Memory processes could account for a significant part of the variance in language performances of hearing-impaired children. However, the circumstance in which the performance of hearing-impaired children can be nearly the same as the performance of hearing children remains relatively little studied. Thus, a group of pre-school children with congenital, bilateral hearing loss and a group of pre-school children with normal hearing were invited to participate in this study. In addition, the hearing-impaired participants were divided into two groups according to their working memory span. A language disorder assessment test for Mandarin-speaking preschoolers was used to measure the outcomes of receptive and expressive language of the two groups of children. The results showed that the high-span group performed as good as the hearing group, while the low-span group showed lower accuracy than the hearing group. A linear mixed-effects analysis showed that not only length of rehabilitation but also the memory span affected the measure of language outcome. Furthermore, the rehabilitation length positively correlated with the measure of expressive language only among the participants of the high-span group. The pattern of the results indicates that working memory capacity is one of the factors that could support the children to acquire age-equivalent language skills. A number of demographic, instrument and medical factors, such as socioeconomic status, parent-children interaction, use of hearing devices and participation in rehabilitation programs, have been identified to relate to language functioning in children with hearing impairment . HoweverFor ordinary children, growth in language skills depends partly on working memory ability . ChildreLanguage skills can be divided into receptive and expressive abilities, and the two types of language skills dissociate to some extent . For aveA set of previous studies have shown that children with congenital hearing loss have lower scores on the tests of working memory than do children with normal hearing . MoreoveOn the other hand, congenital hearing loss is not limited to specific language populations. The prevalence of bilateral hearing loss is similar across regions worldwide (1/1000 to 3/1000), according to a report by In order to test the above hypothesis, this study was designed to collect information about span of working memory and outcome of the two types of language skills from a group of Mandarin-speaking children with mild to profound hearing impairment. The children’s language performances were also compared with another group of hearing children. Most importantly, data analysis was conducted with the purpose of examining the influence of the memory span on the relationship between rehabilitation and outcomes of the two types of language skills in the hearing-impaired children.The participants were 39 children with congenital, bilateral hearing loss and 20 children with normal hearing. All of the participants attended kindergarten. The hearing children and 37 of the hearing-impaired children were in regular classes. The hearing children were from three different schools, and those who went to the same school were also classmates. The schools of the hearing-impaired children were different from one another. Two of the hearing-impaired children were from two schools for the hearing impaired. The chronological age of the hearing-impaired children ranged from 5:0 to 6:1 (mean = 5:5), and the number of girls and boys were 18 and 21. Children with multiple disabilities were excluded from this study. All of the hearing-impaired children were wearing hearing aids before and at the time of attending the present study. The age of the hearing participants ranged from 5:11 to 6:5 (mean = 6:2), and the number of girls and boys were 10 and 10. All of them were native speakers of Mandarin, and according to their school teachers, none of the hearing participants was found to have sensory, neurological or psychological disorders. This study was carried out in accordance with the recommendations of IRB Review Guidelines, Chang Gung Medical Foundation Institutional Review Board (IRB No. 103-3749D) with written informed consent from the parents of the participants.All of the hearing-impaired children have joined a rehabilitation program that adopted an auditory-verbal approach. For each of the children, once the rehabilitation program began, an Ongoing Assessment Form was used to keep track of the child’s development of comprehensive and expressive language. The form was originally designed by When the hearing-impaired child began to be able to comprehend and respond to spoken words, the child’s working memory span was monitored and documented in each session of rehabilitation. The technique used to evaluate the child’s memory span was the same as the “Secret Code” activity described by n = 20). Otherwise, the children were assigned to the low-span group (n = 19). Results of a Kruskal–Wallis test indicated that the memory span of the high-span group  and the memory span of the low-span group  differed significantly (p < 0.01). On the other hand, the age of the high-span group  and the age of the low-span group  were similar (p = 0.06), and the hearing group was older than the high-span group (p < 0.01) and the low-span group(p < 0.01). The number of children in each level of hearing loss was counted for each group of memory span. As listed in Table 1, the levels of hearing loss ranged from mild to profound in both groups of the participants. In addition, information about the length of rehabilitation in months was collected for each of the hearing-impaired participants.A previous normative study indicated that 6-year-old Taiwanese children could recall 5–6 items in the WISC-III Digit Span sub-test . TherefoThe revised version of the Language Disorder Assessment for Preschooler (LDAP-R) was administered to the participants of this study. The assessment instrument is the most widely used standardized test to evaluate receptive and expressive language skills of pre-school children in Taiwan . The LDAThe sub-test of receptive language consists of 37 items. In 23 of the receptive items, each of the participants was presented with a picture of multiple different objects. Then the participants heard a sentence and were asked to point out the object described by the sentence. In 10 of the items, the participants listened to a short story and answered questions about who, where, when and what. In three of the items, the participants listened to a question like “Which is the place for eating, a restaurant or a store,” and gave their answers by making a judgment. In one of the items, the participants were asked to act out a sentence like “Turn your head, and then open your mouth.”The sub-test of expressive language consists of 46 items. In 24 of the expressive items, the participants were presented with a picture and were asked to describe what happened in the picture as much as they could. In 11 of the items, the participants answered questions of common knowledge. In six of the items, the task was sentence repetition, and in another five of the items, the participants were asked to repeat a description of a picture.The test was administered individually in a room. The room was kept as quiet as possible while background noise was mainly from an air conditioner. The noise level was at 41 dB(A), measured by a sound level meter . Before the test began, the purpose of this study and the tasks that the participants were going to perform was fully explained to the parents or the caregivers of the participants. The participants consisted of children with hearing impairment and children with normal hearing. Therefore, the test giver also made sure that the participants were comfortable to take the test. By following the manual of the assessment instrument, the sub-tests were administered in a fixed order in which the receptive sub-test was administered before the expressive sub-test for every participant. It took the participants about 20–30 min to finish the test.For each participant, the percentage of items that were corrected answered in each of the two sub-tests was calculated independently, and the results were taken as accuracy. Then the accuracy values from each of the two hearing-impaired groups were compared, respectively, to the accuracy values from the hearing group. Because the majority of the participants were sampled from an atypical developmental population and the group size in this study was relatively small, box-plots were used to present the distribution of the accuracy for each group of the participants. In addition, the Kruskal–Wallis test was used to conduct the statistical testing of difference between the hearing group and the hearing-impaired groups.In order to examine whether or not the effect of rehabilitation on the measure of language outcome was influenced by the children’s working memory span, a linear mixed-effects model analysis (LMM) was performed for the hearing-impaired group. The LMM analysis has been used in some previous studies to examine possible relationships among multivariate cognitive and linguistic measures from the same participants in different conditions . TherefoAll of the statistical analyses were performed by using The following report of the participants’ performances was organized according to the type of the sub-tests. The results from the test of receptive language were reported before the results from the test of expressive language. Then, whether or not the memory span influenced the relationship between rehabilitation and the two measures of language outcome was reported.Figure 1, the low-span group showed lower accuracy than the high-span group and the hearing group. In most of the participant in the low-span group, the accuracy was lower than 75%. On the contrary, the accuracy was higher than 75% in more than half of the participants from the high-span group and the hearing group.As shown in 2 = 9.27, p < 0.01). Results of planned comparisons confirmed that the difference between the low-span group and the hearing group was significant (p = 0.03). On the other hand, the accuracy was similar between the high-span group and the hearing group (p = 1.00). The pattern of the results verified the idea that the memory span of the children with hearing loss has an influence on the children’s performances in language comprehension.The result of statistical testing showed that there were significant group differences . Results of planned comparisons showed that the high-span group and the hearing group were not different from each other statistically (p = 0.39). In contrast, the group of low memory span showed lower accuracy than the group with normal hearing (p < 0.01).As shown