAs, lincRNAs, or miRNAs in the ectoderm group; b Top ten most mutated mRNAs, lincRNAs, or miRNAs in the mesoderm group; c Top ten most mutated mRNAs, lincRNAs, or miRNAs in the endoderm group; d Top ten ectoderm, mesoderm, or endoderm-specific mutated genes. e Disease type of cases with the mutated RP11-65L 19.4, PPP1R2P9, AP000345.1, C16orf95, SNORA71E genes.Additional file 5: Fig. S5. Analysis of mostly mutated gene sites. a An Venn diagram analysis of the three groups; b Top ten ectoderm-specific mutation sites; disease type of cases with the missense NRAS Q61K and Splice Acceptor GATA3 X309_splice mutation sites. c Top ten mesoderm-specific mutation sites; disease type of cases with the deletion-3 prime UTR ADAR, UTR BHLHE40, UTR ZFX. d Top ten endoderm-specific mutation sites; disease type of cases with the frameshift CEP350 N2889Ifs*10, 3 Prime UTR CHIC2.Additional file 6: Fig. S6. Analysis of mostly mutation sites within PIK3CA and TP53 genes. aPIK3CA E545K, H1047R; bTP53 R175H, R248Q, R273C.Additional file 7: Fig. S7. Mutation analysis of mesoderm markers. a ectoderm; b mesoderm; c endoderm.Additional file 8: Fig. S8. Mutation analysis of ectoderm markers. a ectoderm; b mesoderm; c endoderm.Additional file 9: Fig. S9. Expression analysis of three germ layer markersAdditional file 10: Fig. S10. Survival analysis of three germ layer markers. a OS for ectoderm; b DFS for ectoderm; c OS for mesoderm; d DFS for mesoderm; e OS for endoderm; f DFS for endoderm.Additional file 11: Fig. S11. Mutation profile of Notch signaling pathway. a ectoderm; b mesoderm; c endoderm.Additional file 12: Fig. S12. Mutation profile of TGFβ signaling pathway. a ectoderm; b mesoderm; c endoderm.Additional file 13: Fig. S13. Mutation profile of Wnt signaling pathway. a ectoderm; b mesoderm; c endoderm.


Nigella damascena (Ranunculaceae) are tiny, regional protrusions covered by tightly arranged, non-secretory polygonal epidermal cells with flat, smooth and reflective surface, and are clearly visible even under ultraviolet light and bee vision. We also show that genes associated with cell division, chloroplast development and wax formation are preferably expressed in pseudonectaries. Specifically, NidaYABBY5, an abaxial gene with ectopic expression in pseudonectaries, is indispensable for pseudonectary development: knockdown of it led to complete losses of pseudonectaries. Notably, when flowers without pseudonectaries were arrayed beside those with pseudonectaries, clear differences were observed in the visiting frequency, probing time and visiting behavior of pollinators , suggesting that pseudonectaries serve as both visual attractants and nectar guides.Pseudonectaries, or false nectaries, the glistening structures that resemble nectaries or nectar droplets but do not secrete nectar, show considerable diversity and play important roles in plant-animal interactions. The morphological nature, optical features, molecular underpinnings and ecological functions of pseudonectaries, however, remain largely unclear. Here, we show that pseudonectaries of  Nigella damascena pseudonectaries, and their adaptive function in attracting specific pollinators to concealed nectaries with visual cues.Interspecies interactions, including those between plants and pollinators, can involve deception. The authors characterize the molecular development of  Nectaries can be floral or extrafloral and structural or nonstructural, depending on where they are located and how complex they are7. Floral nectaries with distinct structures are of particular interest to plant taxonomists and evolutionary biologists because they are usually tightly associated with pollination and because pollination is always the prerequisite of successful sexual reproduction10. In angiosperms, floral nectaries show considerable diversity in number, location , and morphology , and are believed to be results of parallel or even convergent evolution11. Despite the diversity, it is widely accepted that the nectary has been one of the most influential key innovations that promoted the diversification of many plant lineages14. Meanwhile, it has been found that, to secrete nectar, nectary cells should be able to synthesize and transport sugar and other necessary components17; genes regulating the formation and proper functioning of nectaries, such as CRABS CLAW (CRC) of the YABBY family, STYLISH1(STY1), STY2, and LATERAL ROOT PROMORDIUM (LRP) of the STY family, and SWEET9 of the SWEET family, therefore, are indispensable (at least in eudicots)20.Nectaries, the highly specialized glands or tissues that secrete nectar, are widespread in flowering plants  and play key roles in plant–animal interactions23. Like