Using RNA interference, we show that ASH plays a pivotal role in the development of both bristles and grooming combs in water striders. Our data suggest that the ASH locus may have contributed to the adaptation to water surface lifestyle through shaping the hydrophobic bristles that prevent water striders from wetting and allow them to exploit water surface tension.The semi-aquatic bugs  conquered water surfaces worldwide and diversified to occupy puddles, ponds, streams, lakes, mangroves and even oceans. Critical to this lifestyle is the evolution of sets of hairs that allow these insects to maintain their body weight on the water surface and protect the animals against wetting and drowning. In addition, the legs of these insects are equipped with various grooming combs that are important for cleaning and tidying the hair layers for optimal functional efficiency. Here we show that the hairs covering the legs of water striders represent innervated bristles. Genomic and transcriptomic analyses revealed that in water striders the  TheseDrosophila, the four genes of the achaete–scute family encode basic helix–loop–helix factors that are master regulators of bristle development [Achaete (ac), scute (sc) and lethal of scute (l'sc) have redundant proneural functions and promote the formation of neural precursors, whereas asense (ase) is expressed in neuronal precursors. Loss-of-function mutant flies for ac and sc display a notum devoid of bristles [l'sc leads to the loss of body bristles whereas ectopic expression of l'sc in the fly notum produces additional bristles [cis-regulatory organization [In the fly elopment ,15. Achabristles . Similarbristles . All foubristles ,19. Genenization .achaete-scute complex (AS-C) in insects contained two genes: an ase gene and an achaete–scute homologue (ASH) gene which has undergone independent duplications in the Diptera [Tribolium castaneum, Tc-ASH is required to promote neural precursor formation and could play the role of multiple duplicated proneural ac–sc genes present in species such as Drosophila [Tc-ase is highly conserved between Drosophila and Tribolium suggesting a conserved ancestral function for asense genes [ac–sc genes show a conserved genomic structural organization between Diptera and Coleoptera [cis-regulatory regions prevent separation of the genes.The ancestral  Diptera . In the osophila . Moreovese genes . In addileoptera . It is wac–sc gene family could have occurred during the evolution of the Gerromorpha lineage. To address this question, we investigated the genomic organization of ac-sc genes in 14 species of the Gerromorpha in comparison with other insect lineages. We generated and compiled a unique dataset of AS-C proteins across insects, including available transcriptomes and genomes of early-diverging lineages. Furthermore using RNA interference (RNAi), we examined the role of this locus in the development of hydrophobic bristles in multiple species of Gerromorpha. Our findings suggest that the gene ase was lost in the Gerromorpha lineage and the only remaining member of the ac–sc family plays a key role in the formation of various bristles in this group of insects.Given the key role that hydrophobic bristles play in water strider biology, we hypothesized that changes to the 2.(a)Drosophila melanogaster genes as a probe. Species names are indicated by the following prefixes Aae: Aedes aegypti, Aca: Aplysia californica, Aga: Anopheles gambiae, Ame: Apis mellifera, Apa: Aquarius paludum, Bge: Blattella germanica, Bmo: Bombyx mori, Bta: Bemisia tabaci, Caq: Catajapyx aquilonaris, Cca: Ceratitis capitata, Cle: Cimex lectularius, Cpa: Cylindrostethus palmaris, Csa: Cupiennus salei, Dci: Diaphorina citri, Dma: Daphnia magna, Dme: Drosophila melanogaster, Dpo: Dendroctonus ponderosae, Dpu: Daphnia pulex, Eda: Ephemera danica, Eaf: Eurytemora affinis, Foc: Frankliniella occidentalis, Gbu: Gerris buenoi, Hha: Halyomorpha halys, Hst: Hydrometra stagnorum, Htu: Husseyella turmalis, Hvi: Homalodisca vitripennis, Hvu: Hydra vulgaris, Lde: Leptinotarsa decemlineata, Ldi: Limnoporus dissortis, Lfr: Limnogonus franciscanus, Lfu: Ladona fulva, Llu: Limnephilus lunatus, Lmi: Locusta migratoria, Mam: Microvelia americana, Mdo: Musca domestica, Mex: Medauroidea extradentata, Mfu: Mesovelia furcata, Mhe: Metrobates hesperius, Mlo: Microvelia longipes, Mmu: Mesovelia mulsanti, Mmus: Mus musculus, Nlu: Nilaparvata lugens, Nvi: Nasonia vitripennis, Oci: Orchesella cincta, Ocu: Oiovelia cunucunumana, Ofa: Oncopeltus fasciatus, Pac: Paravelia conata, Pap: Pyrrhocoris apterus, Pba: Pogonomyrmex barbatus, Pbr: Platyvelia brachialis, Pbu: Paravelia bullialata, Pdu: Platynereis dumerilii, Phu: Pediculus humanus, Pve: Pachypsylla venusta, Ran: Rhagovelia antilleana, Rob: Rhagovelia obesa, Rpr: Rhodnius prolixus, Rze: Rhagoletis zephyria, Sma: Strigamia maritima, Smi: Stegodyphus mimosarum, Sst: Stridulivelia strigosa, Ste: Stridulivelia tersa, Tca: Tribolium castaneum, Tlo: Triops longicaudatus, Xla: Xenopus laevis, Zne: Zootermopsis nevadensis. The novel sequences generated for this analysis have been deposited in the EMBL database with specific accession numbers .Phylogenetic markers were identified in available genomes or transcriptomes by tBLASTn using a set of selected (b)xML v.8 [Γ model. One hundred bootstrap replicates were conducted for support estimation. Bayesian phylogenetic analyses were performed using MrBayes 3.2 [Γ model. We ran two chains for at least 1 000 000 generations and removed the first 250 000 generations as burn-in. The different nucleotide sequence alignments and tree files are available from the Dryad Digital Repository at: http://dx.doi.org/10.5061/dryad.rc454pc [Nucleotide sequences were aligned with MUSCLE , manuallxML v.8  under thayes 3.2  under th.rc454pc .(c)Ancestral reconstruction of the number of grooming combs on the different legs was performed using R software. ML methods were adapted to discrete characters  and the(d)For transmission electron microscopy (TEM), adult tarsi were transversally cut into several pieces and fixed in 2% glutaraldehyde in 75 mM sodium cacodylate buffer (pH = 7.3) overnight at 4°C. Samples were washed in cacodylate buffer and post-fixed in 1% osmium tetroxide for 1 h. Samples were then dehydrated in ethanol and embedded in epoxy resin. Samples were sectioned on a Leica UC7 ultramicrotome, stained with uranyl acetate and lead citrate and imaged with a Philips CM120 TEM at 80 kV. For scanning electron microscopy (SEM), adult bugs were fixed in 4% paraformaldehyde : heptane (ratio 1 : 3) for 20 min at room temperature. Samples were examined by using a Hitachi S800 SEM.(e)Dissected embryos were fixed in 4% paraformaldehyde : heptane (ratio 1 : 3) for 20 min at room temperature, washed several times in cold methanol and then rehydrated through a methanol series to phosphate buffered saline-Tween 20 0.05%. Embryos were prehybridized for an hour at 60°C in hybridization buffer  prior t(f)ASH using parental RNAi was conducted following the protocol described in [Mesovelia mulsanti embryos were placed at 30–31°C which allowed them to develop faster. Technically, this method shortens the delay between the injection of the double-stranded RNA and the late expression of ASH transcripts.Gene knockdown of ribed in . To obta3.(a)Gerris buenoi (Gerridae), leg tarsi are covered with at least two types of ‘hairs’ based on their size and that are regularly arranged along the leg (a). This feature is shared by the microveliid Microvelia americana (b) but not the mesoveliid Me. mulsanti where only one ‘hair’ type can be detected (c). Differences in the density of hairs are also observed across species (a–c). SEM images show a cuticle protuberance at the base of each hair (f), reminiscent of the socket cell of innervated bristles in insects [g). TEM imaging on leg hairs revealed the presence of an outer dendritic segment underneath every single hair . We therefore conclude that the numerous structures found on Gerromorpha legs are innervated bristles.The legs of water striders are covered by layers of hairs of different sizes, which form a cushion between the leg and the water surface thus preventing the leg from breaking surface tension ,9,10,30. the leg a. This fmericana b but notdetected c. Differ species a–c. SEM ach hair f, remini insects  (figure (b)Grooming, or active cleaning of body parts with specialized grooming structures, is a critical activity in the life of insects . GroominHydrometra stagnorum which has one grooming comb in the foreleg and none in the midleg. The reconstruction of the plesiomorphic state predicts that the common ancestor of Gerromorpha had two grooming combs in the forelegs and a single one in the midlegs. This result suggests that the loss of one of the two grooming combs of the forelegs and that of the midlegs occurred in the lineage leading to Hy. stagnorum G. buenoi and Homalodisca vitripennis), one thysanopteran genome  and one blattodean genome (Blattella germanica). Strikingly, we failed to detect asense in the genome of G. buenoi, Ho. vitripennis and F. occidentalis species. To confirm the absence of asense in these genomes, we performed synteny analysis, and we took advantage of the fact that the extended genomic locus of the whole AS-C complex exhibited a set of conserved features across the Holometabola  and Cytochrome P450–4g1 (Cyp4g1) and are present in the same position in many other insects [y and Cyp4g1 that constitute the boundary markers in the Holometabola have not been identified in the vicinity of the AS-C in the three non-holometabolan genomes we investigated  are conserved across insects. This result suggests that asense was lost in the lineage leading to the Hemiptera  and the Thysanoptera , and this loss is not associated with a reshuffling of the surrounding genomic locus.The structures covering the legs of the Gerromorpha being innervated bristles, it is reasonable to consider bristle specification genes as candidates to explain the specific characteristics of these leg bristles. The analysis of the AS-C gene complex is essential to understanding the contribution that proneural genes have made to the evolution of epidermal structures in insects. Whereas AS-C genes have been extensively investigated in flies, little is known about the molecular evolution of these genes across insects. To evaluate how conserved the extended genomic locus is in other insect genomes, we conducted both content and synteny analyses of this locus in two hemipterans asense, we searched for putative orthologues of asense in a broader sampling among insects. Using publicly available insect sequence datasets [ASH and asense genes in most insect lineages  and Thysanoptera (thrips) (Condylognatha). Both of these groups have sucking mouthparts and their monophyletic relationship was recently confirmed [asense gene loss in insects.To further confirm this lineage-specific loss of datasets ,44 alongonfirmed . This fi(e)ASH by in situ hybridization in G. buenoi, Me. mulsanti and Mi. americana. At mid-embryogenesis, Mmu-ASH is expressed in ectodermal cell clusters throughout the central nervous system (CNS), especially in the head and abdominal segments . These cell clusters are likely to correspond to the presumptive neural precursors, suggesting that Mmu-ASH acts as a proneural gene in Me. mulsanti. Because we also detected CNS expression for Mam-ASH and Gbu-ASH (data not shown), it is reasonable to conclude that ASH has a proneural function in all Gerromorpha. These expression domains mirror those of ac-sc genes known in Coleoptera [To determine the role of the unique AS-C gene during development in the Gerromorpha, we investigated the expression of segments a,b. Thesleoptera , Dipteraleoptera ,47 and Lleoptera .Figure ASH expression in transverse stripes in all legs in post-katatrepsis embryos. In G. buenoi, the stripes are first sharp and narrow (e) before the domain of expression expands throughout leg segments (f). The expression of Gbu-ASH eventually becomes restricted to numerous dots in the legs , each dot likely to prefigure the position of a future bristle. This expression pattern recapitulates only the early, but not the late, expression of ac and sc found in D. melanogaster legs [D. melanogaster, the predominant expression from 3 to 5 h after puparium formation is in discontinuous transverse stripes that encircle the tarsal segments. At approximately 5 h ac and sc expression in longitudinal stripes begins to appear in conjunction with the transverse stripes, and by 6 h the longitudinal stripes in each segment replace the transverse stripes [G. buenoi, Me. mulsanti or Mi. americana.We also detected d narrow e before segments f. The exthe legs g,h, eachter legs . In D. m stripes . We haveASH is restricted to discrete regions such as the distal tip of the tarsus  that prefigures the location of the future claw.In late embryos, the expression of Mam-ASH, d that pr(f)M. mulsanti, we observed altered bristle development in 82% of ASH RNAi-treated embryos (n = 32 out of 39), ranging from a weak to a dramatic reduction in the number of bristles in the thorax and the abdomen (a–d), as well as in the legs (data not shown) in late embryos. The variability we observe in the knockdown phenotype is a consequence of the RNAi technique as reported in previous studies in the Gerromorpha [Mi. americana, only mild alterations of bristle development have been obtained , showing missing bristles in the first two thoracic segments . In G. buenoi, we observed an altered phenotype in 97% of ASH RNAi-treated embryos (n = 208 out of 214), consisting of reduced bristle number on the whole body (g–j). ASH depletion, even subtle, consistently affected abdominal bristles , thoracic and head bristles secondarily and leg bristles . The variability in RNAi leg phenotype also ranges from mild to strong . The bristles that are missing or altered are not consistently the same ones between RNAi-treated embryos. However, all bristles can be affected independently of their location. It is reasonable to hypothesize that ASH controls the specification of all leg bristles and that the variability we see results from the partial efficiency of knockdown methods to silence the ASH gene.We used RNAi to deplete ASH function in the embryos. In  abdomen a–d, as wromorpha ,50,51. Isegments e,f. In Gole body g–j. ASH bristles g,h, thorbristles i,j. The ASH, as revealed by RNAi, is the control of grooming comb development in all legs. Whereas wild-type G. buenoi embryos have two grooming combs on the forelegs, and one comb on mid and hindlegs . Previous studies have shown that the knockdown of the gene Ultrabithorax (Ubx) does not affect grooming combs on any of the legs in the Gerridae [Ubx RNAi leads to the development of an ectopic hindleg comb in Microvelia and Mesovelia both of which lack hindleg combs otherwise [ASH repression by Ubx. Further experiments would be required to test this possible interaction between Ubx and ASH genes.Another important role of hindlegs , ASH RNAGerridae ,52. Howetherwise . This suASH RNAi-treated embryos also lack claws (j). This phenotype is observed for the first time in the context of an altered ac–sc complex. In Drosophila, mutation of one or several genes of the ac–sc complex does not affect the tarsal claw. However, the proneural gene amos, whose postembryonic expression prefigures the anlage of the innervated tarsal claw, is thought to be involved in the formation of sensory organ precursors in the tarsal claw [amos prevents bristle formation through the repression of scute function in Drosophila [ASH and amos act in the same developmental network during tarsal claw development in Gerromorpha.ck claws j. This psal claw . Previouosophila . As a fu4.ASH in bristle and grooming comb specification in Gerromorpha. We identified unexpected changes at the genomic ASH locus. Our results suggest that changes in the regulation of the gene ASH could have been associated with the adaptation of semi-aquatic bugs to the water surface and their subsequent diversification.The evolution of a higher density of hydrophobic leg hairs accompanied the invasion of water surface by semi-aquatic bugs. Variation in the density of bristles and the number of grooming combs found among the Gerromorpha might have evolved as an adaptation to the diversity of habitats these bugs live in . We have


Activities of daily living (ADL) are often used as predictors of health and function in older persons. This systematic review is part of a series initiated by the European Network for Action on Ageing and Physical Activity (EUNAAPA).To assess psychometric properties of ADL instruments for use in older populations.Electronic databases  were searched, using MeSH terms and relevant keywords. Studies, published in English, were included if they evaluated one or more psychometric properties of ADL instruments in community-dwelling older persons aged 60 years and older. Combination scales with IADL were excluded. This systematic review adhered to a pre-specified protocol regarding reliability, validity, and responsiveness.In total, 140 articles describing more than 50 different ADL instruments were included. Ten instruments which were applied in minimally three different articles of good quality (clear descriptions and adequate design according to the protocol), were evaluated for reliability, validity and responsiveness; each received a summary score. The four instruments with the highest scores were the Functional Autonomy Measurement System (SMAF), 5-items Katz list , Functional Independence and Difficulty Scale (FIDS) and the Barthel Index.Critical reflection is essential to avoid unnecessary modifications and use of instruments that have not been documented to be valid or reliable.Based on this systematic review, we recommend the SMAF, 5-item Katz, FIDS and Barthel index as ADL measures for research and care practice in older populations.The online version of this article (10.1007/s40520-018-1034-6) contains supplementary material, which is available to authorized users. In community-dwelling, older persons screening and assessing the ability to conduct activities of daily living (ADL), such as getting out of bed, toileting, bathing, dressing, grooming, and eating are frequently used. These measures are applied to detect early onset of disability and are key factors for care management   were searched from their inception to August 2012 and updated in November 2016. Using MeSH terms and relevant keywords, six semantic categories were entered: “ADL, Questionnaire, Age (60 years and older), Setting (community dwelling), Reproducibility, Validity”. Reference lists of review articles and included papers were scanned to identify further potential studies. The search was restricted to English language and peer-reviewed journal articles (see Appendix 2 for a comprehensive overview).To be included a study had to meet the following five criteria: (1) investigate at least one of the mentioned psychometric properties of an ADL instrument ; (2) measure (B)ADL in a separate (sub)scale; (3) include a population 60 years of age or older or with a mean age above 65 years or separately reported on this age group; (4) address community-dwelling older persons, and (5) have a sample size of at least 30 participants.Studies were excluded if they did not utilize a separate ADL scale; if the instrument used was developed for populations with specific diseases; or if the ADL scale had less than three items; or was rated inadequate for reporting reliability, validity, and/or responsiveness.Five independent reviewers performed abstract scanning, selection of full-text articles, and data extraction. Disagreements that could not be solved by discussion between two reviewers were judged by one of the other reviewers. Full-text papers were obtained if the inclusion criteria could be clearly determined from the abstract or eligibility was not sure. In the case where further information was needed authors of articles were contacted. The five independent reviewers read the full text articles and rated them as eligible or not.The overall quality of eligible studies was based on three domains ;  and rateTo evaluate the strength of evidence for the psychometric properties of instruments, the domains ‘Quality’, and ‘Quantity’ were used. Quality of the evidence could be positive or negative for individual studies and was rated by the reviewers according to the checklist as presented in Table Finally, the evaluation of the psychometric properties found in quality articles were summarized in Table Studies including ADL instruments report predictive, discriminant, construct and concurrent validity (see Table The literature search identified 6070 abstracts (5440 + 630 in the update 2016). After screening the abstracts (abstract lists with full references available on request), 1139 full papers (including 65 articles in the update) were identified and 1078 articles obtained and further screened for inclusion or exclusion (Dropbox was used as a filesharing database). The flow of the selection process can be viewed in Fig. N = 56)  ) 11–17, , for twoFour instruments with a minimum rating of three times a ‘plus’ in Table p < 0.01) revealing a good construct validity and responsiveness [53]. The results for the ADL scale in the telephone survey questionnaire were not as good revealing an ICC of 0.73 (CI 0.48–0.84) [51] Hebert and colleagues warn that “a survey method (note: they used a telephone survey) is not valid for generating accurate estimates of disabilities to plan services or determine budget requirements for responding to the needs of a population”.The SMAF  was the only instrument with very good ratings in all three domains . The SMAF is a 29-item scale based on the World Health Organization classification of disabilities. It measures functional ability in five areas: activities of daily living (ADL) , mobility (6 items), communication (3 items), mental functions (5 items), and instrumental activities of daily living (IADL) (8 items). Each item is scored on a 4-point scale from 0 (independent) to 3 (dependent) for a maximum score of 87. For every item that has a rating of 1 or higher , the human resources (help or supervision) required to support the level of disability in this specific area and the stability of these resources were evaluated. The SMAF must be administered by a trained health professional , who scores the individual after obtaining the information by questioning the subject and proxies or by observing . A test–p < 0.05) showing a reasonable reliability. Covinsky et al. [42] found that “the number of ADL reported 2 weeks before hospital admission was significantly associated with mortality 1 year later”. This data also supports a reasonable predictive validity. LaPlante [CS coefficient