The relationship between lung dose-volume histogram (DVH) parameters and radiation pneumonitis (RP) associated with induction concurrent chemoradiotherapy (CCRT) followed by surgery in patients with non-small cell lung cancer (NSCLC) is unclear, particularly when concerning irradiation of the whole lung prior to resection. We performed this study to identify factors associated with grade ≥ 2 RP in such patients.Patients who received induction CCRT  between May 2003 and May 2017 were reviewed. The mean lung dose (MLD) and the percentage of the lung volume that received ≥5 Gy (V5) and ≥ 20 Gy (V20) were calculated. Factors associated with the development of grade ≥ 2 RP were analyzed.p = 0.007, 0.002, and 0.004, respectively). Moreover, an MLD ≥10 Gy and lower lobe location were significant predictors of grade ≥ 2 RP on multivariate analysis . The cumulative incidence rates of grade ≥ 2 RP at 6 months were 15.7 and 45.6% in patients with MLDs < 10 Gy and ≥ 10 Gy, respectively, and were 23.5 and 55.6% in patients with upper/middle lobe- vs. lower lobe-located tumors, respectively.One hundred and eight patients were included in this study, 34 (31.5%) of whom experienced grade ≥ 2 RP. A V20 ≥ 21%, an MLD ≥10 Gy, and a lower lobe tumor location were significant predictors of grade ≥ 2 RP on univariate analysis (MLD and lower lobe location were predictors of grade ≥ 2 RP in patients who received induction CCRT. It is necessary to reduce the MLD to the greatest extent possible to prevent the occurrence of this adverse event. Definitive concurrent chemoradiotherapy (CCRT) is considered a standard therapy for patients with locally advanced stage III non-small-cell lung cancer (NSCLC); the concurrent administration of docetaxel and cisplatin has shown promising results . HoweverRadiation pneumonitis (RP) is a notable adverse event after thoracic radiotherapy. A recent breakthrough phase III trial of durvalumab after CCRT showed significantly longer progression-free survival in patients administered durvalumab than in those administered a placebo . HoweverWhile previous studies have investigated the correlation between the dose-volume histogram (DVH) parameters and RP in patients who received definitive CCRT –12, therIn the present study, we investigated the relationship between whole-lung DVH parameters and the development of grade ≥ 2 RP in patients receiving induction CCRT for NSCLC.Data from patients with histologically confirmed NSCLC who received induction CCRT between May 2003 and May 2017 at our institution were reviewed retrospectively. Induction CCRT mainly administered to patients with resectable bulky N2–3 tumors, although some patients also underwent this procedure based on the surgeon’s discretion. The eligibility criteria for this study were as follows: radiotherapy administered at a dose of 46 Gy in 23 fractions, chemotherapy with cisplatin/docetaxel administered concurrently with radiotherapy, and the completion of the preplanned surgery after induction CCRT. All procedures followed were in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Written informed consent was obtained from each patient before treatment. Moreover, through notifications displayed in the outpatient ward and on the institution’s website, patients were provided the option to opt-out of this study. The institutional review board of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Okayama University Hospital approved this study .Physical examination, chest radiography, chest and abdominal computed tomography (CT), brain magnetic resonance imaging, bone scans, and fluorodeoxyglucose positron emission tomography-CT were performed to obtain detailed data for staging, which was determined using the 7th edition of the TNM classification of malignant tumors. Treatment planning for radiotherapy was performed using 2–10 mm-thick CT scans obtained at 2–10 mm intervals while patients were in the supine position without respiratory arrest and tumor tracking. The gross tumor volume (GTV) included the primary tumors and clinically diagnosed metastatic nodal stations. The clinical target volume included the GTV with a 5–10 mm margin plus the non-metastatic subcarinal and ipsilateral hilar nodal stations; prophylactic nodal irradiation to the non-metastatic nodal stations was not performed after 2015. The planning target volume included the clinical target volume with a 5–10 mm margin and consideration for the internal and setup margins. The internal margin was determined at the discretion of the attending doctor based on X-ray fluoroscopy images. All patients underwent 3-dimensional treatment planning using the Xio computer software version 4.8.0  with a superposition dose calculation algorithm for heterogeneity correction. The prescribed dose at the isocenter was 46 Gy, with 2 Gy per fraction once daily, using a 10 MV photon beam delivered by a linear accelerator .The chemotherapy regimen was cisplatin/docetaxel in all patients, and was concurrently administered with radiotherapy based on the OLCSG 0007 trial . The preThe DVH parameters of the total lung minus the gross tumor were considered. Lung contouring was performed automatically using the CT-based threshold; the trachea and bronchi were manually excluded. The RP grade was determined according to the Common Toxicity Criteria for Adverse Events (CTCAE) version 4.0. The