nectaries, pseudonectaries also show considerable variation in size (from ~0.01 to ~0.5 cm), number , location , color , and morphology 33. Yet, unlike nectaries, which have been investigated extensively in many aspects, pseudonectaries have not attracted sufficient attention until very recently34. Nevertheless, it has been shown that, acting as visual attractants or nectar guides, pseudonectaries of at least some plants play key roles in flower–animal interactions35. Pseudonectaries of Lopezia (Onagraceae) and Pelargonium (Geraniaceae), for example, can attract or guide their favorite pollinators  to proper positions and assist them to find the hidden nectary, suggestive of functional importance33. Pseudonectaries of carnivorous plants  and deceptive flowers  also function to deceive the visiting insects37. Some studies also tried to understand the evolutionary histories of pseudonectaries24, yet the available data are still insufficient for a general picture. Meanwhile, due to the lack of suitable study systems, little is known about the morphological nature, optical features, developmental process, molecular underpinnings, and ecological functions of pseudonectaries.In addition to nectaries, many plants produce false nectaries, or pseudonectaries, that imitate or mimic nectaries or nectar droplets but do not secrete nectarNigella, Trollius, Eranthis, and Xanthorhiza), and show considerable diversity in number, colour, morphology, and functions39  and many other functional technologies are applicable41. This suggests that the hypotheses related to pseudonectary development and evolution, if any, can be tested.The family Ranunculaceae is an excellent system for the study of pseudonectary development and evolution, for three reasons. First, pseudonectaries, especially those that are located on petals, have been documented in at least four genera , seems to be important for pseudonectary development: knockdown of it led to the complete losses of pseudonectaries and, therefore, significantly decreased the attractiveness of the petals. Our results not only provide a comprehensive portrait of pseudonectaries, but also clarify the differences between pseudonectaries and nectaries.Here, by using N. damascena appear to be long-stalked, hair-bearing, doubly geniculated, and vertically bilabiate structures  of petal developmentmis Fig. . Then, dmis Fig. . Notablymis Fig. , suggestmis Fig. , suggestmis Fig. .Fig. 3ThGOLDEN2-LIKE1 (GLK1) and CYTOKININ-RESPONSIVE GATA FACTOR 1 (CGA1)43, and the gene ontology (GO) categories that were enriched include “photosynthesis”, “chlorophyll biosynthetic process”, and “response to cytokinin”  and 2943 (169 TFs) up- and down-regulated genes, respectively 45, chloroplast development 43, wax formation 46, and leaf morphogenesis 47  by using DESeq2and 2943 69 TFs upand 2943 69 TFs upand 2943 69 TFs up)47 Fig. . BecauseNidaYAB5; Fig. YAB5)24 and because ectopic expression of adaxial/abaxial genes have been shown to play key roles in the formation of outgrowths on leaf-like structures52. To gain some insights into its function, we first performed detailed mRNA in situ hybridization studies -treated flowers  and TRV2-NidaYAB5-treated flowers with weak and moderate phenotypic changes, TRV2-NidaYAB5-treated flowers with strong phenotypic changes no longer produce pseudonectaries, whereas all other parts of the petals remain largely unaffected , although the contribution of bumblebees (Bombus lucorum) and wasps (Polistes dominulus) were also substantial (about 10% of the recorded times of visitations). When flowers with and without pseudonectaries  were arrayed side by side  are protrusive, emerald, glistening, and nonsecretory structures that mimic nectar drops but do not produce and secrete nectar. We also found that under UV light and bee vision, pseudonectaries are shiny and reflective, whereas all other parts of the petals and the flower are black or dark green. This, together with the observation on many other plants56, suggests that protrusion, coloration, reflectivity, and being nonsecretory may be the most important features that characterize pseudonectaries. Specifically, being protrusive, colorful, and reflective makes pseudonectaries visible and attractive to specific pollinators , whereas being nonsecretory makes pseudonectaries functionally distinct to real nectaries.In this study, by conducting careful morphological, anatomical, and micromorphological studies, we uncovered the morphological nature of pseudonectaries. We found that pseudonectaries of 56. Some other authors, however, insist that in addition to optical attraction, pseudonectaries can tell pollinators the position of hidden