in The pattern of the results showed the advantage of the hearing-impaired children with high memory span. The hearing-impaired children could perform as good as the hearing children in language comprehension and production when they have an enough span of temporary memory to process linguistic inputs. In addition, the variation was large in the low-span group while the variation was relatively small in the high-span group. It suggested that the children in the high-span group were less likely to deviate from the typical trajectory of language development than those in the low-span group.p-value of the random effect, Likelihood Ratio Tests of the full model with the effect in question against the model without the effect in question were conducted.Linear mixed-effects models, as described above, were constructed by using memory span and sub-test type as the fixed effects and by using rehabilitation length as the random effect. Visual inspection of residual plots showed that deviations from homoscedasticity or normality were not noticeable. To attain the F = 6.77, p = 0.01]; (2) the sub-test type was not significant ; (3) the effect of rehabilitation length was significant (p < 0.01). Overall, the results indicated that both the rehabilitation length and the memory span affected the measure of language outcome.The results of the LMM analysis and the likelihood ratio tests showed the followings: (1) the memory span was a significant predictor of the language outcomes [r = 0.53, p = 0.02) but not the low-span group . The correlation between the rehabilitation length and the accuracy in the receptive sub-test was not statistically significant either in the high-span group  or the low-span group .In order to verify whether or not the effect of rehabilitation on the measure of language outcome was influenced by the children’s working memory span, Pearson’s correlations between the rehabilitation length and two measures of language outcome were computed respectively for the high-span and low-span groups. The accuracy in the expressive sub-set positively correlated with the rehabilitation length in the high-span group (The results of the present study support the idea that working memory capacity affects outcomes of language learning of Mandarin-speaking children with hearing loss. For the children with hearing loss and high memory span, their performances were almost as good as the hearing children, not only in receptive language but also in expressive language. In contrast, the children with hearing loss and low memory span showed lower scores than the hearing children in the receptive and expressive language tests.The above finding suggests that the outcome of language learning of the hearing-impaired children depend partly on the children’s working memory capacity. The children with hearing loss could efficiently assess task-relevant information with the help of a large span of working memory. As a result, they are at an advantage when taking the language tests than those who have a small span of working memory. It is consistent with the literature which shows that there is a positive correlation between memory function and language development among ordinary children . In otheHowever, for children with hearing loss, the outcome of language learning could be more sensitive to the children’s working memory capacity. Some previous studies have shown that children with congenital hearing loss have lower scores on the tests of working memory than do hearing children . When thCapacity of working memory could be enhanced by systematic training and learning . AccordiThis study also has implication for the design of rehabilitation plans for children with hearing impairment. Recently, a question of whether or not measures of working memory should be incorporated into hearing aid decision was raised . WorkingIn summary, the results of the present study verify the hypothesis that language acquisition of Mandarin-speaking children with hearing loss is under the influence of working memory capacity. Not only acquisition of receptive language but also acquisition of expressive language relates to the specific memory ability. Sufficient working memory capacity is one of the factors that could support the children to acquire age-equivalent language skills. However, whether or not working memory capacity acts as a mediator needs to be verified in future research.Both authors contributed equally to this work: ML shaped the design and conception of the study, performed data analysis and wrote the first draft; P-HC designed the study, carried out data acquisition and edited several sections.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, this relationship appeared to differ by age . Thus, in younger participants , IGF-1 was associated with lower odds of hearing impairment  after adjustment for a range of potential confounders. Among people ≥60 y (n = 2990) there was a non-significant ‘J’-shaped association. Our observational evidence that higher levels of IGF-1 appeared to confer some protection against hearing impairment in some older adults warrants replication in other prospective cohort studies.Insulin-like Growth Factor 1 (IGF-1) is associated with cardiovascular disease, itself a risk factor for hearing impairment, and, in animal studies, molecular evidence suggests a role for IGF-1 in hearing function. However, the link between IGF-1 and the occurrence of hearing impairment is untested in population-based studies of humans. A total of 4390 participants aged ≥50 y  from the English Longitudinal Study of Ageing provided serum levels of IGF-1 in 2008 and again in 2012. Hearing acuity was assessed by an objective hearing test (HearCheck handheld device) in 2014 when the prevalence was 38.2%. In the full cohort, IGF-1 was not associated with subsequent hearing impairment (OR For example, in the United States3 and in the United Kingdom4, around two-thirds of people older than 70 years report hearing loss. As well as being an important disease entity in its own right, it has marked economic, social, and health ramifications. Thus, poor hearing has a deleterious impact on physical functioning5, cognitive functioning6, social and family life7 and mental health8. In the US, aggregate societal costs, including not only medical and assistive device costs but also reduced work productivity, were estimated to be $4.6 billion over the lifetime of affected people9. With the condition essentially incurable, the focus becomes identification of potentially modifiable risk factors.Age-related hearing loss (presbycusis) is the most common cause of adult auditory deficiency and it is a major cause of disability11 and selected cardiovascular disease risk factors – smoking12, diabetes13, overweight14 – have been associated with hearing impairment both in cross-sectional14 and longitudinal studies12. Lower levels of IGF-1 have been shown to be associated with elevated rates of cardiovascular disease15. Second, molecular data from animal studies suggest a role for IGF-1 in the development and protection of hearing function17. Rescue of hair cells from apoptosis via IGF-1 receptor-mediated actions or by downregulation of pro-apoptotic gene expression, and regulation of glucose transporters in the outer cells have been proposed as underlying mechanisms18. Third, we have recently shown that physical stature, which is related to IGF-1 levels20, is related to hearing impairment21, with taller individuals less likely to experience hearing loss, which is consistent with results of two other studies23. While lower height in itself is of course not a risk factor for hearing impairment, the pre-adult characteristics that it proxies - inadequate nutrition, illness and poverty – may be.Evidence from a range of scientific disciplines implicates Insulin-like Growth Factor 1 (IGF-1) in the aetiology of hearing loss. First, atherosclerosisprima facie case for a link between circulating levels of IGF-1 and hearing impairment, to our knowledge, this relationship has not been examined in a well-characterised, free-living population of humans. This was the purpose of the present study in which we utilised data on repeat measurement of IGF-1 levels and subsequent objectively measured hearing impairment in the English Longitudinal Study of Ageing (ELSA)24.Despite a strong 24 at recruitment in 2002/3 (wave 1). Data are collected biennially using computer-assisted personal interviews (CAPI) and self-completion questionnaires, with additional nurse visits every 4 years for the assessment of biomedical data. Ethical approval for all data collection was granted from NHS Research Ethics Committees under the National Research and Ethics Service25 and all participants provided informed consent. All methods were performed in accordance with approved guidelines and regulations.ELSA is an on-going, prospective cohort study of a representative panel of men and women living in England aged ≥50 yearsThe ‘baseline’ of the present study was Wave 4 (2008). The analysis was based on the sample with complete data both at baseline (2008) and at follow-up 2014). As indicated in Fig. 4. As ind26. Circulating IGF-1 values were reported as whole numbers (ranging from 3 to 200) expressed as nanomoles per litre (nmol/L). Total IGF-1 includes IGF-1 that is bound to IGF binding proteins (99%) and free IGF-1.Serum total IGF-1 was measured for the first time in ELSA in 2008 (Wave 4) - which represents study ‘baseline’ herewith - and 2012 (Wave 6) when blood samples were drawn from participants at rest who had not eaten, smoked, or drunk alcohol in the preceding 30 minutes. IGF-1 was batch-assayed with the DPC Immulite 2000 method™ device27. This screening tool has shown good sensitivity (ranging from 78% to 92%) and acceptable to good specificity (ranging from 62 to 95%) in two evaluation studies comparing results of the screener to pure tone audiometry28. This is a simple, low-cost, handheld appliance which produces a fixed series of three pure high frequency (3 kHz) tones and three mid-frequency (1 kHz) tones. Participants who wore hearing aids were asked to remove them for the test. With the interviewer holding the HearCheck™ device against the participant’s left ear, they were asked to indicate when they heard a tone when a 1 kHz frequency sound was made at three decreasing intensities . This was followed by three tones  for a 3 kHz sound. Both tests were then repeated for the right ear. The main outcome, hearing impairment, was defined as hearing fewer than 6 tones in the best hearing ear, i.e. a hearing threshold of 20 dB HL at 1 kHz or 35 dB HL at 3k Hz. A secondary outcome considered in sensitivity analysis was “moderate to severe” hearing impairment, defined as hearing threshold of 35 dB HL at 1 kHz or 55 dB HL at 