of scalability). We interpreted this as good construct validity. Responsiveness of the Katz 5 item ADL list was questionable except in groups with visual and cognitive impairment [46].The 5 item Katz list has reasonably good reliability, validity and responsiveness on average as reported in seven articles. However, the content of the five items could differ; two articles reported using “grooming” instead of “transferring”. Also, the wording for inventory items were not consistent. For example, we found eating to be described by both eating and feeding and toileting to be described as toileting or using the toilet. The inconsistent terminology also occurred for the Katz 6 and 7 item versions and the unspecified version of the Katz. Besides the items, also the wording of answer categories could be different (or was not mentioned). Kosloski et al. [48] reported a test–retest coefficient of 0.82 . The FIDS is not as sensitive to ceiling effects compared to the Barthel Index in healthy and in frail older people [65].A few recent articles concerned the development of the Functional Independence and Difficulty Scale (FIDS), a Japanese ADL instrument with 14 items [64–66]. This scale was validated in a Japanese population and showed good reliability . In addition, the range of the scoring was different (for example 0–100 or 0–20). Korner-Bitensky et al. [28] reported similarities in the face-to-face version and phone version > 90% (ICC 0.89). However, the phone version was unreliable in moderate and severe disabled patients. Thygese et al. [29] reported a Cronbach’s The remaining instruments listed in Table https://en.wikipedia.org/wiki/Construct_validity). The field of psychometric research needs more consensus about the use of these terms.Our aim was to assess ADL instruments for use in older community-dwelling populations on psychometric properties. We identified the SMAF, 5-item Katz, FIDS and Barthel index as the four most valid and reliable ADL instruments in this field. To our knowledge, this is the first systematic review of ADL instruments utilized in community-dwelling older persons, and we regard this as a strength. After evaluating more than 6000 abstracts, and, more than a 1000 full text articles, we conclude that psychometric information is mostly not sufficiently included. Reporting of the psychometric information spanned from no information at all to very specific information, which made the evaluation a very hard effort. Additionally, different terms were used in the articles  and the operational definition of these terms did sometimes not match the definitions provided in Table Only around 10% of the articles reported on the used ADL instrument appropriately and provided some valuable data about their reliability and validity in an older population. In total, we found 51 different ADL instruments, sometimes as remarkable as the Prayer ADL list . UnexpecIt took us several years to evaluate all full text articles that we found, but it is still possible that more information about available instruments could be found in other articles or publications (see for instance our footnote on the NHIS ADL in Appendix 3). It is possible that other research was not found given the specific inclusion and exclusion criteria. Therefore, we regard this systematic review as a not all-inclusive overview of the field.Additionally, because 60 years was set as a minimum, we excluded some interesting articles such as that of Reijneveld et al. . This stLaPlante  describeAs we chose to evaluate ADL (sub)scales and to omit combined ADL/IADL scales, we could not make any conclusions on combined lists. Perhaps this was not an optimal choice as La Plante  concludeThe widely used OECD disability indicator  containsThe Barthel Index is a well-known instrument for measuring ADL in patient populations. We found that it could also be used in older population with reasonable reliability and good responsiveness. Unfortunately, validity issues were not mentioned in the articles that we read while a review (not included in our search) by Sainsbury et al.  revealedThe six included articles on the SMAF were written by only two first authors (respectively Hebert and Desrosiers) between 1988 and 2012. As Hebert was the developer of this list, this may have been led to a bias in our review process, because some articles contained the same information. However, as all these articles were of good quality we think that we can safely recommend this instrument. Hebert [53] reveals also that the SMAF ADL could explain 57% of the variance in healthcare costs in community-dwelling older people, showing its usefulness in screening.Frequently, we found articles concerning the OARS scale  scales (for a good overview see Choi and Pak ). The prInstruments developed for patient populations were excluded. We encountered a review concerning ADL lists for dementia patients by Sikkes  that conAn older review by Law and Letts  in the oIn conclusion, only a few well-documented valid and reliable measures for ADL in populations of community-dwelling older people exist. Critical reflection in this field is necessary to avoid unnecessary modifications and the use of instruments that have not been documented to be valid and reliable. We recommend the SMAF and the FIDS in ADL screening and assessment. The Barthel Index and the 5-item list Katz inventory may be used with care concerning wording and items included.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 22 KB)Supplementary material 2 (DOCX 19 KB)Supplementary material 3 (XLSX 15 KB)


Hearing loss in newborns and children is a public health concern, due to high prevalence and negative effects on their development. Early detection and intervention of childhood hearing loss may mitigate these negative effects. Population-based newborn hearing screening programs have been established worldwide to identify children at risk for congenital hearing loss and to follow children at risk for late onset or progressive hearing loss. This article presents the protocol for a systematic review that aims to review the risk factors associated with permanent hearing loss in children, including congenital, early, or late onset. Risk factors associated with progressive hearing loss will be investigated as a secondary aim.Scientific literature from the following databases will be investigated: MEDLINE, Ovid MEDLINE(R) Daily and Ovid MEDLINE(R), Embase, and CINAHL. The primary outcome is a permanent bilateral or unilateral hearing loss with congenital onset or onset during childhood (birth to 18 years). The secondary outcome is progressive hearing loss. Studies must report data on risk factors associated with permanent hearing loss; risk factors may be present at birth or later and result in immediate or delayed hearing loss. Randomized controlled trials, quasi-experimental studies, nonrandomized comparative and non-comparative studies, and case series will be included. The risk of bias will be assessed using the Qualitative Assessment Tool for Quantitative Studies (McMaster University). If aggregation of data is possible for a subsection of studies, we will pool data using meta-analysis techniques. If aggregation of data is not possible, a qualitative synthesis will be presented. We will assess the quality and strength of the overall body of evidence using the Grading of Recommendations Assessment, Development and Evaluation (GRADE). The systematic review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) recommendations.The resulting information will inform the update of a provincial audiological surveillance protocol for the Ontario Infant Hearing Program and will be applicable to early hearing detection and intervention (EHDI) programs worldwide.We have registered the protocol in the International Prospective Register of Systematic Reviews (PROSPERO), registration number CRD42018104121.The online version of this article (10.1186/s13643-019-1073-x) contains supplementary material, which is available to authorized users. Permanent hearing loss affects at least 1 to 2 per 1000 children at birth and has severe consequences for their development . The negIn addition to screening newborns for hearing loss, there is a need to monitor children who are at risk of developing hearing loss after the neonatal period –13. PrioChildren at risk for late onset hearing loss need to be closely monitored, even after passing a screening test, to ensure identification as early as possible and to avoid delays in treatment, unnecessary costs to the healthcare system, and additional parental stress , 20. It In 2007, the Joint Committee on Infant Hearing (JCIH) updated the list of risk factors associated with permanent congenital, delayed onset, or progressive hearing loss in childhood . Due to The overall aim of this systematic review is to synthesize evidence on risk factors related to neonatal, early, and late onset hearing loss as well as progressive hearing loss in children. We will investigate all risk factors that have an immediate or delayed effect on hearing. The resulting information will inform surveillance protocols and will be applicable to EHDI programs worldwide.The review aims to answer the following primary research question: what risk factors are associated with permanent hearing loss in children? Risk factors related to congenital, early, and late onset hearing loss will be examined.A secondary objective of this review is to investigate the risk factors associated with progressive hearing loss in children with congenital, early, or late onset hearing loss.This systematic review protocol has been developed according to the Preferred Reporting Items for Systematic review and Meta-Analysis Protocols (PRISMA-P) , nonrandomized studies without comparison group , and case series. Consequently, we will exclude from this review case reports/case studies (less than five cases). Since we anticipate a very large number of articles, we have essentially extended the “case study” to include < 5 cases as we judged that these small cohorts would provide little additional information. We will also exclude editorials, viewpoints/opinion, literature reviews, conference abstracts, guidelines, recommendations, consensus papers, qualitative research, and gray literature, such as reports, thesis, and notes.Studies not directly related to the review questions but that present pertinent information about the relationships between risk factors and hearing loss will also be included, as long as they meet the review inclusion criteria .https://www.ncbi.nlm.nih.gov/mesh/). The age limit is because only the childhood onset of hearing loss is targeted by the research question. We will include articles on children and adults only if results are presented separately for children; we will accept an upper range of the study group of approximately age 21 years (young adults), when the majority of participants are children.All children with permanent bilateral or unilateral hearing loss will be included. For this review, a child is defined as age birth to 18 years. In most healthcare systems, pediatric care targets children from birth to adolescence (defined as less than 19 years), which is concordant with the Medical Subject Heading (MeSH) definition , toxoplasmosis, admission to a neonatal intensive care unit, ototoxic medications, and a family history of hearing loss . Risk faRisk factors associated with temporary