diagnostic criteria for radiation pneumonitis were as follows: Grade 1: Asymptomatic; clinical or diagnostic observations only; intervention not indicated; Grade 2: Symptomatic; medical intervention indicated; limiting instrumental activities of daily living (ADL); Grade 3: Severe symptoms; limiting self-care ADL; oxygen indicated; Grade 4: Life-threatening respiratory compromise; urgent intervention indicated; and Grade 5: Death. The MLD and the percentage of the lung volume that received more than 5 Gy (V5) and 20 Gy (V20) were analyzed as DVH parameters; the relationships between these parameters and the incidences of grades ≥2 RP were investigated via univariate analysis with Fisher’s exact test as well as multivariate analysis using the Cox proportional hazards model. Before performing Fisher’s exact test, we used the median cutoff value to convert continuous parameters to binomial ones.p < 0.05; factors found to be significant on univariate analysis were subjected to multivariate analysis. The cumulative incidence rate of RP was determined using the Kaplan-Meier method stratified by factors found to be significant on multivariate analysis. The R software, version 3.2.0  was used for all statistical analyses.Statistical significance was defined as One hundred and eight patients were included in this study; their characteristics are shown in Table p = 0.269, 0.386, and 0.404, respectively). Figure A total of 30, 43, 32, and 3 patients experienced RP with grades 0, 1, 2 and 3, respectively. The median period from completion of induction CCRT to the onset of grade ≥ 2 RP was 7.6 weeks (range: 4.3–56 weeks). Of the 35 patients with grade ≥ 2 RP, 6 experienced this toxicity before surgery. All 6 patients developed G2 RP. The mean values of V5, V20, and MLD were 31.6, 21.3%, and 10.5 Gy in the group that developed ≥2 RP before surgery and 37.7, 24.8%, and 12.1 Gy in the group that developed ≥2 RP after surgery, respectively. There was no significant difference between the two groups . There was a tendency for RP to occur more easily in the group with reduced residual lung volume than in the group with normal residual lung volume (p = 0.064). Patient age, sex, performance status, smoking status, resection range, forced expiratory volume in 1 s, and V5 were not significantly associated with the development of grade ≥ 2 RP. On multivariate analysis, the MLD and tumor location were found to be significantly associated with grade ≥ 2 RP . According to the cut-off value established by Tsujino et al. [The results of univariate and multivariate analyses of factors associated with grade ≥ 2 RP are shown in Table o et al. , the cump = 0.6 and 0.5, respectively).Figure RP often occurs in patients undergoing definitive radiotherapy for NSCLC, although grade 5 RP is rare , 9, 12. ’s data. Dang et al. found that the MLD is a predictor of grade ≥ 2 RP using multivariate logistic regression analysis, with an odds ratio of 1.42  [MLD is also an important predictor of RP in patients undergoing definitive CCRT. Barriger et al. reported that the rates of grade ≥ 2 RP were 2.2 and 19% in patients with MLDs < 18 Gy and > 18 Gy, respectively . Palma e28–1.58) . Hence, Takahashi et al. investigated the relationship between RP and DVH parameters among patients with NSCLC treated with induction CCRT and surgery, and found that a V20 to the resected lung  of ≥12% was a significant predictor of grade ≥ 2 RP . However’s study, fatal RP (which occurred in 1.9% of patients) was found to be associated with tumor location, with 1, 0, and 5% of RP-related fatalities occurring in patients with tumors in the upper, middle, and lower lobes, respectively (p = 0.007) [Previous investigators have reported that a lower lobe tumor location is predictive of grade ≥ 2 RP development in patients receiving definitive chemoradiotherapy (CRT) –11. For p < 0.01) and MLD  compared to the latter [p = 0.039) [Various methods have been tried to improve radiotherapy dose distribution. Intensity-modulated radiation therapy (IMRT) is advantageous over 3-dimensional conformal radiotherapy in terms of avoiding adjacent organs-at-risk. A prospective phase I study found that IMRT was associated with a global decrease in normal tissue exposure compared to 3-dimensional conformal radiotherapy, and that the former as associated with a significant reduction in V20  .In the near future, it might also be possible to use proton beam therapy to deliver a sufficient radiation dose without increasing lung toxicity. Berman et al. compared dose distributions between IMRT and intensity modulated proton therapy in 10 patients with pathologic stage IIIA NSCLC . The CT To the best of our knowledge, our study is the first to show that MLD and tumor location are significant predictors of RP in patients undergoing induction CCRT for NSCLC. However, our study had certain limitations. First, it was a retrospective analysis in which certain patient details were unknown; for example, Kocak et al. reported that RP scoring was difficult in 28% of their patients because of confounding medical conditions . Second,MLD and lower lobe location were predictors of grade ≥ 2 RP in patients with NSCLC who received induction CCRT. Therefore, it is necessary to reduce MLD during radiotherapy planning to the greatest extent possible to prevent the occurrence of this adverse event.