3k Hz. Data on use of hearing aid was recorded as ‘Yes, most of the time’, ‘Yes, sometimes’, and ‘No’. The vast majority of participants did not wear a hearing aid , while 8% wore a hearing aid most of the time and 6% some of the time.At wave 7 (2014), all study members were invited to take part in an objective hearing test unless they reported use of a cochlear implant or had an ear infection. Hearing acuity was tested with the Siemens HearCheckParticipants underwent a face-to-face CAPI at baseline (2008), including a question to self-rate the participant’s hearing (including hearing aid if wears one) on a 5-point scale (‘poor’ to ‘excellent’). Self-reported hearing impairment at baseline was defined as rating hearing as ‘poor’ or ‘fair’.29. Educational attainment was classified as low (compulsory schooling), medium (up to high school diploma) and high (university degree or higher). Height and weight were measured directly during the nurse visit (concomitant with the blood test) and body mass index (BMI) was defined as weight (kg) divided by height squared (m2).Covariates were also assessed through CAPI at baseline (wave 4). Self-reported health behaviours considered as covariates in the present analyses included smoking status  and leisure-time physical activity (5 levels from low to high). Cognitive function was assessed using a battery of tests: memory measured using a word-list learning test (immediate and delayed), and executive functioning ascertained using a word-finding task . A summary score of memory functioning was used as covariate for cognitive function. General health was self-rated on a 5-point scale ranging from ‘poor’ to ‘excellent’. To account for change in cognitive function and in general health between baseline and assessment of hearing, we also created a change variable equal to the difference between the score at baseline and at follow-up. Depression symptoms were ascertained during the CAPI using the eight-item Center for Epidemiologic Studies Depressive CES-D scale and depression was defined as anyone scoring 4 or above30, as reproducibility of IGF-1 over 4 months to 15 years can be only moderate leading to attenuation of the association if based on a single measurement33. Baseline characteristics were compared across IGF-1 quintiles (sex-specific) by analysis of covariance. To summarise the relation between IGF-1 levels and hearing impairment, we computed odds ratios (ORs) and 95% confidence intervals using logistic regression analyses. In Model 0, we adjusted the association for baseline age (continuous) and sex. In Model 1, we further adjusted the ORs for height, smoking status (3 categories), BMI (continuous), cognitive function (continuous), educational level , physical activity (5 levels) and self-rated health (5 levels) – all statistically significant predictors of hearing impairment. In the main analysis, we used self-reported hearing impairment at baseline as a covariate in Model 2 , to account for pre-existing hearing impairment. In a secondary analysis, we excluded participants who self-reported hearing impairment at baseline , focusing on a sample likely free of hearing impairment at baseline (hearing acuity rated as very good or excellent) to model the ‘incidence’ of new cases of hearing impairment. We performed three additional sensitivity analyses using 1) the average of two IGF-1 measurements only (excluding participants with only one measurement); 2) IGF-1 measured at baseline (2008) only; 3) modelling moderate to severe hearing impairment.We used the average of the two IGF-1 measurements at both wave 4 and 6 when available, or either of these if only one was available: 747 were based only on wave 4 measurement, and 558 only on wave 6 measurements, hence 3085 (70%) were the average of wave 4 and wave 6 measurements. Taking the average of two measurements can limit fluctuations due to measurement error or biologic variability and lead to more precise estimates of the relationship with the outcome34 and tested nonlinearity by using the likelihood ratio test, comparing nested models with a linear or linear and cubic spline terms. Computations were made using the SAS macro %lgtphcurv935 for the logistic model and the p-value for curvature was reported .To test for a linear trend, we also modelled IGF-1 as a continuous variable. We examined potential nonlinear relations using restricted cubic spline transformations36, we used this as our threshold. All statistical analyses were performed using SAS software version 9.3 .We explored the association of IGF-1 levels with hearing impairment according to the age at IGF-1 measurement and gender. With age-related hearing impairment being a condition whereby the majority of people are first affected around the ages of 60At baseline, the mean (SD) age of the population was 64.2 (8.0) years, ranging 50 y to 90 y, and comprised 55% women. Compared to the participants who were included in the analytical sample, people who were not included were older , but the sex ratio was similar.The average serum IGF-1 value was somewhat higher in men (17.1 [5.2] nmol/L than women (15.4 [5.0] nmol/L). Baseline characteristics across quintiles of IGF-1 are presented in Table In the 2014 survey, of 4390 participants there were 1680 cases of objectively measured hearing impairment . The association between IGF-1 levels and subsequent hearing impairment are shown in Table When we excluded the participants who self-reported poor, fair or good hearing at baseline to get closer to modelling the ‘incidence’ of hearing impairment among participants free of hearing impairment at baseline, there were 541 incident cases in 2176 participants. These participants were younger (mean age 62.9 y), more often women (60%) and displayed overall a more “favourable” health and behaviour profile. The results in these analyses also showed no significant association overall but a strong interaction with age was detected (p = 0.005). In the younger group (<60 y), the association was negative too and somewhat stronger than in the main analysis, with an OR of 0.38  when comparing the upper to the lower quintile of IGF-1. In the older group, evidence of a ‘J’-shape was clearer with a p-curvature = 0.02 in the fully adjusted model and increased odds of hearing impairment were found in the top IGF-1 quintile compared to the bottom , may have led to residual confounding and biased the associations. Moreover, we did not have information on noise exposure which would have been an important factor to take into account. We also used an average of two measured of IGF-1 taken 4 years apart or only one measurement if only one was available, in order to improve accuracy and increase sample size. This may have attenuated the results but sensitivity analysis including only participants with both measurements led to similar conclusions but not statistically significant associations, and similarly when we only used IGF-1 measurement in 2008. We also had to exclude an important part of the sample who had missing data (26% of participants with complete baseline data did not have a follow-up measurement) and, compared to those included, excluded participants were older, less educated, engaging in less healthy behaviours  and more likely to develop hearing impairment, hence also potentially biasing the observed estimates. However, even if those included participants are different from the general population, as long as there is enough variability in the measurements, generalisability of the associations should not be entailed. The point has recently been made in the UK Biobank study which has a response rate of only 5.5%46. Some studies have compared exposure-disease associations according to survey participation status, showing similar magnitudes. For example in the UK Health and Lifestyle Surveys, 29% of participants did not participate in resurvey 7 years later, but the associations between baseline cardiovascular risk factors such as hypertension and cardiovascular mortality did not differ by participation status47. We also acknowledge that this study does not strictly assess incidence of hearing impairment as hearing was tested objectively only at follow-up, while we used questionnaire data to assess self-rated hearing at baseline. Finally, we measured total IGF-1 levels, which are considered a crude estimate of the biologically active hormone, whereas free IGF-1, which represents less than 1% of total IGF-1, is believed to be the most active form and to have greater physiological and clinical relevance than total IGF-141. However, results from animal studies showing a role in the development and maintenance of hearing function also measured total IGF-117, and total IGF-1 has been used as a biomarker in most epidemiological studies in relation to chronic disease15.The main strength of this study is that it is a large, nationally representative sample of older people living in England with good follow-up. IGF-1 levels were measured at two separate occasions, and taking the average of these two measurements is a way to limit bias in the relationship with hearing impairmentIn people at the lower end of the older-aged spectrum (aged 50 to 60 years), higher levels of IGF-1 were associated with lower odds of hearing impairment and most particularly in participants free of hearing impairment at baseline, where a moderately high effect size was found. This is the first population-based study to show a linear relationship between IGF-1 and hearing impairment. We found significant 14% decrease odds for a relatively small increase in IGF-1 (5nmol/L). The ‘J’-shaped relationship observed at older ages may partly reflect the association of IGF-1 with vascular health. With currently no existing cure for hearing loss, identification of relevant risk factors remains a priority. More observational studies are needed to confirm our results before considering the possibility of testing low-dose supplementation in IGF-1 for long-term prevention of hearing impairment.Supplemental Figures 1-3