conductive hearing loss will be excluded. Articles reporting “infants at risk” will not be eligible; the specific risk factor(s) must be identified.For studies that include a comparison group, the comparator will be either (1) no risk factors or (2) a comparison between one risk factor and other factors. Due to the nature of studies in this field, it is anticipated that most studies will not include a comparison group.The primary outcome is permanent bilateral or unilateral hearing loss in childhood. Permanent hearing loss refers to sensorineural hearing loss or conductive loss that is structural in nature and expected to last for 6 months or more . Therefoi)Congenital and early onset is defined as hearing loss present at birth or diagnosed within the first 3 months of life. This definition is consistent with the JCIH (2007) benchmark of diagnosis by 3 months of age .ii)Late onset is defined as the occurrence of hearing loss, typically after 3 months of age, after a normal hearing screen or confirmation of normal hearing through audiologic assessment.We will categorize the outcomes according to the onset of hearing loss:The flexibility in these definitions allows for known variations in the classification of onset of hearing loss in the literature. Studies that report exclusively on temporary conductive hearing loss or change in hearing following surgical procedures  are not targeted by this review.The diagnosis of hearing loss must be confirmed by appropriate assessment methods at the time of the study. Results from automated tests used for hearing screening (such as automated otoacoustic emissions or automated auditory brainstem responses) will be excluded. Outcomes based solely on parental report of hearing loss will also be excluded. Hearing loss defined as “requiring hearing aids/amplification” will be accepted as a permanent hearing loss only if assessed by audiologists or specialized professionals, based on appropriate tests.The secondary outcome of interest for this review is progressive hearing loss. Any amount of progressive hearing loss as defined by the authors will be included. All other inclusion criteria detailed under the primary outcome will apply.The search strategy was developed in collaboration with a health sciences librarian experienced in systematic reviews. The following databases will be searched: MEDLINE including Epub Ahead of Print, In-Process & Other Non-Indexed Citations, Ovid MEDLINE(R) Daily and Ovid MEDLINE(R) (1946 to July 30 2018), and Embase (1946 to July 30 2018) using the Ovid interface. CINAHL will be searched from inception on July 23, 2018 (database coverage dates not stated). Searches will be limited to the cohort, case control, or randomized controlled trials using existing filters. We will consider exposure of all risk factors outlined in the provincial surveillance protocol for the Ontario IHP . We willRestrictions will apply to language and publication date. We will consider papers published only in English since 1990, that include birth cohorts ≥ 1985. Articles in other languages will be screened only at the broad screening level. The date limit was set to correspond broadly with the implementation of UNHS programs.http://www.covidence.org). Prior to the title and abstract screening, a screening form will be developed based on the inclusion and exclusion criteria and will be tested among reviewers with a subset of articles. Titles and abstracts will be assessed by two independent reviewers (BV and EF or DN) for potential relevance; the reviewers will resolve any conflicts  and involve a third reviewer if required. At the second stage, two independent reviewers (BV and EF or DN) will screen all potentially relevant full-text articles . Disagreements will be resolved by consensus or a third member of the research team.The study selection involves three stages, managed within covidence Study designPopulation characteristics Details of control or comparison groups (if available according to the study design)Type of assessmentExposure—risk factors (including any author-specific definitions)Onset of hearing loss Progressive loss (including author-specific definitions)Outcome data (including author-specific definitions of hearing loss)Using study-specific data forms, pre-determined data will be extracted for each study. Data extracted will include:One researcher will extract all information, which will be verified by a second reviewer. Discrepant findings will be resolved through consensus or a third reviewer when required.https://methods.cochrane.org/bias/resources/cochrane-risk-bias-tool). Other study designs will be assessed using the Qualitative Assessment Tool for Quantitative Studies . This tool, developed at McMaster University, provides an overall methodological rating  based on an appraisal of eight domains: selection bias, study design, confounders, blinding, data collection methods, withdrawals and dropouts, intervention integrity, and analysis.The risk of bias assessment will be conducted by one researcher and verified by a second. Randomized control trials (RCT) will be evaluated using the Cochrane Risk of Bias Tool . Other classifications to be considered are the types of hearing loss  and age of onset (preschool versus school age).I2 statistics; for the interpretation of I2, a rough guide of low (0–25%), moderate (25–50%), substantial (50–75%), and considerable (75–100%) heterogeneity will be used [The feasibility of meta-analysis will be determined based on the similarity of clinical population, same risk factors, type of outcomes, and study designs. If meta-analysis is possible for some outcomes, we will use a random effects model to aggregate results. For continuous data, we will compute mean or standardized mean differences with 95% confidence intervals (CIs). For dichotomous data, risk ratios with 95% CIs will be computed, where appropriate. Statistical heterogeneity will be evaluated using  be used ; potenti be used .We will examine the following variables in subgroup analyses. Subgroups will be specific to age at onset  and to specific conditions or treatments such as cCMV infection, meningitis, and pediatric cancers treated with ototoxic medication.http://www.gradeworkinggroup.org) [After data extraction and analysis, we will assess the quality and strength of the overall body of evidence using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) tool (oup.org) . To use The primary objective of this review is to collect and synthesize information on risk factors associated with congenital, early, or late onset permanent hearing loss in children. It is widely acknowledged that children with congenital or neonatal risk factors need to be tested during the neonatal period and be closely monitored for late onset hearing loss. Our question is of importance because most newborn hearing screening and surveillance programs are based on the 2007 JCIH factors. This list was released more than 10 years ago, and its development process was not as accurate and transparent as in a systematic review. In addition, this list is modified in several programs suggesting that it does not apply to their context or practice or it is not followed in its entirety –32.We intentionally developed a search strategy and included numerous study designs to capture as many risk factors as possible. Based on other reviews, it is expected that the majority of studies will be descriptive and include small samples or cohorts of children with hearing loss. Our aim is to provide recommendations for policy makers and clinics based on the best available evidence. Consequently, limiting this review to high-quality methodological designs such as randomized controlled trials or large cohorts would limit the evidence for decision-making. Because it is intended to gather evidence for clinical practitioners and policymakers, we are convinced that all types of data are useful, on the condition that their methodological strengths and weaknesses are assessed, and integrated into the results.The selection of publication date for inclusion in this systematic review was a balanced decision. It has to be long enough to include well-known risk factors that have not been widely investigated recently, such as congenital rubella syndrome. On the other hand, it has to integrate the latest knowledge due to the evolution of healthcare and treatments over time. For example, care for premature newborns in neonatal intensive care units has changed and practice in the administration of ototoxic drugs is better controlled, which has a positive impact on acquired hearing loss. Consequently, the systematic review aims to integrate both knowledge that has not been recently investigated and new scientific data on the possible association between risk factors and hearing loss. Only risk factors included in the review articles will be addressed, that is, risk factors not included in articles covered by our comprehensive search strategy will not be considered for hearing loss in children.We anticipate that the results from this systematic review will guide decision-making in the Ontario IHP and will be applicable to UNHS programs worldwide.Additional file 1:PRISMA-P 2015 Checklist. (DOCX 30 kb)Additional file 2:Search strategy to be used. (PDF 330 kb)


When rTL was categorized into quartiles according to the distribution of rTL among controls, individuals with the longest telomeres had increased odds of adenoma when compared to individuals with shortest telomeres . This study suggests that long telomeres in normal colon tissue are associated with increased colorectal cancer risk.Telomere length has been associated with risk of several cancers. However, studies of the relationship between telomere length and colorectal cancer risk have been inconsistent. This study examined the relationship between telomere length in normal colon tissue and the prevalence of colorectal adenoma, a precursor to colorectal cancer. This nested case-control study consisted of 85 patients aged 40 to 65 undergoing a screening colonoscopy: 40 cases with adenoma(s) detected at colonoscopy and 45 controls with normal colonoscopy. During the colonoscopy, two pinch biopsies of healthy, normal appearing mucosa were obtained from the descending colon. Relative telomere length (rTL) was quantified in DNA extracted from colon mucosa using quantitative real-time PCR. Logistic regression was used to assess the relationship between telomere length and adenoma prevalence and estimate odds ratios and 95% confidence intervals. rTL was significantly longer in colon tissue of individuals with adenomas compared to healthy individuals ( Telomeres play an important role in maintaining chromosomal stability and genetic integrity ,2. The eBoth short and long telomeres have been implicated in carcinogenesis. Telomere length limits uncontrolled cellular proliferation; normally, tumour suppressor mechanisms ensure that once telomeres reach a critical length, apoptosis or cellular senescence is triggered . This prThe role of telomere length in the etiology of colorectal cancer is not well understood. Studies which have examined telomere length in blood leukocytes and colorectal cancer risk have been inconsistent, reporting no relationship –40, incrColorectal tumourigenesis involves a dysplastic progression from aberrant epithelium to adenomatous polyp (adenoma) to colorectal cancer (adenocarcinoma) ,55. ThisIn summary, the