nectar or pollens, thereby guiding the visitation31. While these two scenarios are not mutually exclusive, there is a third viewpoint, which postulates that pseudonectaries function to distract undesirable visitors from rewards intended for pollinators53. In this study, we not only examined the optical features of pseudonectaries but also conducted controlled experiments. We found that pseudonectaries are indeed visible to pollinators , and that pseudonectaries from all the petals of a flower form a concentric circle, in which the two from each petal mark the entrance of the nectary chamber. When flowers without pseudonectaries were arrayed beside the ones with pseudonectaries, clear differences were observed in the visiting frequency, probing time and visiting behavior of the pollinators. This suggests that pseudonectaries can not only optically attract pollinators but also help them find the hidden nectar, thereby guiding their visitation. The distraction scenario of pseudonectaries, however, cannot be supported or rejected in this study.Several scenarios have been proposed for the ecological functions of pseudonectaries. Based mainly on morphological observations, many authors believe that, by forming protrusive, colorful, and glistening structures that mimic nectaries, nectar droplets or even pollinators, pseudonectaries serve to optically attract pollinators22. However, in many of these cases, it would become difficult even for the preferred visitors and real pollinators to find and reach the nectar. The formation of pseudonectaries, hence, would be one of the best strategies to attract preferred visitors without losing the hidden nectar.It is interesting that plants bearing pseudonectaries usually also produce real nectaries. If the functions of pseudonectaries are to attract pollinators and help them find the hidden nectar, why do plants hide their nectar? One explanation is that exposed nectar that can be easily foraged by both preferred and nonpreferred visitors and/or quickly dry off, thereby causing the waste of energy. The plants, therefore, have evolved various strategies to hide their nectar. Indeed, in most of the plants with floral pseudonectaries, nectar and real nectaries are very well hidden, either in the spurs or pockets of petals or in the tube formed by corollaCGA1, CYCP2;1, and YUCCA10), for example, seem to be required for the outgrowth of pseudonectaries, while those associated with chloroplast development  and wax formation  are indispensable for the formation of optical features46. Genes involved in nectary development, such as orthologs of STY1/2 and LRP, however, are not required, suggesting that pseudonectaries indeed have nothing to do with nectaries. In addition, we found that the abaxial gene NidaYAB5 is a key regulator of pseudonectary formation; knockdown of it not only led to complete losses of pseudonectaries but also eliminated all cell types associated with pseudonectories. It is possible that the gene was initially ectopically expressed on the adaxial surface to promote outgrowth but then controls all aspects of pseudonectary development by regulating its downstream genes. Notably, consistent with the widely accepted theory of leaf-like structure formation51, ectopic expression of NidaYAB5 can explain the reason why pseudonectaries were formed in the adaxial side of the lower lip of the petal.It is interesting that, by conducting extensive transcriptomic and functional studies, we identified the genes and networks that likely play key roles in the formation of the various aspects of pseudonectaries. Genes involved in cell division and cell expansion , were sown in soil (vermiculite:nutrient soil = 2:1) and grown under conditions of 24 °C, 60% relative humidity, and a 12-h-light/12-h-dark photoperiod.Seeds of N. damascena were dissected and photographed with a Nikon Model C-DSS230 stereomicroscope assembled with a Nikon digital camera DXM1200F . For micro-CT scanning, typical mature petals of N. damascena were mounted in a plastic box with wet absorbent paper paved at the bottom to prevent dehydration and were subject to take the high-resolution three-dimensional (3D) images (16-bit .tif format) by using a micro-CT scanner . Three-dimensional reconstructions were then performed with NRECON v.1.6 to remove the noise and convert the slices into an 8-bit .bmp format. Regions of interest were further selected by using CTAN v.1.10 and saved as datasets. The datasets were then loaded into CTVOX v.2.2 for the manipulation of 3D surface-rendered models. The internal sections of interest within the petal model were explored with the cutting/clipping shape editor and were captured as screenshots for