GJB2 is prevalent in East and Southeast Asians and may lead to mild-to-moderate hearing loss with reduced penetrance. To investigate the pathogenic mechanism underlying this variant, we generated a knock-in mouse model of homozygous p.V37I by an embryonic stem cell gene targeting method. Auditory brainstem response test showed that the knock-in mice developed progressive, mild-to-moderate hearing loss over the first 4–9 months. Overall no significant developmental and morphological abnormality was observed in the knock-in mouse cochlea, while confocal immunostaining and electron microscopic scanning revealed minor loss of the outer hair cells. Gene expression microarray analysis identified 105 up-regulated and 43 down-regulated genes in P5 knock-in mouse cochleae (P < 0.05 adjusted by the Benjamini & Hochberg method), among which four top candidate genes with the highest fold-changes or implication to deafness Fcer1g, Nnmt and Lars2 and Cuedc1 were verified by quantitative real-time PCR. Our study demonstrated that the homozygous p.V37I knock-in mouse modeled the hearing phenotype of the human patients and can serve as a useful animal model for further studies. The differentially expressed genes identified in this study may shed new insights into the understanding of the pathogenic mechanism and the phenotypic modification of homozygous p.V37I.The homozygous p.V37I variant in  GJB2 are the most frequent, accounting for 20–30% of cases in many countries and ethnic groups2Hearing loss is the most common sensory disorder in humans, affecting approximately 2–3% of childrenGJB2 encodes the gap junction subunit protein connexin 26 (Cx26). It comprises 226 amino acids with 4 transmembrane domains. Together with over 20 other connexin subunit proteins, Cx26 forms hexameric hemichannels (connexons) on opposite cellular membranes, allowing intercellular communication between adjacent cells. In cochlea, Gjb2 is expressed in the non-sensory cells and structures including the supporting cells, the stria vascularis, the spiral ligament and the spiral limbus4Gjb2 was implicated in many auditory processes including potassium recycling, nutrient and energy supply, generation of endocochlear potential and maintenance of the endolymphatic homeostasis in the inner earGJB2  have been reported causing recessive non-syndromic deafness DFNB1 (MIM# 220290). The hearing loss associated with DFNB1 was highly variable, ranging from birth to adulthood in age of onsets and from mild to profound in degree of severity. Based on the deleterious impact on the protein structure, most of the GJB2 mutations can be categorized into truncating mutations  that often lead to null alleles of GJB2 and non-truncating mutations  that only affecting single or multiple amino acids. Genotype-phenotype correlation studies showed that the degree of hearing loss associated with non-truncating mutations in GJB2 was significantly less severe than that with truncating mutationsGJB2, is of our particular interest as it is prevalent in East and Southeast Asians, with allele frequencies of 0.062 in Chinese Hans, 0.043 in Thai, 0.01 in Japanese and 0.006 in Korean810In vitro studies showed that Cx26 with truncating mutations cannot target to the cell membrane and has severely deteriorated gap junction activity1013GJB2 has been shown to be strongly associated with mild-to-moderate hearing loss. This variant, however, has reduced penetrance which was estimated to be 17%To date, over 300 mutations in MIM# 22020. The heGjb2, which exhibits a rather early and severe hearing phenotype. Targeted ablation of Gjb2 in mouse inner ear resulted in elevation of the hearing thresholds by 40–50 dB at postnatal day 21 (P21), stalled postnatal development of the organ of Corti as the tunnel of Corti and the Nuel’s space were never opened, and massive cell death of the sensory epithelium in the middle and basal turns of cochlea15Gjb2, which may help to better understand why homozygous p.V37I is associated with milder degree of hearing loss than truncating mutations and what factors may contribute to the incomplete penetrance of homozygous p.V37I.The pathogenic mechanism underlying the milder degree of phenotype and incomplete penetrance of the homozygous p.V37I variant remained unclear. So far, animal models of DFNB1 have only been established for conditional null alleles of P < 0.05) increase of the hearing thresholds at 4, 24 and 32 kHz. This increase was statistically significant since 38 weeks at 8 and 16 kHz. Our results showed that the homozygous p.V37I knock-in mice modeled the progressive, mild-to-moderate hearing loss in human patients.Hearing thresholds of the homozygous p.V37I knock-in and wild-type mice were consecutively monitored at frequencies of 4, 8, 16, 24 and 36 kHz between age 6 and 50 weeks by auditory brainstem response (ABR) tests . ElevateThe gross cochlear development and morphology in the homozygous p.V37I knock-in mice appeared normal . ScanninGjb2 leads to eventual progressive hearing loss, we performed a gene expression microarray analysis in randomly chosen P5 knock-in and wild-type mouse cochleae. A total of 105 up-regulated and 43 down-regulated genes were identified in the knock-in mouse cochleae  and down-regulated genes  with the highest fold changes as well as a gene (Lars2) previously implicated in syndromic deafness Perrault syndrome. The up-regulation of Fcer1g, Lars2, Nnmt and down-regulation of Cuedc1 were confirmed by qRT-PCR , while the down-regulation of Slc30a10 and Gpr126 were not  among all reported variants associated with hearing loss. It was estimated that over 4 million people among East and Southeast Asians will be homozygous for p.V37I and genetically susceptible for mild-to-moderate hearing lossThe p.V37I variant in Gjb2. In comparison with the Gjb2 knock-out mice, whose hearing thresholds were elevated by 40–50 dB at P21In this study, we generated a knock-in mouse model for the homozygous p.V37I variant in Gjb2 leads to eventual progressive hearing loss, we performed an inner ear gene expression microarray analysis for the P5 knock-in and wild-type mice. A total of 148 genes showed significant up-regulation (n = 105) or down-regulation (n = 43) in the knock-in mice Lars2 encodes mitochondrial leucyl-tRNA synthetase. Mutations in many genes encoding this type of aminoacyl-tRNA synthetases, such as HARS2, KARS, NARS and LARS2, have been reported to be associated with non-syndromic or syndromic hearing loss222324Gjb2 may contribute to the hearing loss in the knock-in mice.Though late-onset hearing loss was associated with homozygous p.V37I in both human patients and the mouse model, many p.V37I homozygous patients with late-onset hearing loss exhibited subclinical hearing loss  at birth, implying that the pathophysiological process can be accumulative and begin in much earlier age-in mice , which p qRT-PCR . Fcer1g,GJB2 is to discover the factors that may contribute to its incomplete penetrance, which was estimated to be 17%In summary, our study generated a homozygous p.V37I knock-in mouse model that modeled the representative hearing phenotype in human patients and provided a valuable tool for further functional studies of this variant. One intriguing question about the homozygous p.V37I variant in Gjb2 genomic region  were assembled into the targeting vector . A loxP-neo cassette was inserted into the intron 1 of Gjb2 in this vector  cell gene targeting as previously describeds vector . The p.VABR tests were performed in wild-type (n = 10) and homozygous p.V37I (n = 10) mice every four weeks between age 6 weeks and 50 weeks as previously described Mouse cochleae were harvested and fixed with 2.5% glutaraldehyde for overnight at 4 degrees Celsius. After decalcification with 0.1 M EDTA for 14 days, the cochleae were fixed with 1% OsO4, dehydrated with ethanol of increasing grades, infiltrated with acetone and final resin, placed in capsules contained embedding medium and heated at 70 °C for 9 hours. Semithin sections of cochleae were cut in thickness of 5 μm, stained with methylaniline blue and observed under microscope DMI 3000 .Mouse cochleae were fixed with 2.5% glutaraldehyde for 2 hours, washed three times in PBS and decalcificated with 0.1 M EDTA for 2 hours. The lateral wall was carefully removed and the hair cells were exposed on the modiolus. The cochleae were then fixed with 1% OsO4, dehydrated with ethanol of increasing grades, immersed into the mixture of alcohol and iso-amylacetate followed by iso-amylacetate, dehydrated, coated with gold-palladium and observed under scanning electron microscopy TM-1000 .Mouse cochleae were perfused and fixed with 4% paraformaldehyde for 4 hours at room temperature. After decalcification with 0.1 M EDTA for 14 days, the organs of Corti were microdissected under a microscope. F-actin was stained with rhodamine phalloidin (1:100) to identify the hair cells at the basal, middle and apical turns of the cochlea under a Leica TCS SPE confocal microscope (Leica Microsystems).P values were calculated by t-test with unpaired unequal variance (Welch) and adjusted for multiple testing by the Benjamini & Hochberg methodGene expression microarray analysis was performed in homozygous p.V37I knock-in and wild-type mice at P5 (n = 6 each). Cochleae without the vestibular part of the inner were harvested from the sacrificed mice and immediately immersed in the RNAlater RNA stabilization reagent . Total mRNA samples were extracted using the TRIzol reagent  and reverse-transcribed to cDNA using the PrimeScript™ RT Master kit . Hybridization was performed using the Illumina MouseWG-6 v2 Expression Beadchip system , which contains 45200 probes per array product. Normalization algorithms were used to transfer sample signals to minimize the effects of variation arising from nonbiological factors. Analysis of gene expression profiles was completed by the Illumina BeadStudio module application (Illumina). A twofold or greater difference in the intensity of gene expression between the mutant and wild-type mice was sought. Up-regulated or down-regulated genes were cataloged. The Fcer1g, Nnmt, Cuedc1, Slc30a10, Gpr126 and Lars2 was further evaluated by SYBR green-based qRT-PCR in mouse inner ear cDNA samples of knock-in and wild-type mice (n = 6 each). qRT-PCR primers and probes of each gene were designed using ABI Primer Express Software v2.0 . qRT-PCR analysis of the cDNA samples was performed using the ABI Prism 7700 sequence detection system (Applied Biosystems) following the standard procedure. Data were analyzed using the ABI Prism 7500 SDS software (Applied Biosystems). Quantitative expression data of each specific target were obtained for each cDNA sample. Expression of Gapdh was used as endogenous normalization control. The comparative threshold of cycle (CT) method was used to determine any difference in target expression between the homozygous p.V37I knock-in and wild-type mice. All values were log2 transformed before statistical anaylsis.The differential expression of P values were presented as the result of the two-tailed analysis. For gene expression microarray analysis, P values were calculated by t-test with unpaired unequal variance (Welch) and adjusted for multiple testing by the Benjamini & Hochberg methodFisher’s exact test was used to compare the ABR thresholds of the knock-in and wild-type miceHow to cite this article: Chen, Y. et al. Characterization of a knock-in mouse model of the homozygous p.V37I variant in Gjb2. Sci. Rep.6, 33279; doi: 10.1038/srep33279 (2016).