role of telomere length in colorectal carcinogenesis warrants further examination as results from studies of the association between telomere length and colorectal cancer risk remain equivocal. Within an individual, telomere length varies across tissues ; thus, iThis case-control study was nested in a cross-sectional study which recruited patients undergoing a screening colonoscopy at a regional endoscopy centre in Kingston, Ontario, Canada. Screening colonoscopies were performed on patients with a positive family history of colorectal adenoma or cancer, patients who had a positive fecal occult blood test (FOBT), and patients at average risk for colorectal cancer. Between 2009 and 2012, healthy male and female colonoscopy patients aged 40 to 65 were invited to participate in the cross-sectional study . Participants in the cross-sectional study consisted of 444 patients who provided written informed consent. Complete study design details and subject eligibility and exclusion criteria can be found elsewhere .Briefly, consenting patients were asked to complete a self-administered research questionnaire prior to their colonoscopy. At the time of colonoscopy a fasting blood sample and two pinch biopsies of colonic tissue were collected for biochemical and genetic analyses. Biopsies were obtained from healthy, normal appearing mucosa in the descending colon at least 10 cm from any abnormalities and the two biopsies were taken 10 cm apart from one another. Anthropometric data and colonoscopy results were obtained from medical records. The study was approved by the Queen's University Health Science Research Ethics Board.Of the 444 patients who consented to participate in the cross-sectional study, 48 were excluded due to incomplete colonoscopies, 50 were excluded due to a diagnosis of colorectal cancer, hyperplastic polyps or inflammatory bowel disease; a further 12 participants were excluded as a result of insufficient DNA for analyses. The final sample available for this study consisted of 334 patients.Participants were selected for this case-control study based on the pathology reports of the screening colonoscopy. Any colorectal abnormality detected during colonoscopy was removed and assessed by a gastrointestinal pathologist using standard diagnostic criteria. The case group consisted of patients who were found to have one or more colorectal adenomas at colonoscopy. Of the 110 patients with adenomas, 40 were randomly selected as the case group for this study. Patients with no colorectal abnormalities detected at colonoscopy comprised the control group. Of the 224 patients with normal colonoscopy, 45 were randomly selected as controls for this study.Colonic tissue samples obtained from each participant were placed immediately into 5 Prime cell lysis solution (Intermedico) and stored at -20°C until DNA extraction. Genomic DNA was isolated from colonic tissue using 5 Prime ArchivePure DNA Tissue Kits (Intermedico). Extracted DNA was stored in a solution of 10 mmol/L Tris; 1 mmol/L EDTA,  and kept at -20°C. Prior to rTL analyses DNA from the two pinch biopsies was pooled and was quantified using by UV absorbance at 260 nm and diluted with molecular grade water  to a concentration of 2 ng/μL.et al. [2 (Sigma-Aldrich); 1 mmol/L DTT (Sigma-Aldrich); 0.2 mmol/L each dNTP (Life Technologies), 0.025 U/uL AmpliTaq Gold polymerase (Life Technologies) and either 500 nM telomere primers, or 300 nM albumin primers. Using 96-well plates, 15 μL of master mix was added to 20 ng of DNA in wells, such that the final qPCR reaction volume was 25 μL. Thermal cycling followed the protocol described by Lan et al. [rTL was measured by quantitative PCR (qPCR) using the method developed by Cawthon with several minor modifications . Telomeret al. ) and albet al. ) were man et al. . Meltingq values of all samples and amplification efficiencies of both the telomere and the albumin (single copy gene) product [q) for each sample was ≤ 0.3 [LinRegPCR software (2014 version) was used to determine C product . qPCR efas ≤ 0.3 .Telomere length was expressed as a relative T/S ratio. The quantity of telomere (T) product was compared to the amount of albumin (S) product in each sample and were determined relative to a reference DNA sample using the Pfaffl  method. t-tests or χ2 tests. To examine the association between rTL and colorectal adenoma prevalence, rTL was categorized into quartiles based on the rTL distribution among the controls. Logistic regression was used to examine the relationship between rTL  and the prevalence of colorectal adenomas. To assess the possibility of a U-shaped relationship, a quadratic term of rTL was included in the model. Model fit was assessed using the likelihood ratio test. For all analyses in which rTL was represented as a continuous variable, rTL was standardized for ease of interpretation and to minimize collinearity in regression models with higher order terms. Factors related to adenoma risk  were assessed in order to describe the study population. The main analyses did not adjust for any of these factors as they were considered upstream of telomere length in the hypothesized causal model. However, results of analyses adjusted for age are also presented. SAS software version 9.4 (SAS Institute Inc.) was used to conduct all statistical analyses.Means and frequencies of selected characteristics for cases and controls were calculated and compared using p = 0.02), smoking (p = 0.004), and weakly related to older age (p = 0.13) and the use of non-steroidal anti-inflammatory drugs (p = 0.08). No relationship was observed between colorectal adenoma prevalence and obesity, or alcohol consumption. There was a weak inverse correlation between age and colon tissue rTL .The characteristics of the study population are shown in p = 0.008). Analyses of the relationship between rTL quartile and adenoma prevalence . Odds of colorectal adenoma prevalence were lowest for those with the shortest rTL. Compared to individuals with average rTL (standardized rTL = 0), those with rTL one standard deviation above the mean (standardized rTL = 1) had a 69% increase in the odds of colorectal adenoma prevalence (p = 0.007). Results presented are not adjusted for covariates; adjustment for age did not meaningfully change results.The shape of the relationship between telomere length and adenoma prevalence was found to be linear. Logistic regression models with rTL as a continuous variable indicated a positive dose-response relationship . InclusiThis nested case-control study assessed the relationship between colon tissue rTL and the prevalence of colorectal adenomas. Results from this study indicated a linear relationship between rTL in normal appearing colon tissue and the prevalence of colorectal adenomas. Compared to those with average rTL, those longer rTL had increased odds of having a colorectal adenoma. The odds ratios observed in this study provide evidence of direction, strength and statistcial significance of the relationship between rTL and colorectal adnoma risk, however these odds ratios do not provide an estimate for the prevalence risk ratio or incidence ratio.Similar to what has been reported elsewhere, a weak negative correlation was observed between colon tissue rTL and age in this study. The relationship between age and rTL appears to be generally consistent across a number of tissues ,5,70–73.et al. [Epidemiologic evidence on the relationship between colon tissue telomere length and colorectal adenoma prevalence is limited. To the best of our knowledge, only one other study has measured telomere length in normal colon tissue of both patients with adenomas or cancers and healthy controls . Fernandet al.  also repThe relationship between telomere length and colorectal risk has largely been studied by measuring telomere length in peripheral blood leukocytes and there is considerable heterogeneity in results across studies . ProspecThis study was limited by a small sample size. The larger study from which cases and controls were selected included 334 participants who were eligible for this study, but due to resource contstraints we were only able to measure telomere length in the colon tissue of 85 individuals. As a result of the small sample size, confidence intervals for estimates were wide. Future studies with large sample sizes are needed to validate these results. Nevertheless, the results of this study are biologically plausible.p-value of 0.15). Adjustment for covariates did not result in substantial changes to the estimated effect of rTL on adenoma prevalence and the conclusions remained the same. Previous studies have suggested that the relationship between telomere length and adenoma risk is modified by age and sex [Traditional risk factors of colorectal cancer are thought to be upstream of telomere length in the causal pathway and therefore were not considered potential confounders in this study. A sensitivity analysis which evaluated age, sex, BMI, alcohol consumption and pack years of smoking as covariates in the statistical model, found only sex and pack years of smoking to be important covariates  can easily be missed. The miss rate of adenomas is generally thought to be no more than 10%. Failure to detect adenomas among colonoscopy patients may have resulted in individuals being wrongly classified as having a normal colonoscopy and may have biased the results of this study. The detection rate of adenomas in this study is expected to be high as all colonoscopies were performed at a single clinical centre by experienced academic gastroenterologists whose detection rate is well within the acceptable range.It is important to consider the time frame during which telomere length may affect the development of colorectal cancer. Currently, it is not known at which point(s) during the adenoma-carcinoma sequence telomere length may pose a risk. An advantage of using colorectal adenoma, rather than adenocarcinoma as an endpoint, is that the time period from exposure and the occurrence of the outcome is shortened; this shortened time frame may allow a relationship to be more easily detected. It was assumed that telomere length is relatively stable, such that measuring telomere length at time of adenoma diagnosis would represent an appropriate exposure window. It is unlikely that adenoma would affect telomere length in distant normal appearing tissue, so reverse causation is not a concern. However, the use of adenoma prevalence as an outcome has the potential to cause bias. Prevalence is a function of incidence and duration, so the prevalence odds ratios of the relationship between rTL and colorectal adenomas may be subject to incidence prevalence bias. It is possible that telomere length affects the length of time for progression through the adenoma-carcinoma sequence. If this were the case, the results from this study would be biased.Given what is known about the important role of telomere length in chromosomal stability and regulating cellular proliferation, it is plausible that long telomeres in colon tissue increase the risk of colorectal cancer. Our results, which indicate that increased telomere length is related, in a dose-response manner, to an increased prevalence of colorectal adenomas, strengthen the conclusions of the only other paper which has addressed the same research question with a similar study design . NeverthS1 Table(XLSX)Click here for additional data file.