Solanum lycopersicum L.) against attack by Pectobacterium carotovorum subsp. carotovorum (Pcc), the causal agent of soft rot diseases, were studied. The expression of some tomato defense genes were evaluated by real‐time PCR quantification analysis, 24 and 72 hr after actively growing tomato plants were inoculated with Pcc. These included: MYB transcriptor factor, ethylene response element‐binding protein, suppressor of the G2 allele of Skp1, cytochrome P450, small Sar1 GTPase, hydroxycinnamoyl‐CoA:quinate hydroxycinnamoyl transferase, pathogenesis‐related protein 1a, endo‐1,3‐beta‐glucanase, chitinase, proteinase inhibitor, defensin, CC‐NBS‐LRR resistance protein, and phenylalanine ammonia lyase. The results showed dynamic transcriptomic changes, with transcripts exhibiting different expression kinetics at 24 and 72 hr to confer resistance to tomato against Pcc infection.Defense responses of tomato ( Pectobacterium carotovorum subsp. carotovorum (Pcc). The results showed dynamic transcriptomic changes, with transcripts exhibiting different expression kinetics at 24 and 72 hr to confer resistance to tomato against Pcc infection.Targeting various disease response‐related genes, real‐time PCR was performed on tomato leaf tissue sampled 24 and 72 hr postinoculation with  The main objective of this study was therefore to investigate the effects of inoculation of tomato leaves with Pcc on tomato defense responses based on the relative expression of selected genes.Tomato is a host of 2P. carotovorum subsp. carotovorum (Pcc) (105–108 CFU/ml) suspended in inoculation buffer , and control plants were mock inoculated with the buffer. Pectobacterium cells suspended in the inoculation buffer were pressure infiltrated . The extracted RNA was treated with DNase I (Thermo Scientific). From the total RNA, cDNA was synthesized using a RevertAid™ Premium First Strand cDNA synthesis kit . For accurate calculation of relative gene expression by qPCR, the input RNA was standardized to the same single concentration for cDNA synthesis across all treated and untreated samples.MYB transcription factor, ethylene response element‐binding protein (EREBP), suppressor of the G2 allele of Skp1 (SGT1), cytochrome P450, small Sar1 GTPase (SAR1‐GTPase), hydroxycinnamoyl‐CoA:quinate hydroxycinnamoyl transferase (HQT), pathogenesis‐related protein 1a (PR1), endo‐1,3‐beta‐glucanase (PR2), chitinase (PR3), proteinase inhibitor (PR6), Defensin (PR12), CC‐NBS‐LRR resistance protein, and phenylalanine ammonia lyase (PAL). Information on the primers used appears on Appendix Table elongation factor 1‐alpha and actin8 used as references genes. Datasets were statistically compared between non‐treated samples and treated samples at each time point using one‐way analysis of variation with the statistical analysis software GraphPad inStat 3 (GraphPad Software). The confidence level of all analyses was set at 95%, and values with p < .05 were considered significant.During the cDNA synthesis, control reactions lacking reverse transcriptase (RT), referred to as a non‐RT control, were set up to check the samples for DNA contamination. The non‐RT contained the same amount of total RNA as the experimental samples, with the oligodT primer, dNTPs, reaction buffer, ribolock except RT. The products from the cDNA synthesis reactions were used for the qPCR and all the control reactions lacking RT samples exhibited no DNA contamination by showing no amplification. Quantitative real‐time PCR was performed using a rotor Gene‐3000A instrument (Qiagen) and the SensiFAST SYBR No‐ROX Kit (Bioline) according to the manufacturer's instructions to quantify the expression of defense‐related genes 3P. carotovorum subsp. carotovorum (Pcc)  Table . The folc) Table a–m.Myb, EREBP, CytP450, HQT, PR3, PR6, and CC‐NBS‐LRR were significantly upregulated with a maximum expression of 14‐fold observed with Myb. The lower significant increases with two‐ to fivefold changes were observed on the expression of CytP450 and CC‐NBS‐LRR  defense responses to pathogen attack , to confer resistance by multiple R genes recognizing distinct avirulent P. parasitica or Pseudomonas syringae pv. tomato isolates , the first enzyme in the phenylpropanoid pathway linking primary metabolism to secondary metabolism  defense response to pathogen attack (Alfano et al., Pcc infection of the tomato triggers the expression of a number of the genes selected, which is an indication of their involvement in defense. However, this preliminary finding requires further investigation such as the use of knockout tomato mutants to comprehensively assess gene function and the defense response.The transcript of HQT, an important defense component (Mhlongo, Piater, Steenkamp, Madala, & Dubery, y Figure a,f. A pry Figure . Our resThe authors declare that they have no conflict of interest.ATD was involved in plant inoculation, sample collection, and performed the real‐time PCR and analyzed and interpreted gene expression data. LBT was involved in concept formulation and project administration. KN was involved in concept formulation, preparation of bacterial inoculum, plant inoculation, and sample collection. ATD, LBT, and KN were all involved in manuscript preparation each writing a section. Review and editing was done by all.None required.All data generated or analyzed during this study are included in this published article.