This study investigated the effects of leisure and social activities on the ability of middle-aged adults to maintain activities of daily living (ADL), and whether performing these activities alone or with others contributed to the ability to perform ADL. The study used nationally representative longitudinal data of 22,770 adults in Japan, aged 50–59 years, who did not have limitations in performing ADL at the beginning of the 5-year survey period. The study considered six activity categories: two leisure activities  and four social activities . Multiple logistic regression analysis was used to examine the relation between participation in these categories at baseline and difficulties in ADL at the 5-year follow-up. The association between the extent of social interaction during these activities  and difficulties in ADL was also investigated. The analysis yielded significant negative correlations between “exercise or sports” and difficulties in ADL for both men and women, and between “hobbies or cultural activities” and difficulties in ADL for women. However, these significant relationships occurred only when activities were conducted “with others.” The present findings might help prevent deterioration in middle-aged adults’ performance of ADL in Japan. Although the Japanese have a relatively long lifespan, the gap between mean life expectancy and healthy life expectancy  is still 9.13 years for men and 12.68 years for women [Healthy life expectancy of Japanese individuals is calculated based on activity limitations in the Comprehensive Survey of Living Conditions (CSLC) , a natioA number of studies found that leisure activities  and social activities (e.g. volunteering and community activities) help maintain ADL in older adults. For example, a 4-year longitudinal study in Japan reported that participation in hobby activities decreased the risk of functional disability among older adults . AnotherIn addition, the presence of others participation in these activities may contribute to preventing deterioration in ADL performance (ADL deterioration). A study of older adults in Japan found that even when exercise was performed no more than once a week, the incidence of functional disabilities among those participating with others was lower than among those who exercised alone . This fiHowever, no research has simultaneously examined the effects of middle-aged adults’ leisure and social activities on the maintenance of ADL; nor have studies investigated whether the effects of these activities are affected by the presence of other persons.Our previous study investigated the effect of leisure and social activities on middle-aged adults’ mental health status, and considered the impact of the presence of other persons participating in the activity as an additional variable, using nationally representative longitudinal data from the Longitudinal Survey of Middle-aged and Elderly Persons (LSMEP)  conducteThis study used data extracted from the LSMEP, a nationwide, population-based survey, conducted in Japan by the MHLW each year since 2005. Respondents to the survey were extracted randomly through stratified two-stage sampling. First, 2,515 districts were selected at random from the complete set of 5,280 districts surveyed in the CSLC, conducted in 2004 by the MHLW. Subsequently, 40,877 residents of each selected district were randomly chosen from individuals aged 50–59 years, in proportion to the district’s total population.In 2005, the first year of the survey, questionnaires were dropped off at the respondents’ homes by enumerators who returned to collect the self-reported surveys several days later. From the second year of the survey, the delivery method of the questionnaire changed from a “drop-off” to mail, and respondents returned the questionnaire by mail; the questionnaire was sent only to those who answered the questionnaire either last year, the year before, or both. The LSMEP did not recruit any new respondents since the survey’s first year.We used data from the first and sixth surveys, in 2005 and 2010. Of the 40,877 people who received the original questionnaire, 34,240 responded to the survey in 2005 (response rate: 83.8%). Of these, we excluded 2,322 respondents with missing values regarding difficulties in ADL in 2005 and 2,737 respondents who had difficulties in ADL as of 2005 that could have prevented them from engaging in leisure and social activities, such as exercise or sports, leaving 29,181 eligible baseline respondents. Then, these respondents were contacted each year, provided they continued to complete the survey. As a result, the study sample comprised 22,770 respondents who completed the survey in 2010 (follow-up rate: 78.0%).We obtained official permission to use the LSMEP from the MHLW based on Article 33 of the Statistics Act. We obtained the data in a fully anonymized and de-identified form. An ethical review was not required, based on the Japanese government’s Ethical Guidelines for Epidemiological Research .Subjects’ difficulties in ADL were evaluated by responses to the question: “Do you have any difficulties in activities of daily living as follows: walking, getting up from a bed or a floor, sitting down and standing up from a chair, dressing, washing one’s hands or face, eating, toilet use, bathing, going up and down stairs, and carrying shopping bags?” Dichotomized responses (yes/no) were recorded.go, working with potted plants, traveling)” and “exercise or sports ,” and four types of social activities were assessed: “community events (e.g. neighborhood associations functions),” “support for children (e.g. assistance for children’s clubs),” “support for elderly individuals ,” and “other social activities.” Respondents were asked whether they had participated in each activity during the year prior to the survey. For each question, those answering “yes” and “no” were categorized as active and inactive, respectively. Active respondents were asked to indicate the way in which they primarily participated in each activity: “by oneself,” “with families or friends,” “with co-workers (including former co-workers),” “in a neighborhood community association,” or “in a non-profit organization or public-interest corporation.” Respondents were permitted to select more than one response option. For the present study, respondents were categorized into three groups for each activity: “by oneself,” “with others” or “both” .As with our previous study , two typDemographic and socioeconomic status included age , gender, living arrangement , father, mother, father-in-law, and mother-in-law, respectively), job status (employed or unemployed), personal income, and family care provision, which was evaluated by the question: “do you provide care for your family?” with dichotomized responses (yes/no).Respondents’ answers regarding the presence of chronic diseases  were rated on a dichotomized scale (yes or no).Mental health status was assessed using the Japanese version of the K6 scale , a screeHealth behaviors included smoking status (“smoker” or “non-smoker”) and alcohol drinking status (“drinker” or “nondrinker”).We used multiple imputation by chained equations to handle missing data in this study. Analysis of imputed datasets reduces the effects of potential biases introduced by missing data . This meTo investigate the relationship between the six types of leisure and social activities in the baseline survey and difficulties in ADL in the follow-up survey, two kinds of multiple logistic regression models were applied. Model 1 included the six types of activities  separately, as independent variables. Because the proportion of people who engaged in “support for children,” “support for elderly individuals,” and “other social activities” was low, the statistical power of these items was likely to be low. Therefore, the independent factors in Model 2 were two types of leisure activities  and a summarized index referred to as “social activities” indicated involvement in at least one of the four social activities. Furthermore, we conducted chi-square analyses and individual t-tests to compare the characteristics of active and inactive respondents with regard to leisure activities .Multiple logistic regression analyses were also performed to examine the impact of the sociality of activities  on reducing difficulties in ADL. We used leisure and social activities that had positive effects in reducing difficulties in ADL in the prior logistic regression analyses as independent variables. Demographic and socioeconomic status , health status , and health behaviors  at baseline were controlled in the multiple logistic regression analyses. Logistic regression analyses, chi-square analyses, and individual t-tests were performed by gender because the proportion of respondents reporting participation in leisure and social activities in the baseline survey and limitations in ADL in the follow-up survey differed according to gender.p < 0.05. All statistical analyses were performed using IBM SPSS version 23.0.The level of significance for all analyses was set at A total of 17,856 respondents answered questions to all variables used in this study (78.4% of respondents in this study). The number of missing cases of each variable before multiple imputation by gender and by age (50–54 years old or 55–59 years old in the baseline survey) is shown in Descriptive statistics of the sample’s characteristics after multiple imputation are shown in p < 0.001) in Model 1. Model 2 also found a significant relationship between “exercise or sports” and fewer difficulties in men’s ADL .p < 0.01) and “exercise or sports”  decreased the risk of difficulties in ADL at follow-up in Model 1. In contrast, “support for children”  and “support for elderly individuals”  increased this risk. Similarly, Model 2 showed that “hobbies or cultural activities”  and “exercise or sports”  decreased the risk of difficulties