Cardiac development is a dynamic process and sensitive to environmental chemicals. Triclosan is widely used as an antibacterial agent and reported to transport across the placenta and affect embryonic development. Here, we used human embryonic stem cell- (hESC-) derived cardiomyocytes (CMs) to determine the effects of TCS exposure on cardiac development. After TCS treatment, the differentiation process was significantly blocked and spontaneous beating rates of CMs were also decreased. Transcriptome analysis showed the dysregulation of genes involved in cardiogenesis, including GATA4 and TNNT2. Additionally, DNA methylation was also altered by TCS exposure, especially in those regions with GATA motif enrichment. These alterations of transcriptome and DNA methylation were all associated with signaling pathways integral to heart development. Our findings indicate that TCS exposure might cause cardiomyocyte differentiation toxicity and provide the new insights into how environmental factors regulate DNA methylation and gene expressions during heart development. Cardiac development is a dynamic process, which occurs with complex transcriptional programs and signaling pathways . Cardiomin vitro model to investigate the molecular processes of embryonic cardiac development. Recent data indicate that this differentiation process recapitulates the similar developmental pattern of embryonic cardiogenesis in vivo [Human embryonic stem cells (hESCs), with their ability to differentiate into cardiomyocytes (CMs) in culture, serve as an  in vivo . hESC-deμg/L levels (range: <2.3–3620 μg/L) [μg/L (~0.1 μM) [μg/L (~1 μM) in maternal urine during pregnancy [Triclosan (TCS), as broad spectrum antibacterial agents, is widely used in household and personal care products (PCPs) such as hand soaps, toothpastes, and deodorants. It is one of the most frequently detected and highly concentrated chemicals in the environment and humans . TCS has20 μg/L) . Studies20 μg/L) , 10. Hig~0.1 μM) . The maxregnancy . The stuIn the present study, to investigate whether TCS exposure could induce cardiac toxicity during embryo development, a hESC-based cardiac differentiation model was used to explore the potential effect of TCS. Interestingly, we found that TCS exposure inhibited the differentiation of hESCs into CMs and spontaneous beating rates of CMs. Through gene expression and DNA methylation analysis, we observed that TCS exposure affected the CM marker gene expression and DNA methylation. Our findings will provide epigenetic mechanism information on the cardiotoxicity of TCS.H9 hESCs, purchased from the Institute of Biochemistry and Cell Biology at Shanghai, Chinese Academy of Sciences, were seeded onto 1% matrigel-coated 6-well plates in mTeSR1 medium  to 80–90% confluence. H9 hESCs were cultured and differentiated into CMs by using a monolayer-based directed differentiation protocol as previously reported . BrieflyμM was added on day 0 of differentiation. H9 were subjected to a 21-day differentiation procedure with TCS or vehicle control  treatment. The final concentration of TCS used in the differentiation assay was set based on our preliminary cytotoxicity assay result.TCS (≥97.0%) was obtained from Sigma-Aldrich Co. . To evaluate the impact of TCS on CM genesis, TCS at a concentration of 1 α-actinin was performed. For cardiac differentiation rate assessment, the NKX2.5 expression levels were detected. Briefly, CMs were fixed with paraformaldehyde and permeabilized with 0.1% Triton X-100. Then CMs were incubated with primary antibody cTnT  together with antibody α-actinin  or antibody NKX2.5  at 4°C overnight, followed by secondary antibody incubation. Signals of individual and merged image detection were performed using the fluorescence microscope . The fluorescence density of cells in each group was quantified and calculated by using ImageJ software. The relative intensity values were compared between the control and exposure groups. In total, cardiac differentiation capability, the morphology, and the beating rates of CMs were examined and compared between the TCS-treated and control groups. All experiments were repeated at least three times, and the images provided represent typical results.Changes in morphology were examined and photographed under a microscope every day. Spontaneous beating rates of CMs were recorded using a video-based camera system under the inverted microscope. To characterize the structure of H9-derived CMs, immunostaining for cardiac troponin T (cTnT) and sarcomeric p value greater than 0.01 in one or more samples were removed. We also excluded probes that mapped to more than one location in a bisulfite-converted genome or overlapped with the location of known SNPs. Methylation data were processed using the ChAMP package [β value and ranges from 0 (unmethylated) to 1 (methylated).At day 20 of differentiation, cells in culture were subsequently enriched by a commercial CM purification kit . Then the purified CM DNA was collected. Infinium MethylationEPIC BeadChips were used for the determination of methylation levels of more than 850000 CpG sites. Bisulfite-treated DNA sample was processed according to the protocol supplied by Illumina. The BeadChips were scanned with the Illumina HiScan SQ scanner, and raw data were imported to the GenomeStudio to extract the intensities. Probes located on the sex chromosomes and those that had a detection  package . The sig package . At eachRaw data were processed by ChAMP . DiffereAt day 20 of differentiation, cells in culture were subsequently enriched by a commercial CM purification kit. CM RNA was extracted by RNeasy Kits  and treated with DNase I  according to standard protocols. RNA sequencing was done in Novagene using TruSeq stranded mRNA library preparation . Briefly, intact RNA was fragmented, end repaired, adapter ligated, and PCR amplified following the Illumina protocol. Libraries were sequenced by Illumina HiSeq 2000. After quality control, sequence data were processed with STAR  and normt-test. All statistical analyses were performed using SPSS software, version 16.0 . Differences at p < 0.05 were considered as statistically significant. For DNA methylation and RNA-seq, all statistical tests were conducted in R (version 3.1.1). For DNA methylation and gene expression validation, all data are expressed as the mean ± standard deviation (SD) and Student's t-test was adopted to estimate the significance of the differences between the TCS-treated and the control groups.Dates for the effect of TCS on cardiac differentiation were expressed as the mean ± standard error of mean (SEM). Statistical comparison between the TCS-treated and the control groups was determined by Student's in vitro. The first beating cluster of cells was observed between day 8 and day 10. We observed that greater than 80% CMs were obtained at the end of differentiation in the control group, while around 60% CMs were obtained in the TCS-treated group. The changes in the morphology of hESCs during differentiation were examined and photographed under a microscope. The cardiac structure can be evaluated by cTnT and α-actinin. During differentiation, the sarcomere structure was visualized by α-actinin and cTnT immunostaining in H9-derived CMs. Results showed that most cells in culture were positive for cTnT and α-actinin. No significant differences in morphology were observed between TCS-treated cells and the control cells. Immunolabeling of these myofilament proteins indicates that well-organized sarcomeric structures were similarly developed in both the TCS and control groups , including 917 upregulated and 1246 downregulated DEGs with fold change > 2 and FDR < 0.05, were identified using DESeq2 comparing the TCS group with the control group Figures . RepreseDNA methylation is the process of adding a methyl group to C5 position of the cytosine by DNA methyltransferases (DNMTs), which is a crucial epigenetic modification during CM differentiation . To inveNext, we analyzed conservation of TCS-related DMRs and the underlying DNA sequences of these DMRs were conserved across placental mammals , which iμg/L (~1.4 μM) TCS caused reduction in heart rate and resulted in a more substantial impact on end-diastolic volume, stroke volume, and ejection fraction in zebrafish [μg/L (~1 μM) TCS caused cardiovascular toxicity in zebrafish and 1 μM TCS could inhibit cardiogenesis in mouse embryonic stem cells. However, the relationship between TCS exposure and heart development defects is not well understood. By integration analysis of transcriptome and DNA methylome, we found that TCS could block the formation of CMs from hESCs through affecting the CM-related gene expressions and DNA methylations. Our findings highlight the TCS effects on key genes involved in heart development.Human heart development requires fine tuning of CM-related genes, as well as other critical genes, and is sensitive to environmental factors . As a wiebrafish . ResultsμM TCS was used as the test concentration. This is also the concentration used in most experiments that examine the effect of TCS on cells. Exposed to TCS, cardiac differentiation rates were significantly affected. Additionally, the spontaneously beating rates of CMs were also reduced at the TCS group. In agreement with these phenotypes, the mRNA level of ACTC1 was significantly decreased after TCS exposure. ACTC1, the cardiac α-actin gene, has been reported to play roles in sporadic congenital heart disease (CHD) [In our previous study, we already found that TCS exposure affected the proliferation of mESC . In the se (CHD) . Moreovese (CHD) . Knockinse (CHD) .DNA methylation is one of several epigenetic mechanisms that regulate gene expressions . We sougOverall, we have shown that the hESC-derived CM model enables quantitative screening of the potential cardiotoxicity of environmental chemicals by analyzing the changes of CM viability, contractility, gene expression, and epigenetic modification. Researchers can efficiently detect both the morphological and genetic toxicities of the cardiac toxicants in a short-term experiment. One limitation in our study is that we utilized a single hESC line. Differences in cell induction conditions and the epigenetic factors might affect the differentiation competence of hESCs into CMs, which might limit their ability to accurately predict cardiotoxicity. In a future study, using different hESC lines and multiple time point assays is needed to provide more insights into chemicals' cardiotoxicity.In summary, by combining the DNA methylome and transcriptome analysis after TCS exposure, we observed the dynamic changes in transcriptome and DNA methylome in CMs. Our findings open new avenues in how TCS exposure affects the heart development and provide new insights into how environmental factors regulate DNA methylation and gene expressions.


Current data suggest that approximately 466 million people (5.0%) of the world’s population have disabling hearing loss, therefrom, 34 million children, impacting their quality of life. To provide estimates on the prevalence of hearing loss on a national level, we reviewed the epidemiological literature addressing hearing loss in children and adolescents living in Germany as an example for a Western country.clinicaltrials.gov. Study selection, data extraction, and methodological assessment were carried out by two reviewers.We searched Medline, Web of Science, Cochrane Library, ScienceDirect and LIVIVO to identify published data. Furthermore, we manually searched websites of relevant institutions and journals not listed in electronically and searched for ongoing studies and/or not yet published data in In total, 11 reports provided data with sample sizes ranging from 310 up to more than 14 million children and adolescents. Prevalence data were collected by interviews (self-assessments), using pure-tone audiometry or the international classification of diseases (ICD-10) coding and ranged from 0.1 to 128 per 1000 children. Although the estimate of the prevalence of hearing loss goes down, when the threshold was raised, generating a comprehensive and coherent set of estimates proved challenging owing to clinical heterogeneity including variation in age, the study setting, the definition of hearing loss and the assessment method. Moreover, representativeness  was often impaired owing to estimates lacking currentness  or selected (patient) data and may not be typical for a more general population.In conclusions, this work raises public awareness of the high prevalence of hearing loss, highlights issues associated with epidemiological research and is of great importance for researcher and those who use epidemiological data to inform clinical and political decision making. Hearing loss is the fourth highest cause of disability globally . CurrentOverall, it is assumed that half of all cases of hearing loss in children could be prevented through public health measures. Particularly, early detection  and therapeutic management are crucial to minimize the impact of hearing loss on a child’s development  and prospects for personal