Active school travel (AST) is influenced by multiple factors including built and social environments, households and individual variables. A holistic theory such as Mitra’s Behavioural Model of School Transportation (BMST) is vital to comprehensively understand these complex interrelationships. This study aimed to assess direct and indirect associations between children’s AST and environmental, household and child factors based on the BMST using structural equation modelling (SEM).Data were drawn from Neighbourhoods for Active Kids (NfAK), a cross-sectional study of 1102 children aged 8–13 years (school years 5–8) and their parents from nine intermediate and 10 primary schools in Auckland, New Zealand between February 2015 and December 2016. Data were collected using an online participatory mapping survey (softGIS) with children, a computer-assisted telephone interviewing survey (CATI) with parents, and ArcGIS for built environment attributes. Based on the BMST a conceptual model of children’s school travel behaviour was specified for SEM analyses (‘hypothesised SEM’), and model modification was made to improve the model (‘modified SEM’). SEM analyses using Mplus were performed to test the hypothesised/modified SEM and to assess direct and indirect relationships among variables.N = 542; Root mean square error of approximation = 0.04, Comparative fit index = 0.94, Tucker-Lewis index = 0.92). AST was positively associated with child independent mobility, child-perceived neighbourhood safety, and parent-perceived importance of social interaction and neighbourhood social environment. Distance to school, and parental perceptions of convenience and concerns about traffic safety were negatively associated with AST. Parental fears of stranger danger were indirectly related to AST through those of traffic safety. Distance to school and child independent mobility mediated relationships between AST and child school year and sex.The overall fit of the modified SEM was acceptable  contains supplementary material, which is available to authorized users. Active travel  can be a convenient and regular way for children to accumulate physical activity. Children’s physical, psychological and social health and cognitive development benefit from active travel through opportunities to accumulate physical activity, interact with friends and nature, and spatially navigate their neighbourhood –3. In moThere is a clear need for reducing the use of motorised transportation in favour of active travel. The school trip is one area where such changes could be achieved. In New Zealand (NZ) a majority of schools have zoning regulations, providing children who live inside the zone an absolute right to enrol at the school –7. This A wealth of studies have collectively demonstrated the complex nature of children’s AST , 9–13. TA conceptual model specifically for children’s school travel behaviour, the Behavioural Model of School Transportation (BMST) was developed by Mitra . The BMSThe application of theories such as BMST can highlight the structure of mediated relationships between variables such as built environments and safety , 18, 23.Yu and Zhu , 25 utilThe purpose of this paper is: 1) to develop and test a new model for use in children’s school travel behaviour, and 2) to assess direct and indirect associations between children’s AST and environmental, household and child factors based on Mitra’s BMST  using SE to devel to assesNew Zealand is characterised as a highly suburbanised nation with a total population of 4.9 million in 2018 of which 13% were children aged 5–14 years . AucklanNeighbourhoods for Active Kids (NfAK) is a cross-sectional study conducted in Auckland that uses a child-centred approach to measuring and describing relationships between the built environment and a range of children’s activity behaviours and health outcomes. Information was collected using an online participatory mapping survey  with children, a computer-assisted telephone interview  with parents, and geographic information systems (GIS) for built environment attributes. Design and methods of the full study are described in detail elsewhere .Briefly, children aged 8–13 years (school years 5–8) and their parents from nine intermediate (middle/junior high) and 10 contributing primary (elementary) schools across nine neighbourhoods in Auckland, NZ participated in the study between February 2015 and December 2016. Schools were selected based on a matrix of school decile , child-specific school walkability   and chilhttps://maptionnaire.com) [A softGIS survey (ire.com) –34 was uire.com) , 34. TraInformation about observed variables including description of variables, type of variables , code or scale of variables, and descriptive statistics is summarised in Additional file How do you usually get to school?” with responses being ‘walk’, ‘bike’, ‘scooter (non-motorised)’, ‘public bus, train or ferry’, ‘car, motorbike, scooter or taxi’, and ‘another way ’. School travel mode was dichotomised to active travel  and passive travel . Public transport (including school bus) was considered passive travel in this study. While public transport involves both active and passive travel modes , it Child’s school year (grade), sex and ethnicity were reported by schools or their parents/caregivers, and included in analyses as covariates. School-travel-related physical activity was assessed using Actigraph GT3X+ accelerometers  during the 8:00 am-9:00 am commuting period on weekdays  . Raw datρ = 0.29, p < 0.001) [ρ = 0.18, p < 0.001) [α = 0.85) [Traffic