in ADL, whereas social activities  were associated with increased risk.With regard to women, “hobbies or cultural activities” . Similarly, among women, both “hobbies or cultural activities” and “exercise or sports” were significantly and negatively correlated with difficulties in ADL only when it was performed with others .Then, associations between social aspects of respondents’ engagement in “hobbies or cultural activities” or “exercise or sports” and difficulties in ADL were examined . The “inIn the previous Japanese study using LSMEP data, we showed that middle-aged women’s and men’s participation in leisure activities such as “hobbies or cultural activities” and “exercise or sports” with others was related to mental health status at the 5-year follow-up . In thisOur findings are consistent with several longitudinal studies that have reported that exercise or sports prevent ADL deterioration among older adults –26 and mFurthermore, comparisons of active and inactive respondents, determined by whether they participated in leisure activities, yielded significant results. Active individuals were less likely to smoke and more likely to be in good mental health. In fact, poor mental health  and smokImportantly, the present study indicated that participating in leisure activities with others might help maintain ADL. The findings suggest that only two baseline factors, participating in hobbies or cultural activities (among women) and exercise or sports (for both genders) with others were associated with reduced deterioration in ADL at follow-up. One possible explanation is that social relationships may enhance the benefit gained from these activities. One study showed that middle-aged individuals with consistently high levels of social relationships over a 34-year period leading up to old age had a lower risk of disability in their later years compared to those whose social relationships that decreased with time or remained at constant low to medium levels . AnotherNonetheless, these results may be influenced by differences in the sample size. The number of respondents participating in leisure activities with others was much larger than any other group. Although there were no significant relationships between engaging in leisure activities both “alone” and “with others” and difficulties in ADL, the sample size of this group was particularly small, implying that these results need to be considered carefully.On the other hand, men’s and women’s participation in any of the four categories of social activities at baseline was not associated with limitations in ADL at follow-up. However, some studies targeting older adults have found such a relationship; for example, two longitudinal studies indicated that volunteering was associated with decreased functional dependency , 12. ThiFurthermore, women who engaged in social activities, especially those who support children and elderly individuals, may be more likely to have difficulties in ADL than those who did not engage in these activities. Previous studies have demonstrated a high prevalence of musculoskeletal pain—especially lower-back pain—in women with family-caregiving  or childThe present study has several limitations related to the research process and sample. First, the most important limitation of this study is that it cannot claim a causal relationship between participation in leisure and social activities and maintenance of ADL. It is possible that causal relationships between these factors are the inverse of our hypothesis. Therefore, it is thus plausible that those who were more proficient in ADL at baseline were more likely to engage in various activities. Thus, directionality should be analyzed in future studies. Second, despite using multiple imputation to avoid the impact of missing variables, selection bias may exist. About 6,000 people did not respond to the baseline questionnaire, and about 6,000 respondents had dropped out at the end of 5 years. This limitation should be considered when generalizing the findings. Third, although we used multiple imputation methods to reduce the effect caused by attrition bias , it is iDespite the above-mentioned limitations, the present study has several strengths. It used a large sample size and nationally representative data, and therefore reduced the effect caused by selection bias. Further, our study examined a wide range of leisure and social activities; it is more comprehensive than previous studies on middle-aged adults in Japan. Finally, in addition to our previous study, which recommends effective ways of promoting mental health, the present findings suggest that it is more effective for middle-aged adults to engage in leisure activities with others.In conclusion, the present study found that exercise or sports for both genders and hobbies or cultural activities for women may have positive effects on ADL maintenance among middle-aged adults in Japan when these activities are performed with others. These findings might be useful when designing and developing strategies to encourage maintenance of ADL among middle-aged adults in Japan.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


This study revealed that two novel proteins in rice – LPL2 and LPL3 – homologous to the SCAR/WAVE subunits PIR/SRA1 and NAP1, respectively, control epidermal cell morphogenesis through actin reorganization. less pronounced lobe epidermal cell2-1 (lpl2-1) and lpl3-1, whose leaf and stem epidermis developed a smooth surface, with fewer serrated pavement cell (PC) lobes, and decreased papillae. The lpl2-1 also exhibited irregular stomata patterns, reduced plant height, and short panicles and roots. Molecular genetic studies demonstrated that LPL2 and LPL3 encode the PIROGI/Specifically Rac1-associated protein 1 (PIR/SRA1)-like and NCK-associated protein 1 (NAP1)-like proteins, respectively, two components of the suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (SCAR/WAVE) regulatory complex involved in actin nucleation and function. Epidermal cells exhibited abnormal arrangement of F-actins in both lpl2 and lpl3 expanding leaves. Moreover, the distorted trichomes of Arabidopsis pir could be partially restored by an overexpression of LPL2. A yeast two-hybrid assay revealed that LPL2 can directly interact with LPL3 in vitro. Collectively, the results indicate that LPL2 and LPL3 are two functionally conserved homologs of the SCAR/WAVE complex components, and that they play an important role in controlling epidermal cell morphogenesis in rice by organising F-actin.Filamentous actins (F-actins) play a vital role in epidermal cell morphogenesis. However, a limited number of studies have examined actin-dependent leaf epidermal cell morphogenesis events in rice. In this study, two recessive mutants were isolated:  The shape of plant cells, as determined by cell wall expansion, is critical for organ and tissue development and morphogenesis during plant growth . In the Oryza sativa L.) is a model monocotyledon plant because of its small, well-mapped genome size, shared single-nucleotide polymorphisms, and improved genetic transformation technology  mediate leaf trichome development and morphology  affect epidermal cell differentiation. Knockout of these two genes leads to higher stomatal density and rapid water loss in rice (Rice (Zea mays) BRICK1 (BRK1), BRK2, and BRK3 genes are involved in epidermal cell morphogenesis and cell division  and cortical ﬁlamentous actin (F-actin) polymerization and stabilization in plants . SeveralTUT1/OsSCAR1, which encodes the same SCAR-like protein. This protein plays a significant role in activating the Arp2/3 complex to regulate actin polymerization  regulatory complex, and the actin-related protein 2/3 (Arp2/3) complex signaling pathway . The ArpOryza sativa ssp. japonica cv. Zhonghua 11, ZH11) and T-DNA insertion library seedlings with ZH11 ecotype background were cultivated in the greenhouse of Lanzhou University , with 12-h light period/day, 60%–80% relative humidity, and day/night temperature of 32/22 °C. lpl2-1 was screened and back-crossed into ZH11 three times prior to use. Two T-DNA insertion mutant lines lpl2-2 (RMD_04Z11HY54) and lpl2-3 (RMD_04Z11CR30) were ordered from the Rice Mutant Database, China (http://rmd.ncpgr.cn/introduction.cgi?nickname). The location of T-DNA insertions in lpl2-2 was further verified by PCR using LPL2 ﬂanking primers (LPL2-2FP-F and LPL2-2FP-R) and primer PFRB4-RB. We verified T-DNA of lpl2-3 using primers LPL2-3FP-F, LPL2-3FP-R and NTLB5-LB. lpl3-1 (ACNB06) was ordered from CIRAD, France (http://orygenesdb.cirad.fr/index.html), and its T-DNA insertions were verified by PCR using primers LPL3-FP-F/-R and LPL3 T-DNA speciﬁc primer (LPL3-SP). The pir T-DNA insertion mutant SALK_106757  were performed using methods from our previous report .lpl2-1 (Oryza sativa ssp. japonica) and 9311 (Oryza sativa ssp. indica) hybrids were selected using dental resin impressions for mapping. The published RM-series rice simple sequence repeat (SSR) markers (http://www.gramene.org/) were used to map the mutant gene. The locus was roughly mapped between RM4108 and RM4352 on the long arm of chromosome 3 by the primary location. Subsequently, the locus was fine mapped onto the ~98-kb region between two markers RM14374 and RM14380 using 880 lpl2-1 homozygote. The candidate gene LPL2 was identiﬁed by sequence analysis of all genes on the region.The F2 population progenies of LPL2 was ampliﬁed using primers OE-LPL2-F and OE-LPL2-R with the KpnI and SpeI restriction enzyme sites, respectively (Supplementary Table S1), with reverse-transcribed cDNA as a template. Then the LPL2 ORF was subcloned into an overexpression (OE) binary vector POX driven by maize ubiquitin promoter (from Liu Yaoguang laboratory) to obtain OE-LPL2, which was introduced into lpl2-1 mutant embryonic calli by Agrobacterium tumefaciens-mediated transformation  of t plants . PhenotyLPL2 