showing. For SEM, typical petals of N. damascena at different developmental stages were fixed in fresh FAA , followed by dehydration in a graded water–ethanol series, and dried with a CO2 critical-point dryer. After being sputter-coated with gold, the dried petals were examined with a Hitachi S-4800 scanning electron microscope. For histological observation, paraffin and semi-thin section series were performed. For the former, the pretreatment, embedding, and sectioning of petals were carried out as described24 but sections were stained with safranine and fast green. For the latter, petals were fixed in 2.5% glutaraldehyde (pH 7.2) and embedded in Spurr resin. Serial sections were prepared on an ultramicrotome Leica EM UC7 and stained with 0.33% toluidine blue. The sections were photographed with a Leica DM5000B light microscope. The cell layer of the pseudonectary and lobe areas in the median transverse semi-thin sections was counted directly, and the width of three representative cells in each area was measured using tpsUtil and tpsDIG260. Significance evaluation  between adjacent stages was evaluated using the Wilcoxon rank sum test.For stereomicroscope photographing, typical mature petals of http://sourceforge.net/projects/gftbox) with two coordinated networks: the Polarity Regulatory Network and Growth Rates Regulatory Network62. The initial state was a mesh which consists of 36,000 finite elements with a grid showing the deformation. The elastic growth rate across the whole tissue is isotropic (Kpar = Kpar2 = Kper), and is higher in the region where pesudonectaries will be formed. Code is available upon request.The pictures in Fig. N. damascena41 and the Reads Per Kilo bases per Million reads (RPKM) values were calculated by RSEM63. The quality of all the 12 transcriptomes was reflected by reads mapping rates (83.19–84.57%) and Pearson correlation coefficients between triplicates (0.99–1.00). The genes showing RPKM ≥ 1 in at least one part are defined as expressed. Meantime, a gene is considered as specifically expressed if its RPKM ≥ 1.0 in a specific part but <1.0 in other parts. A gene is regarded as preferably expressed if its RPKM in a specific part is at least 1.5-fold higher than those in any other parts. For GO analysis, the protein sequences of genes that are preferably expressed in Part III were BLAST against the Arabidopsis thaliana non-redundant protein database with an E value cut-off <1e−1064. The GO terms of each gene were determined according to those of its best hit in A. thaliana. The GO enrichment was carried out using the agriGO program with false discovery rate ≤ 0.0565. Differential expression analyses between Part III and its neighboring areas were performed using DESeq2 package in R44, Wald test, Benjamini and Hochberg’s correction. The cut-off value of fold change and P value were 1.5 and 0.05, respectively. The P values were adjusted using the Benjamini–Hochberg procedure66.The four parts of S9 petals, each with three biological replicates, were subjected to total RNA extraction using the SV Total RNA Isolation System (Promega). A total of 12 libraries were constructed independently for single-end 100-bp-long reads sequencing on Illumina HiSeq2000. The clean reads of 12 samples were separately mapped to the reference transcriptome of NidaYAB5 following the procedure as described24. VIGS was applied to study the function of NidaYAB5. The same gene fragment used for the in situ hybridization was amplified and introduced into the TRV2-based pYL156 vector, which was electroporated into Agrobacterium tumefaciens strain GV3101. The detailed procedure of construct transformation and plant treatment followed the previous study41. A total of three rounds of treatments were conducted with TRV2-NidaYAB5  were arrayed side by side  were determined by using nonparametric Wilcoxon signed rank test.Pollination studies were carried out in an open area of the Institute of Apicultural Research, Chinese Academy of Agricultural Sciences from 9:00 a.m. to 5:00 p.m. in four sunny and calm days of July, 2017. Visitors being able to successfully transfer pollens from pollen sac to stigma were regarded as pollinators, while those that suck nectar but do not transfer pollens were regarded as nectar robbers. The most effective pollinators are the pollinators that have the highest frequency of visitation and longest time of probing. For the experiment, flowers with and without pseudonectaries (i.e., 18 mock flowers and 18 TRV2-ide Fig. . For eacFurther information on research design is available in the Peer ReviewSupplementary InformationReporting summaryDescription of Additional Supplementary FilesSupplementary Dataset 1