safety perception was measured by the summed score of two items with a 4-point Likert scale (Spearman’s < 0.001) , 44. Nei< 0.001) , 44. Ind = 0.85) .Parents/caregivers reported their highest academic qualification, their current employment situation, and number of adults, children aged under 18 years and working cars in their household.What are the main reasons your child gets to school by respective school travel mode?”, and “How important would you say this reason when deciding how your child gets to school?” Reasons were categorised into ‘distance to school’, ‘traffic safety’, ‘stranger danger’, ‘convenience’ and ‘social interaction’. Each reason was first dummy coded as ‘not main reason’ and ‘main reason’. ‘Main reason’ was then rated as ‘not important’, ‘a little bit important’, ‘important’, or ‘very important’.Importance of parent reasons for decision-making on children’s school travel mode was assessed by two items: “Neighbourhood Social Environment was a first-order factor (latent variable) which was collectively measured by three observed variables: neighbourhood safety, neighbourhood cohesion and neighbourhood connection [α = 0.76), neighbourhood cohesion with nine items (Cronbach’s α = 0.80), and neighbourhood connection with five items (Cronbach’s α = 0.85) [nnection . The sum = 0.85) . A 5-poiSoftGIS home location (point) and child-drawn school route (polyline) data were downloaded from the softGIS survey, and imported into ArcGIS 10.2 , Redlands, CA). SoftGIS routes inside the school polygon were trimmed. All softGIS routes were manually cleaned and obviously incorrect softGIS routes  were excluded from further analyses. Distance to school (in metres) along softGIS routes was calculated, and log-transformed. SoftGIS routes were then buffered using a 80 m radius on each side of the street centre line to measure built environment attributes .Active Mobility Environment was a first-order factor (latent variable) which was collectively assessed by four observed variables: residential density, street connectivity, high traffic exposure and low traffic exposure. Residential density was calculated as the ratio of residential dwellings to the residential land area  of 80 m softGIS route buffer [δr is the distance of an individual softGIS route, ∑rδr is the sum of softGIS route distances  [e buffer . Meshbloe buffer . Road ce weight) . Road cl weight) .Structural equation modelling using Mplus version 8.1  was emplN = 19), a multilevel model was deemed inappropriate.Mplus can estimate mixture modelling with cross-sectional data including combinations of continuous, binary, ordinal, and nominal observed variables, and can handle missing data . MultiplActive Mobility Environment and Neighbourhood Social Environment). The construct validity including convergent validity, discriminant validity and reliability of the measurement model was assessed. Convergent validity was assessed using factor loadings , average variance extracted , and construct reliability . Discriminant validity was assessed by a correlation between the latent variables being significantly smaller than 1.0.A measurement model specified observed variables for each latent variable  and direction  dependent relationships to AST  was randomly divided into two groups (Stage 1: N = 543 and Stage 2: N = 542). Chi-square tests and t-tests were conducted using IBM SPSS Statistics v24  to test for differences in observed variables between the two groups. No significant differences were observed between the Stage 1 and Stage 2 groups.A model development strategy was applied to improve the conceptual model of children’s school travel behaviour. Two stages were involved: (1) testing the hypothesised SEM, and (2) developing the SEM through modifications of the measurement or structural models . The SEMThe weighted least squares means and variance adjusted (WLSMV) estimation was used for analysis of categorical outcomes  , 54. To N = 3), living out of the school catchment zone (N = 12), or having missing data for school travel mode (N = 2). Data from 1085 participants were included in analyses. Descriptive statistics for observed variables are presented in Table Figure zone N = 2, or havN = 543). Linear and logistic regressions were conducted to identify non-significant observed variables associated with AST or the other observed variables [A full SEM including the measurement and structural models are illustrated in Additional file ariables . The resActive Mobility Environment was specified by a combination of exploratory factor analysis and theory, comprising four observed variables: residential density, street connectivity, high traffic exposure and low traffic exposure . Results of the construct validity of the measurement model denoted good validity and reliability with standardised factor loadings (λ) ranging from 0.50 to 1.00; AVEs of 0.59 (Active Mobility Environment) and 0.62 ; and CRs of 0.84 (Active Mobility Environment) and 0.82 . A correlation between Active Mobility Environment and Neighbourhood Social Environment was significantly smaller than 1.0 , indicating good discriminant validity.n theory , encompaN = 542). The modified SEM accounted for 94.4% of the variance in AST. Standardised and unstandardised relationships between the observed/latent variables and AST are presented in Fig. The overall fit of the modified SEM was acceptable/adequate with RMSEA = 0.04, CFI = 0.94 and TLI = 0.92 (Stage 2: p < 0.01) and more males than females  were more likely to actively travel to school. Neighbourhood safety , independent mobility , importance of social interaction , and Neighbourhood Social Environment  were significantly and positively associated with AST. Significantly negative associations with AST were found for importance of traffic safety , importance of convenience , and distance to school . Distance to school had the strongest direct association with AST among the observed and latent variables.Children in higher school year . All indicators of Active Mobility Environment  were negatively correlated with distance to school . The pathway from importance of stranger danger to AST was fully mediated by importance of traffic safety (p < 0.05).Specific indirect (mediating) effects from the observed/latent variables to AST are shown in Fig. p < 0.01), sex (p < 0.05) and Neighbourhood Social Environment (p < 0.05) to AST. The pathways from school year (p < 0.05) or sex (p < 0.05) to AST were also partially mediated by independent mobility through neighbourhood safety. Distance to school was a partial mediator of the pathway from school year to AST (p < 0.01).Independent mobility partially mediated the pathways from school year  for use in children’s school travel behaviour, in which the dynamic interrelationships between children’s AST and multiple environmental factors were conceptualised. Direct and indirect associations between children’s AST and the built environment, the social environment, household and child characteristics, and household and child beliefs were comprehensively assessed using SEM. The modified SEM demonstrated acceptable/adequate model fit, explaining 94.4% of the variance in AST. This study shows that children’s AST has a complex structure and demonstrates how multiple factors at the individual level were interrelated. Distance to school and independent mobility had a multifaceted function including making a direct impact on children’s AST but also acting as mediators influenced by child characteristics such as school year and sex. Older  and male children were more likely to actively travel to school than younger (i.e. primary school) and female children. Parental perceptions of convenience, traffic safety and social interactions as well as child perception of neighbourhood safety were mutually associated with children’s AST.Active Mobility Environment was not directly associated with AST but was mediated by distance to school, suggesting that urban environments that support active mobility  can shorten distance to school and encourage AST.In agreement with findings from previous studies, distance to school was strongly associated with AST and increased by school year , 58–61. In NZ, parents tend to choose their children’s primary school based on the quality of school resources and the overall school reputation rather than the accessibility of school  or living within the school zone  , 62. ThiPublic transport is underutilised in NZ for school travel. This study showed 11.2% of the primary and 45.7% of the intermediate children had parental permission to use public transport on their own. However, only 2.9% of the primary and 21.2% of the intermediate school children used public transport to school . Building safe neighbourhoods and supporting parents using a step-by-step approach to improve children’s independent mobility can be practical future interventions , 65. ForChildren were less likely to actively travel to school if their parents prioritised convenience as a reason for choosing their school travel modes. Research has shown that parents of children who use active travel modes and those who use passive travel modes can both perceive their school travel mode as convenient or easy , 66. HowNeighbourhood Social Environment, suggesting the important role of independent mobility for AST in its own right. These findings supported empirical evidence from previous studies that independent mobility is influenced by child’s age, sex, and the quality of neighbourhood environments  [Independent mobility was not only positively associated with AST, but also acted as a mediator between AST and school year, sex, neighbourhood safety and  safety) , 73–78.  safety) . Social  safety) . Future  safety)  to help  safety) . For exa safety)  wherein If parents reported that traffic safety and stranger danger were important for decision-making regarding their children’s school travel mode, children were less likely to actively travel to school. Parental perceptions of traffic safety  and stranger danger  have been recognised as key obstacles to AST , 66, 81.The importance of social interaction was positively associated with AST and physical activity specifically during the morning commute (8:00 am-9:00 am). The findings demonstrated that the choice of AST viewed as an opportunity for social interactions can be coupled with a way to accumulate physical activity. Egli et al.  revealedThis study used a SEM technique to comprehensively understand the complex interrelationships between children’s AST and environmental, household and child factors. To our knowledge, this is the first study to incorporate voices from children (softGIS) and parents (CATI) and objective GIS measures in a model which was a novel and holistic approach and advantageous to test and develop the conceptual model of children’s school travel behaviour. Use of child-drawn routes to school using softGIS to generate route environment measures in the current study is likely to have provided greater specificity of the built environment children actually encounter en-route to school compared to calculating these measures