using primer LPL2-RT-F/R. SYBR Premix ExTaq  and the MX 3050 qPCR System  were used according to the manufacturers’ instructions. The thermal cycling conditions were: 95 °C for 30s, 40 cycles of 95 °C for 15s, and 60 °C for 30s, in a total volume of 20 µl. We used the transcription factor eEF (LOC_Os03g08020) as an endogenous control. The specific primers for different genes are listed in Supplementary Table S1. All experiments were repeated at least three times.qPCR was performed to illustrate the expression of – 4cm were cut carefully into 1 cm-long strips, and then prefixed with 200 µM m-maleimidobenzoyl-N-hydroxyl-succinimide ester (MBS) in PEM buffer , plus 2% (v/v) DMSO and 0.05% (v/v) Triton X-100, for 30min in the dark. Subsequently, MBS solution was removed and replaced by actin ﬁxation solution of 4% (w/v) paraformaldehyde (PFA) in the PEM buffer with 0.1% Triton X-100 and 2% glycerol for 1h. Then the epidermal sheets were rinsed with PEM buffer and extracted in 1% Triton X-100 and 5% DMSO in PEM for 5min, with three repeats. Finally, the 1 cm-long thin strips of leaf tissues were placed into 10% Alexa-Fluor 488 phalloidin dilution (Life Technologies) and stained for 2–4h at 37 °C in the dark. All images were analyzed using a confocal microscope (Olympus FluoView FV1000 MPE).The detailed procedure of actin staining was performed as described previously  and trans-activation domain vector pGADT7 (AD), respectively. The full-length cDNA of LPL3 was also cloned into the AD and BD vector with primer LPL3-Y2H. Then, these two groups of plasmids were transformed into yeast strain Y2HGold to culture and observe according to the manufacturer’s manual (Clontech). These experiments were repeated three times. All primers used are listed in less pronounced lobe epidermal cell2-1 (lpl2-1), which exhibits less pronounced and smooth epidermal PCs, was screened from a rice T-DNA insertion mutant library in T1 progeny. This phenotype was stable, but it did not link with the T-DNA insertion. To analyze the function of the LPL2 gene, the mutant lines without T-DNA were isolated from the T2 progeny, and were backcrossed with wild-type (WT) ‘zhonghua11’  for three generations in succession. After purification, the height of the aerial part and the root length in lpl2-1 were shorter than that of ZH11 during early seedling growth . During the heading period, lpl2-1 was also decreased in height because all the internodes of the mutant were shorter than those of ZH11 . Notably, clearer and smoother epidermal PCs were found in lpl2-1 , suggesting that lpl2-1 was most likely caused by a single recessive mutation. To clone the causal gene for LPL2, lpl2-1 was cross fertilized with ‘9311’, a WT polymorphic indica rice, to construct a small scale mapping population of backcross 9311 F1 (BC3F1). After self-pollination, BC3F2 mapping population was constructed successfully. The candidate gene locus was fine mapped in a ~98-kb region between two known molecular markers (RM14374 and RM14380) in chromosome 3 by map-based cloning (LOC_Os03g05020 (full-length gene nearly 16kb). However, when cloning and sequencing the LOC_Os03g05020 cDNA based on three independent PCR products, the full-length cDNA was composed of 30 exons, and a 3862bp full-length coding region, instead of the predicted mRNA, which comprised 27 exons and 3555bp full-length cDNA from Rice GE  . Based on the new cDNA sequence, we found that the deletion mutation in lpl2-1 led to a frameshift mutation in the 16th exon, and the re-translated 594th glycine was turned into a stop codon , displayed 74% sequence similarity to PIR in Arabidopsis, and identified 27% with human PIR121 . The sequence similarity alignment assay showed that LPL2 is somewhat highly conserved throughout the plant kingdom.All F1 plants of reciprocal crosses between  cloning . SubsequRiceGE/) . Therefoop codon . LOC_Os0LPL2 (OE-LPL2) with the maize ubiquitin promoter in lpl2-1. Twenty individual lines from complemented transgenic line #1 (com #1) to com #20 of OE-LPL2/lpl2-1 were completely rescued, including the reduced plant height and aberrant epidermal cell phenotypes, whereas the control lines with the empty plasmid failed to restore normal phenotypes , were isolated . Therefore, the genetic analysis indicated that these three lpl2 mutants were controlled by the same gene. Altogether, the genetic and transgenic evidence demonstrated that loss-of-function of LPL2 results in an abnormal epidermis and other phenotypes.Furthermore, to verify the function of isolated . Interes1 mutant –F. Consiand ZH11 –J. More Supplementary Fig. S4). To verify this speculation, a pir mutant induced by a T-DNA insertion in Arabidopsis (SALK_106757), which shows various defective trichomes on leaves and stems compared to WT, was ordered . Whereas, when the full-length cDNA of LPL2 was overexpressed into pir plants, driven by a cauliflower mosaic virus 35S promoter (35S-LPL2), most of the T1 generation plants could be partially rescued in terms of the distorted leaf and stem trichomes . In the lpl2-1 epidermal PC margin, however, lesser abundant and prominent cortical F-actin patches were observed . As expected, these results highlighted that the epidermal PC lobe deficiency in lpl2-1 is a result of disorganization of the actin cytoskeleton. These results indicated that LPL2 is a functional SCAR/WAVE protein, which plays an evolutionarily conserved role in controlling epidermal cell morphogenesis in rice, as well as in maize and Arabidopsis.Combined with LPL2 function in encoding a subunit of the WAVE complex, and previous studies on maize and Arabidopsis epidermis,  mutants , B, E, Fhttp://www.ebi.ac.uk/Tools/sss/ncbiblast/), which is a homolog of BRK3 in maize and NAP1 in Arabidopsis. We identified its T-DNA insertion mutants , which exhibited deficient phenotypes similar to those of lpl2-1, including less pronounced and smooth epidermal PCs, moderate dwarfism, and poor fertility . Therefore, the defect of epidermal PC lobes in lpl3-1 was considered to be caused by the abnormal rearrangement of local actin filaments. The full-length LPL3 cDNA was cloned and sequenced; it consisted of 22 exons and a 4080-bp coding region . These results suggested that LPL3 also plays an important role in regulating epidermal cell morphogenesis in rice, and it is another functional SCAR/WAVE protein.Bioinformatics analysis revealed another subunit of the WAVE complex, the NAP1-like protein (LOC_Os08g43130) in rice . This work demonstrated that LPL2 encodes a PIR-like protein, a subunit of the functional SCAR/WAVE complex involving in F-actin nucleation in Arabidopsis, rice, maize, and animals , PAN2, DISCORDIA1, ALTERNATIVE DISCORDIA1, and ZmROP2/ZmROP9 play an extremely important role in regulating the SMC asymmetric division and epidermal PC development. Mutations in these genes will give rise to abnormal SCs and PCs (Meanwhile, in pidermis . While tLPL3 is a homolog of BRK3 in maize, and it encodes an NAP1-like protein, a subunit of the SCAR/WAVE complex in Arabidopsis (lpl3-1 was isolated using a reverse genetic approach; it also showed almost identical epidermal PC features among rice lpl2 and three maize brk mutants (lpl2 and lpl3 indicate that the SCAR/WAVE complex might function via the interaction between LPL2 and LPL3. In Arabidopsis, aberrant trichomes were observed in the nap1 mutant, similar to the phenotypes of pir, because the F-actin cytoskeleton showed a disturbed spatial orientation (tut1/scar1 mutant, although the shape of leaf prickle hairs had no distinction between lpl2, lpl3, and WT (scar1, but the abnormal phenotype of PC lobes was not significant (lpl2 and lpl3. Therefore, the diverse functions of different SCAR/WAVE elements in rice involved in epidermal cell morphogenesis need to be further elucidated.Similarly, lpl2 and lpl3 might lead to reduced adhesive properties of PC walls, and decreased stress tolerance (lpl2 and lpl3. Apart from the observations made with lpl2 and lpl3, maize brks, Arabidopsis pir, nap1, and brk1, also showed abnormalities in F-actin organization (In most dicot leaf epidermises, the formation of lobes and interdigitation is a significant characteristic . Compareolerance . Furthernization .The results of this study revealed that LPL2 and LPL3 are important components for assembling the SCAR/WAVE complex in rice. A yeast two-hybrid assay proved that LPL2 directly interacts with LPL3, consistent with previous findings that NAP1/GNARLED and PIR/KLK/SRA1 can directly interact in plants and animals . MoreoveTUT1/SCAR1 can activate ARP2/3 to promote actin nucleation and polymerization in vitro. Hence, LPL2 and LPL3 should be involved in the signaling pathway of OsRacs-SCAR/WAVE-OsARP2/3. However, which other subunits are a part of the SCAR/WAVE complex, and how they coordinate to work in rice, are still unknown. Consequently, further investigation might focus on the primary signaling pathway of the cytoskeleton, which plays an essential role in epidermal cell morphogenesis and basic plant cell shape by regulating the polymerization of F-actin in rice.It is worth noting that JXB online.Supplementary data are available at Table S1. Primers used in this study.Figure S1. Plant height and root length of ZH11 and lpl2-1 at the seedling stage.Figure S2. Phenotype of spikes in ZH11 and lpl2-1.Figure S3. Supplement of LPL2 cDNA sequence.Figure S4. Amino acid alignment of Arabidopsis PIR, rice LPL2, maize BRK2 and human PIR121.Figure S5. Seedlings and leaf abaxial epidermis of ZH11, lpl2-1, lpl2-1/-2 and lpl2-1/-3.Figure S6. Plant and trichomes of adaxial leaves in Col and pir.Figure S7. The proportion of cells showing actin patches in ZH11 (lpl2-1 ( in ZH11  and lpl2 (lpl2-1 .Figure S8. Analysis of actin cytoskeletal structures in ZH11 and lpl3-1 mature leaf epidermal cells.Figure S9. Amino acid alignment of Arabidopsis NAP1, rice LPL3, maize BRK3 and human NAP1.