growth later in life –8. ThoseData on the prevalence and incidence of hearing loss are, however, often difficult to identify, outdated, or may not reflect the current population. This work aimed to estimate the number of children and adolescents with hearing loss and the proportion of children wearing hearing aids using available data from the general population living in Germany as an example for a Western country. Considering the methodological quality (risk of bias and representativeness) from these prevalence data this approach allows us to highlight potential issues associated with epidemiological research and judge whether there is sufficient evidence-based knowledge in this otologic research area on a national level . This syThe present systematic review was planned and conducted according to rigorous methodological standards, and reported in adherence to the PRISMA statement . An a prWe included published studies and/or other data sources providing estimates on the prevalence  and/or incidence (number of new cases in a defined observation period) of hearing disorder in children and adolescents (up to 19 years of age) living in Germany. Studies addressing exclusively newborns or populations with specific diseases such as children with genetic defects or metabolic disorders were excluded as well as studies conducted prior to 1975 . No restrictions were made in relation to the design of the included studies.www.cochranelibrary.com), ScienceDirect (Elsevier), and LIVIVO. Additionally, we searched the bibliographies of relevant studies to identify further citations. A search was also conducted for ongoing or completed but not yet published studies in clinicaltrials.gov and the German study register (www.drks.de). Furthermore, the websites of different national institutes gathering epidemiological data were searched , the National Association of Statutory Health Insurance Physicians (www.kbv.de), and the German Federal Statistics Office (www.destatis.de)). The search strategy used in Medline (Ovid) is presented in the Additional file A systematic literature search for studies published in German or English language was carried out in May 2017 and an update search was conducted in March 2019. We searched Medline, Medline Daily Update, Medline In Process, and other Non-Indexed Citations (Ovid), Web of Science , Cochrane Library (Two reviewers (PK and CS) screened the titles and abstracts of all reports identified by the searches. Thereafter, full-text copies of all potentially relevant articles were obtained and were assessed for eligibility.(i) key study characteristics ; (ii) prevalence data and / or incidence of hearing loss  as well as information on the proportion of these children with hearing aids or cochlear implants.The following data were extracted independently by the two reviewers mentioned above: (i) the validity of data collection, i.e., whether the prevalence of hearing loss was judged by the respondents themselves , or whether the studies applied standardized procedures ; (ii) specification of the hearing loss, i.e., whether the hearing loss was defined after standardized criteria  or whether no adequate definition was used ; (iii) the completeness of data, i.e., whether all recruited children (whole study sample) were considered when data were analyzed (low risk of bias) or whether data were missing . Data representativeness based on the characteristics of the study sample; i.e., when a selected sample (e.g. children and adolescents from one region or city in Germany) was considered to derive prevalence estimates, representativeness was judged as “low”, whereas data representativeness was judged as “high” when the study included a broad-ranging sample reflecting the entire adolescent population living in Germany.The risk of bias and the representativeness was assessed considering pre-defined criteria which were developed by our group based on other epidemiological research . TherebyOf note, both for data extraction and the methodological assessments, we relied on information provided in the individual study reports. If no judgment could be made owing to missing information (poor reporting), the corresponding item for risk of bias or data representativeness was classified as “unclear”.clinicalstrials.gov [n = 12] and the DRKS register [n = 19]). In total, 2113 references were excluded by title and/or abstract screening because they did not address our research question.The systematic literature search identified 2601 references. Additionally, we identified 63 references by hand searching, including 31 registry entries referring to ongoing or completed and not yet published studies  provided data on the prevalence of hearing disorders in children and adolescents. The study flow diagram is presented in Fig. Table Data on the prevalence of hearing loss - stratified (where possible) by assessment method, age, and definition of hearing loss - are provided in Table Self-reported hearing loss: prevalence of any self-reported hearing loss in adolescents up to 19 years of age ranged from 1.0% (23) to 4.0% (18). Data from screening programs: prevalence estimates based on preschool screening programs (> 30 dB HL) (conducted between 1991 and 2002) ranged from 3.9 to 5.2% (19). Using a threshold of > 20 dB HL impairment, the prevalence estimate in preschool children increased up to 7.4% (20). In comparison, a nationwide screening study estimated a prevalence of 2.4% (> 30 dB HL) and 12.8% (> 20 dB HL) in school children [Data from registries or special facilities: Using the most current data from the German disability registry from 2015, the prevalence of severe hearing loss (defined as “deafness” without providing any threshold) –across all age groups up to 18 years of age- was reported to be 0.01% (17). This estimate is slightly lower than one that was recorded in 1977 suggesting that 0.08% of children at eight years of age show a hearing loss (> 50 dB HL) [ to 4.0% . Data fr0 dB HL) , 30. In 0 dB HL) –28, wherrom 1.0%  to 4.0% 0 dB HL) ). Using 0 dB HL) –28).Only one study including children and adolescents of 14 years and younger provided self-reported data on the prevalence of hearing aids . HearingThe respective assessments are shown in Table Data representativeness  was an issue in most studies: (i) the data collected in registries were provided only by selected physicians –25 and/oOverall, two studies were judged to be at low risk of bias and of high representativeness , 21.The prevalence of hearing loss in the child and adolescent German population ranged from 0.1 to 128 per 1000 children and adolescents. Even though the prevalence of hearing loss decreases, when the hearing threshold is raised , generating a coherent set of estimates proved challenging due to clinical heterogeneity including variation in the age range, the study setting, the definition of hearing loss and the assessment method. As an example, (i) self-reported hearing loss ranged between 1.0 and 4.0%; (ii) estimates for positive findings measured within speech frequencies in screening studies ranged between 7.4 and 12.8% (using a threshold of > 20 dB HL) and 2.4 and 5.2% (using a threshold of > 30 dB HL), respectively; (iii) whereas estimates based on data collected in registries were between 0.01 and 0.10% for profound hearing loss including deafness or at approximately 4.0% when ICD-10 coding were used. Age- and gender-dependent differences in hearing loss have been reported. Although, our review could not identify such differences in dependence of age, owing to the wide age-ranges included in most the studies, the mean gender ratio males/females was 1.2. Only one study provided estimates on “self-reported” coverage with hearing aids. But valid data on the proportion of hearing impaired children not supplied with hearing aids could not be derived from this study. Likewise, data on the proportion of children and adolescents treated with implantable hearing devices and cochlear implants in the general population are also currently lacking. We identified one other systematic review reporting that hearing loss (derived from screening studies) affected approximately 3.0% of all children and adolescents under 20 years of age living in the United States . AlthougThe current systematic review provides prevalence data on hearing loss in the German child and adolescent population. Particularly, it benefits from a thorough and comprehensive literature search including manual searches  to identify data not formally published in an electronic database. Although we considered the risk of bias and assessed representativeness of the available epidemiological data, some issues owing to these assessment methods had to be solved: As an example, there are no well-established tools to assess bias and representativeness of epidemiological studies. Therefore, taking into account published epidemiological literature , we deveOn the other hand, we are aware that our review findings need to be interpreted with caution due to several limitations: First, risk of bias assessment revealed, for some studies, inappropriate methods to capture prevalence data (e.g. by self-assessment using different questionnaires), which in turn could have led to over- or under-estimation of the true prevalence of hearing loss. The impact of this bias on the reported estimates could, however, not be determined, as none of those studies referring to self-reported data ascertained their prevalence estimates using pure-tone audiograms or another thorough diagnostic assessment. Second, in most studies, external validity (representativeness) of the results was potentially impaired owing to prevalence estimates derived from: (i) children and adolescents living in a special region , 26–28; (ii) Moreover, most of the available data were either not representative for the general population of children and adolescents living in Germany or were considered to be at risk of bias limiting their validity. (iii) In view of the negative impact associated with untreated hearing loss, particularly in children and adolescents, as well as the socio-economic costs, a well-done epidemiological study in a more representative population using standardized definitions of hearing loss and objective methods for case ascertainment seems warranted. Such a study would provide reliable age-dependent  data on the prevalence of hearing loss, and would allow estimation of the extent of coverage with hearing aids in the general child and adolescent Western European population. (iv) When valid estimates on the prevalence of hearing loss becomes available, one will be better able to address the major preventable causes of hearing loss, develop and disseminate recommendations to prevent them and build partnerships to develop strong hearing care programs including hearing screening and management.This systematic review set out to estimate the prevalence of hearing loss in the German population as an example of a Western European country. It also highlights issues associated with estimating the prevalence of hearing loss. Therefore, it has not only implications for pediatricians, general practitioners and otolaryngologists, but also for researchers and those who use epidemiological data to inform political decision making. (i) We found that prevalence data varied broadly reflecting different definitions of hearing loss, different methods of case ascertainment and data collection, different settings and different age-ranges. Generation of a comprehensive and coherent set of prevalence estimates was therefore challenging. Additional file 1. Search strategy in Medline. (DOCX 21 kb)