The detection rate of urinary BP-3 in females (12.3%) was higher than that in males (0%) (p = 0.015). Moreover, the detection rate of urinary BP-3 in adolescents (14.1%) was higher than that in children (0%) (p = 0.005). For children, no urinary BP-3 was found. There were no differences in detection rates and concentrations of urinary TCS, BP-3, and BPA between males and females, respectively. For adolescents, urinary BP-3 was only found in the females. Urinary TCS levels in females were higher than those in males (p = 0.047). The present study showed that urinary TCS concentrations in females were significantly higher than those in males, respectively. In addition, BP-3 was only detected in urine samples of female adolescents. Sex and age were the important factors influencing urinary TCS and BP-3 concentrations.The purpose of this study was to determine the levels of urinary triclosan (TCS), benzophenone-3 (BP-3), and bisphenol A (BPA) in 52 children and 71 adolescents. The effects of age and sex on the levels of urinary TCS, BP-3, and BPA were explored, respectively. Results demonstrated the overall detection rates of urinary TCS, BP-3, and BPA were 18.7%, 8.1%, and 49.6%, respectively. The females had higher TCS concentrations than males ( Humans are potentially exposed to several phenolic chemicals in commonly used products that may cause endocrine-disrupting effects. For example, triclosan  has been added to soaps, toothpastes, mouthwashes, deodorants, and cosmetics as a preservative and antiseptic agent ,2. BisphOral and dermal absorption are the major routes of exposure of these phenolic chemicals. Due to their extensive use, the general population is potentially at risk of exposure to TCS, BPA, and BP-3, which result in their wide distribution in human blood, breast milk, and urine, respectively ,18,19,20TCS, BPA, and BP-3 have been reported for endocrine-disruption potentials in a number of in vitro and in vivo studies. In general, BPA and TCS are considered to have a weak estrogenic effect ,31, wherSeveral studies by Howdeshell et al. (1999) indicated that BPA altered the postnatal growth rate and reproductive function in female mice ,36,37,38Coronado et al.  have sugThese phenols are also suspected to affect development in humans. Philippat et al.  indicateHowever, there are likely to be some important variables  that determine whether people contact TCS, BP-3, and BPA-containing products. Relative children and males, adolescents, and females have more chances to use personal care products such as toothpastes, lotions, and cosmetics. The purpose of this study is to explore the effects of age and sex on the levels of urinary TCS, BP-3, and BPA. The elementary school children and college students were selected as our study subjects.13C12-triclosan was obtained from Cambridge Isotope Laboratories Inc. .Triclosan (TCS), bisphenol A (BPA), and benzophenone-3 (BP-3) standards were all purchased from Sigma-Aldrich . HPLC-grade methanol and water, analytical-grade ammonium acetate, formic acid, andβ-Glucuronidase/arylsulfatase were provided by Merck . Morning urine samples were obtained from 52 elementary school children of 11–13 years old and 71 college students of 19–21 years old in southern Taiwan, respectively. Written informed consent was obtained from each subject and their parents. The study procedures were approved by the Institutional Review Board of I-Shou University (ISU-IRB-98-03).13C12-Triclosan. After gentle mixing, samples were added to 50 µL of glucuronidase/sulfatase solution , and the hydrolysis was allowed to proceed at 37 °C overnight. After the incubation, the deconjugated urine sample was diluted with 800 µL to 0.1 M formic acid and centrifuged. The aliquot was injected into the on-line solid phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC)–tandem mass spectrometry (MS/MS).The pretreated and analyzed methods of urine samples were referred to in the procedures, as reported by Ye et al. . At firsThe on-line SPE-HPLC-MS/MS system was constructed from several Agilent 1290 modules coupled to a triple quadrupole Agilent 6430 mass spectrometry . The mass spectrometer was equipped with an electrospray ionization (ESI) interface. The SPE column was a LiChrosphere RP-18 ADS , and the HPLC column was ZORBAX Eclipse XDB-C18 . Methanol and water were used as mobile phases. The procedure for extracting the analytes from the urine involved concurrent SPE and HPLC-MS/MS cycles .The Agilent 6430 mass spectrometry was used in negative mode with the following settings: source gas (nitrogen) with a flow rate of 10 L/min at a temperature of 350 °C, nebulizer pressure of 50 psi, and capillary voltage of 4 kV. The mass spectrometer operating parameters were optimized using Agilent Optimizer Software for the analytes . The limp-value of <0.05 was considered statistically significant for these tests.The data were analyzed using the SPSS 18.0 statistical package. To demonstrate the distribution and disparity of urinary TCS, BPA, and BP-3 levels, only positive samples > LOD were used to calculate the geometric means and ranges, respectively. Mann-Whitney U test was used to evaluate the differences of urinary TCS, BPA, and BP-3 levels between males and females, and between children and adolescents, respectively. Chi-square/Fisher’s exact test was used to compare the differences of detection rates of urinary TCS, BPA, and BP-3 between males and females, and between children and adolescents, respectively. The p = 0.051). The detection rate of urinary BP-3 in females (12.3%) is higher than that in males (0%) (p = 0.015). In addition, the detection rate of urinary BP-3 in the college students (14.1%) is also higher than that in elementary school students (0%) (p = 0.005)  concentrations of urinary TCS, BP-3, and BPA in the detected samples were 59.52 µg/L (n = 23), 19.72 µg/L (n = 10), and 5.81 µg/L (n = 61), respectively. The present study observed that the females had higher TCS concentrations than the males (= 0.005) .For elementary school children, the detection rates of TCS and BPA were 13.5% and 48.1%, respectively. No urinary BP-3 was found. Concentrations of TCS and BPA ranged from <1.56 to 149.64 µg/L, and <0.16 to 22.27 µg/L, respectively. The GM concentrations of urinary TCS and BPA in the detected samples were 61.09 µg/L and 5.61 µg/L, respectively. No differences of levels and detection rates of urinary TCS, BP-3, and BPA between males and females were found, respectively.p = 0.047).For college students, the detection rates of TCS, BP-3, and BPA were 22.5%, 14.1%, and 50.7%, respectively. Urinary BP-3 was only found in the females. Concentrations of TCS, BP-3, and BPA ranged from <1.56 to 198.66 µg/L, <0.16 to 97.02 µg/L, and <0.16 to 190.10 µg/L, respectively. The GM concentrations of urinary TCS, BP-3, and BPA in the detected samples were 58.84 µg/L, 19.72 µg/L, and 5.95 µg/L, respectively. Urinary TCS levels in the females were higher than those in the males for college students  and BP-3 (8.1%) were lower than those available in the literature (74.6–100% and 85.3–98.4%) ,53,54,55p = 0.047). This observation was consistent with the findings of Li et al. [For urinary TCS of the detected samples, the present study observed that the females in the college had significantly higher TCS concentrations than the males  and 71 adolescents of 19–21 years old (<1.56 μg/L) in the present study were lower than those for 314 children of 6–11 years old (5.9 μg/L) and 715 adolescents of 12–19 years old 10.2 μg/L) in the study of Calafat et al. .2 μg/L i, 95 primp = 0.015). No urinary BP-3 was found in the males. This was consistent with the findings of Calafat et al. [For urinary BP-3 of the detected samples, the present study observed that the females had a significantly higher detection rate of urinary BP-3 than the males  and 71 adolescents of 19–21 years old (<0.156 μg/L) in the present study were lower than those for 314 children aged 6–11 years old (17.2 μg/L) and 715 adolecents aged 12–19 years old (20.0 μg/L) in the study of Calafat et al. , respectThe sex-related differences of urinary BPA from the findings of previous studies were not all consistent. He et al.  reportedThe medians of BPA of 52 children of 11–13 years old (<0.156 μg/L) and 71 adolescents of 19–21 years old (<0.156 μg/L) in the present study were similar to those for 922 Chinese population (<LOD μg/L) in the study of He et al. , but lowThe present study showed that urinary TCS and BP-3 concentrations in females were significantly higher than those in males, respectively. In addition, urinary BP-3 was only detected in the female samples of college students. Sex and age were the important factors influencing urinary TCS and BP-3 concentrations, respectively. Further studies are needed to assess the effects of these phenol chemicals on human health, especially for more highly exposed groups.