Camellia sinesis), which is an elite and widely grown cultivar in China and is rich in flavan-3-ols providing with high nutritional value to human health. In our study, metabolic profiling was preformed to identify two conjugates and four aglycones of flavan-3-ols: (−)-epigallocatechin-gallate [(−)-EGCG], (−)-epicatechin-gallate [(−)-ECG], (−)-epigallocatechin [(−)-EGC], (−)-epicatechin [(−)-EC], (+)-catechin [(+)-Ca], and (+)-gallocatechin [(+)-GC], of which (−)-EGCG, (−)-ECG, (−)-EGC, and (−)-EC accounted for 70–85% of total flavan-3-ols in different tissues. Crude ANR enzyme was extracted from young leaves. Enzymatic assays showed that crude ANR extracts catalyzed cyanidin and delphinidin to (−)-EC and (−)-Ca and (−)-EGC and (−)-GC, respectively, in which (−)-EC and (−)-EGC were major products. Moreover, two ANR cDNAs were cloned from leaves, namely CssANRa and CssANRb. His-Tag fused recombinant CssANRa and CssANRb converted cyanidin and delphinidin to (−)-EC and (−)-Ca and (−)-EGC and (−)-GC, respectively. In addition, (+)-EC was observed from the catalysis of recombinant CssANRa and CssANRb. Further overexpression of the two genes in tobacco led to the formation of PAs in flowers and the reduction of anthocyanins. Taken together, these data indicate that the majority of leaf flavan-3-ols in Shuchazao’s leaves were produced from the ANR pathway.Anthocyanidin reductase (ANR) is a key enzyme in the ANR biosynthetic pathway of flavan-3-ols and proanthocyanidins (PAs) in plants. Herein, we report characterization of the ANR pathway of flavan-3-ols in Shuchazao tea ( Camellia sinensis (L.) O. Kuntze] plants. China has cultivated tea for more than 2000 years. During the long cultivation history, numerous elite tea varieties have been cultivated for health benefits. To date, approximately 246 cultivars, such as Longjing and Shuchaozao, are grown for multiple nutraceutical benefits and aroma flavors  and (−)-epigallocatechin-3-O-gallate [(−)-EGCG)]. The formation of (−)-ECG and (−)-EGCG was recently characterized by enzyme investigations. Two enzymes, namely UDP-glucose: galloyl-1-O-β-d-glucosyltransferase (UGGT) and 1-O-galloyl-β-d-glucose O-galloyltransferase (ECGT) [O positon of both (−)-EC and (−)-EGC to form (−)-ECG and (−)-EGCG.The main composition of tea flavan-3-ols has been appropriately characterized by numerous investigations. Primary aglycones of flavan-3-ols include (+)-catechin [(+)-Ca)] and (−)-epicatechin [(−)-EC)] b, and (+e (ECGT) , were isMedicago trucatula and M. sativa)  -EGCG was the highest, from 3.0% to 4.5% -Ca was the lowest, from 0.1–0.3% in five position leaves. The contents of (−)-ECG, (+)-GC, (−)-EC, and (−)-EGC were in a range of 0.9–1.9%, 0.4–2.3%, 0.4–1.1%, and 0.4–0.5%  in the five positional samples, respectively.Among the five positional samples, the buds contained the highest total contents of flavan-3-ols, approximately 9.6% . The 4th position leaves contained the lowest total content, approximately 5.7% . Among six metabolites, except for (−)-EGC and (−)-EC, four metabolites showed an apparent decrease trend from the 2nd leaf to the 4th leaf. This result provides evidence indicating that different quality of tea products is associated with these sample positions.Crude enzyme analysis has been reported being an effective method to demonstrate the ANR pathway in plants . To undeR,3R-2,3-cis stereo types, such as (−)-EC and (−)-EGC, accounted for most of flavan-3-ol metabolites synthesized in leaves described above.To further confirm the chirality of enzymatic products separated by reverse phase column, the major product derived from the incubation of crude ANR extract and cyanidin was isolated on TLC plates according to our previous reports . Based oANR sequences (GenBank: KY615701.1 and GenBank: KY615702.1), which have been curated by NCBI. Based on these two sequences, we isolated two ANR cDNA sequences from new leaves of Shuchazao. In addition to ours reported here, two ANR homologs, namely CsANR1 and CsANR2, were reported from the blister blight-resistant tea cultivar TRI2043 in Sri Lanka . After reaction for five min, samples were measured at 640 nm on a UV spectrophotometer  [Flowers were ground into fine powder in liquid nitrogen to extract anthocyanins and PAs. Anthocyanins were extracted from 100 mg powdered tobacco flower samples as described previously . Anthocy, Japan) . AbsorbeANR and transcription factor genes. Three developmental stages of flowers Ruixue were alabastrum (S1), half opened (bloom) flower (S2) and full bloom (S3). Transcriptomes from these flower stages were sequenced by the Beijing Genomics Institute  [ANR cDNAs were extracted from these transcriptomes to compare its abundance in different tissues. Moreover, all unigenes in these transcriptomes were blasted against transcription factor (TF) database from plant  to annotate potential TFs. The resulting TF genes were further blasted against tree genomes (NCBI Sequence Read Archive Database No. PRJNA381277) [http://www.omicshare.com/tools/Home/Soft/trend) to understand each TF gene expression trend pattern, those TF genes which with the same trend were grouped together to be tagged as one group. As a result, different profile groups were numerated into profile 1, 2, and so on.Recently, eight transcriptomes were sequenced from buds, the 1st leaf, the 2nd leaf, young stem, old stem, and three developmental stage flower samples of two commercial verities, “Pingyangtezao (PYTZ)” and “Ruixue”. Transcriptomes of buds, the 1st leaf, the 2nd leaf, young stem, and old stem of PYTZ were sequenced by Gene Denovo Biotechnology Co. . Although these transcriptomes have not been published, we were allowed to mine transcripts of tea , China) . TranscrA381277)  to unifyA381277) . Transcr


Background: With the aging population, the prevalence of age-related hearing loss will increase substantially. Prevention requires more knowledge on modifiable risk factors. Obesity and diet quality have been suggested to play a role in the etiology of age-related hearing loss. We aimed to investigate independent associations of body composition and diet quality with age-related hearing loss.Methods: We performed cross-sectional and longitudinal analyses (follow-up: 4.4 years) in the population-based Rotterdam Study. At baseline (2006-2014), 2,906 participants underwent assessment of body composition, diet, and hearing. Of these 2,906 participants, 636 had hearing assessment at follow-up (2014-2016). Association of body composition and of diet quality with hearing loss were examined using multivariable linear regression models.Results: Cross-sectionally, higher body mass index and fat mass index were associated with increased hearing thresholds. These associations did not remain statistically significant at follow-up. We found no associations between overall diet quality and hearing thresholds.Conclusions: This study shows that a higher body mass index, and in particular a higher fat mass index, is related to age-related hearing loss. However, whether maintaining a healthy body composition may actually reduce the effects of age-related hearing loss in the aging population requires further longitudinal population-based research. Age-related hearing loss is highly prevalent among older adults ,2, and iOne such risk factor may be obesity, which has been linked to increased hearing thresholds ,5,8–10. It has been suggested that diet quality plays a role in the relation between body composition and age-related hearing loss. Studies observed a relation between food groups, such as fish and carbohydrates consumption, and hearing thresholds –14. HoweWe aimed to investigate the association between detailed body composition (distinguishing between lean mass and fat mass) and age-related hearing loss and between overall diet quality and age-related hearing loss. We mutually adjusted for body composition and diet quality, therefore examining the independent relationship between body composition, diet quality and age-related hearing loss.2 (SD: 4.1) and a median dietary adherence score of 7 (interquartile range: 6-8). Participants had a mean overall frequency hearing threshold of 24.1 decibel (dB) (SD: 12.1). Of the total group of 2,906 participants, 636 participants had a second hearing assessment at follow-up  (nge 5.1) .After adjustment for relevant confounders (model 2), one point higher BMI was associated with a 0.53 dB  increase in hearing thresholds across all frequencies and with 0.42 dB  increase in hearing thresholds in the lower frequencies . AssociaTable 2, 3Body composition and diet quality were not related to change in hearing thresholds at follow-up . Some foThere were no significant interactions (p<0.05) between body composition and sex, diet quality and BMI, and between diet quality and sex. Effect sizes did not differ between men and women (data not shown).In this large sample of community-dwelling individuals, we found that adiposity was associated with increased hearing threshold. Although not statistically significant, these effects estimates were similar at follow-up. We found no associations of overall diet quality with age-related hearing loss.Strengths of our study included the population-based setting, the large sample size, and the standardized assessment of hearing thresholds with pure-tone audiograms and detailed measurement of body composition. Some limitations of this study should also be acknowledged. First, although we adjusted for possible confounders, there still might be residual confounding present. At last, the FFQ relies on an individual’s capacity to recall their dietary behaviour over the past month. Recall bias in dietary behaviour could be a systematic bias.We found that a higher BMI was associated with higher hearing thresholds in our cross-sectional analysis. BMI is an important marker for metabolic diseases , and is The fact that we find an association for FMI and not for FFMI is in line with the common idea that body composition influences hearing thresholds through vascular mechanisms. Hearing thresholds are associated with vasculopathies in metabolic diseases and therefore it is hypothesized that BMI is associated with the development of increased hearing thresholds ,18,23. TWith the growing prevalence of obesity ,26, healIn our longitudinal analysis of body composition and age-related hearing loss, no significant associations were found, but effects estimates remained about the same as in the cross-sectional analysis. The absence of a significant effect might be explained by the fact that relative few people had a hearing assessment at follow-up but more likely, that the time interval might have been too short. To our knowledge, there are only two studies of a longitudinal origin  21], in, in21], In conclusion, this study shows no association of diet quality with hearing loss and that a higher BMI is associated with hearing thresholds. This association with BMI is mainly driven by a higher FMI, suggesting involvement of metabolic and cardiovascular mechanisms, which may affect the cochlear function and suggesting that FMI is a better measure of body composition in age-related hearing loss. Whether a healthy body composition could serve as a preventive strategy for age-related hearing loss and thereby reducing the adverse consequences of hearing loss in older adults requires further longitudinal population-based research.This study was embedded in the Rotterdam Study, a population-based prospective cohort study in the Netherlands . From 20The Rotterdam Study has been approved by the medical ethics committee of the Erasmus MC (registration number MEC 02.1015) and by the Dutch Ministry of Health, Welfare and Sport . All participants provided written informed consent to participate in the study and to have their information obtained from treating physicians.2) [2), and fat-free mass index . In this division, the sum of FMI and FFMI is BMI.Information on body weight and length was obtained by physical examination and BMI was calculated (kg/m2) . A total2) . With thDiet quality was assessed with a validated self-administered semi-quantitative food-frequency questionnaire (FFQ) consisting of 389 items. The FFQ was found to be an appropriate measurement tool for ranking people according to their food intake . As descAudiometric assessment was performed in a soundproof booth by one health care professional . A compuPure tone audiometry was performed to determine hearing thresholds in decibel (dB) hearing level, measured according to the ISO-standard 8253-1 . For both ears, air conduction ) was tested. Masking was performed according to the method of Hood . The best hearing ear was determined by taking the average threshold over all frequencies. When hearing thresholds for both ears were equal, we alternately chose the left or right ear. The low-frequency hearing threshold is the average of 0.25, 0.50, and 1 kHz and the high-frequency hearing threshold is the average of 2, 4, and 8 kHz. We excluded participants with an air-bone gap of 15 dB or more in the better ear to eliminate conductive hearing loss.Information on smoking was collected through self-report and categorized into never, former and current . Educati2, and education. In the second model we additionally adjusted for total brain volume, education, physical activity, smoking (current and past), energy intake, alcohol intake, hypertension, hypercholesterolemia and type 2 diabetes. In the third model for body composition, we additionally adjusted for diet quality score and in the second model for diet quality, we additionally adjusted for BMI. For hearing thresholds at follow-up, the same regression models were used, additionally corrected for hearing levels at baseline and for time between hearing assessments.The association of body composition  and of diet quality  with hearing loss  was examined using multivariable linear regression models. In the first model we adjusted for sex, age, ageWe explored whether associations differed by sex and if effects differed across BMI groups.Missing data on covariates (<1%) were imputed using the multiple imputation algorithm (5 imputations) of SPSS. IBM SPSS statistics for Windows, version 24.0  was used.Data can be obtained on request. Requests should be directed toward the management team of the Rotterdam Study (secretariat.epi@erasmusmc.nl), which has a protocol for approving data requests. Because of restrictions based on privacy regulations and informed consent of the participants, data cannot be made freely available in a public repository.Supplementary Tables S1-S6