using the more common method of GIS-modelled shortest routes . HoweverN > 20 clusters, ideally N > 50) to perform multilevel analyses in SEM.The absence of school cluster analysis and unavailability of observed variables in analyses were limitations of this study. The use of a multilevel model is recommended for data structured by multiple levels . The effect of clusters  and group level  data such as school policies and AST programmes can influence children’s AST. Future research should consider a larger school sample size Additional file 2:Hypothesised indirect relationships between children’s school travel behaviour and the built environment, the social environment, household and child characteristics, and household and child beliefs. (DOCX 95.1 kb)Additional file 3:Table S1. Information about observed variables and their descriptive statistics. (N = 1085) (DOCX 63.7 kb)Additional file 4:The hypothesised full structural equation modelling. (DOCX 561 kb)Additional file 5:Unstandardised estimated coefficients of the structural equation model of children’s active travel to school. Root mean square error of approximation (RMSEA) = 0.04, comparative fit index (CFI) = 0.94, Tucker-Lewis index (TLI) = 0.92. (DOCX 606 kb)


Clinacanthus nutans Lindau leaves (family Acanthaceae) demonstrated peripherally and centrally mediated antinociceptive activity via the modulation of opioid/NO-mediated, but cGMP-independent pathway. In the present study, MECN was sequentially partitioned to obtain petroleum ether extract of C. nutans (PECN), which was subjected to antinociceptive study with aims of establishing its antinociceptive potential and determining the role of opioid receptors and l–arginine/nitric oxide/cyclic-guanosine monophosphate (l–arg/NO/cGMP) pathway in the observed antinociceptive activity.Methanol extract (MECN) of n = 6). The effect of PECN on locomotor activity was also evaluated using the rota rod assay. The role of opioid receptors was determined by pre-challenging 500 mg/kg PECN (p.o.) with antagonist of opioid receptor subtypes, namely β–funaltrexamine , naltrindole  or nor–binaltorphimine  followed by subjection to the abdominal constriction test. In addition, the role of l–arg/NO/cGMP pathway was determined by prechallenging 500 mg/kg PECN (p.o.) with l–arg , 1H- oxadiazolo quinoxalin-1-one , or the combinations thereof (l–arg + ODQ) for 5 mins before subjection to the abdominal constriction test. PECN was also subjected to phytoconstituents analyses.The antinociceptive potential of orally administered PECN  was studied using the abdominal constriction-, hot plate- and formalin-induced paw licking-test in mice (p < 0.05) inhibited nociceptive effect in all models in a dose-dependent manner. The highest dose of PECN (500 mg/kg) also did not significantly (p > 0.05) affect the locomotor activity of treated mice. The antinociceptive activity of PECN was significantly (p < 0.05) inhibited by all antagonists of μ-, δ-, and κ-opioid receptors. In addition, the antinociceptive activity of PECN was significantly (p < 0.05) reversed by l–arg, but insignificantly (p > 0.05) affected by ODQ. HPLC analysis revealed the presence of at least cinnamic acid in PECN.PECN significantly (PECN exerted antinocicpetive activity at peripheral and central levels possibly via the activation of non-selective opioid receptors and modulation of the NO-mediated/cGMP-independent pathway partly via the synergistic action of phenolic compounds. Pain is an essential sensation that plays a vital role as a body’s natural defence system by alerting us to possible tissue injury while nociception is described as “the neural processes of encoding and processing noxious stimuli” that usually leads to pain . The proPain can be modulated by various analgesic drugs that suppress pain signals by acting in various mechanisms on the peripheral (PNS) and central nervous system (CNS) . HoweverClinacanthus nutans (C. nutans) Lindau, known to the Malay as “Belalai Gajah”, is one of the medicinal plants that have received considerable attention from many research groups within the past few years. This small shrub belongs to the Acanthaceae family and commonly grows in tropical Southeast Asian countries. Local communities in the Southeast Asian countries including Malaysia have traditionally used C. nutans for the treatment of various disorders , including pain relief  oxadiazole quinoxaline–1–one (ODQ) were purchased from Sigma–Aldrich , while β–funaltrexamine hydrochloride (β–FNA), nor–binaltorphimine dihydrochloride (nor–BNI), and naltrindole hydrochloride (NALT) were purchased from Tocris Bioscience . Acetic acid and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific . Formaldehyde was obtained from R & M Chemicals . All drugs were dissolved in physiological saline (0.9% [w/v] NaCl) except for PECN, ASA, DZP and MOR, which were dissolved in 10% DMSO (v/v). The vehicle that was used alone had no effects per se on the nociceptive responses in mice. The other solutions  were prepared in 0.9% NaCl. All drugs, chemicals, PECN solutions were administered in 10 mL/kg volumes and were freshly prepared immediately prior to use.The following drugs, namely acetylsalicylic acid (ASA), morphine hydrochloride (MOR), diazepam (DZP), n = 6) were treated with vehicle , negative control), ASA , or PECN  60 min before the administration of phlogistic agent . The number of abdominal constrictions produced was counted from 5 min after acetic acid administration cumulatively for 25 min, as described previously in detail [The animals (n = 6) were treated with vehicle , MOR  or PECN  60 min before the test.In order to evaluate the central antinociceptive effect of PECN, a hot plate test was carried out according to the method previously described in detail by Sani et al. . The anin = 6) received vehicle , ASA , MOR , or PECN  60 min before the intraplantar (i.pl) administration of 5% (v/v) formalin into the right hind paw. The nociceptive response, indicated by the sum of time that the animal spent licking the injected paw, developed between 0-5 min after formalin injection (early phase) and 15–30 min after formalin injection (late phase) were recorded for 30 min.The procedure used was similar to the one used in our previous work . Mice (nn = 6) were treated with the vehicle , DZP , or PECN  60 min before the test. The average time the mice spent to stay on the rota–rod was measured for 120 s at 5, 10, and 15 min after the administration of PECN, respectively.The method used was conducted similarly to the one that was described previously . The appThe possible role of opioid receptor system in the modulation of PECN-induced antinociceptive activity was investigated as previously described . Brieflyl–arg/NO/cGMP pathway to the antinociceptive effect of PECN, the previously described method was adopted from Jiménez–Andrade et al. [n = 6) were pre–treated (i.p.) with 20 mg/kg l–arg (a NO precursor) or 2 mg/kg ODQ (a specific soluble guanylyl cyclase inhibitor) 5 min before the administration of vehicle  or PECN . Sixty minutes after the administration of test solutions, the mice were subjected to acetic acid–induced abdominal constriction test as described above.To investigate the possible contribution of e et al. . The micThe qualitative phytochemical screening of PECN was performed to determine the presence of flavonoids, saponins, triterpenes, tannins, alkaloids and steroids according to the conventional protocols as adopted by Mamat et al. .The HPLC analysis of PECN was conducted at the Laboratory of Phytomedicine, Forest Research Institute of Malaysia, Kepong, Malaysia according to the similar protocols as reported in Zakaria et al. . The HPLp < 0.05.Data are expressed as mean ± standard error of mean (SEM). Except for the hot plate test, the mean differences between the control and treatment groups for the other tests were analysed using the one–way analysis of variance (ANOVA) followed by Dunnet’s post–test . Data for the hot plate test were analysed using the two-way ANOVA followed by the Bonferroni’s post hoc test. In all cases, the differences were considered as significant if p < 0.001) and dose–dependent decrease in the number of abdominal constrictions in comparison to the control group. The percentage of antinociception calculated for the three respective doses of PECN ranged between 63.8 to 77.3%. ASA  also caused significant (p < 0.001) inhibition of abdominal constriction with the percentage of antinociception recorded at approximately 46.8%.The effect of PECN against acetic acid–induced abdominal constriction test in mice is shown in Fig. p < 0.01) changes in response latency to thermal stimulus–induced nociception, which started 60 min after PECN administration and lasted until the end of the experimentation when compared against the control group. Concurrently, MOR given via oral administration at the dose of 5 mg/kg also caused a highly significant (p < 0.001) prolongation of latency response starting at the interval of 60 min and lasted until the end of the experimentation (210 min).The effect of PECN on thermal-induced nociception was assessed using the hot plate test and is shown in Table p < 0.001) reduction in the latency of formalin–induced paw licking in the first phase  of the test as shown in Fig. p < 0.001) reduced the latency of formalin–induced paw licking in the second phase  of the test as shown in Fig. Figure p < 0.001) reduction in the latency spend by the mice to remain on the rota-rod at all interval measured indicating its potential to affect motor coordination.The effect of orally administered PECN on motor coordination of mice was measured using the rota–rod test and is shown in Fig. p < 0.001) reversed after i.p. pretreatment with 10 mg/kg β–FNA, 1 mg/kg NALT or 1 mg/kg nor–BNI, which did not exert any significant (p > 0.05) effect on nociceptive response when tested alone.Effect of PECN on opioid receptors-mediated nociceptive response was determined using the acetic acid-induced abdominal constriction test and is shown in Fig. l–arg/NO/cGMP pathway in the modulation of PECN-induced antinociception is shown in Fig. l–arg or ODQ, alone or in combination with PECN, were assessed using the acetic acid–induced abdominal constriction test. As shown in Fig. l–arg (the NO precursor) alone did not affect the acetic acid-induced nociception but caused significant reversal (p < 0.001) on PECN-induced antinociception when pretreated together. Moreover, ODQ, given alone, caused significant (p < 0.001) attenuation of the acetic acid–induced nociception while pretreatment with PECN caused significant (p < 0.01) reversal of the antinociceptive activity of PECN  = 17.265 min), P2 (retention time (RT) = 18.332 min), P3 (RT = 18.631 min), P4 (RT = 20.136 min) and P5 (RT = 27.494 min). Further analysis demonstrated that the three major peaks showed their λmax values in the region of 222.5–332.6, 192.0–336.2, and 216.6–335.0 nm, respectively, thus, suggesting the presence of phenyl chroman (C6–C3–C6) skeletal structures. As depicted in Fig. The HPLC profiles of PECN were measured at six different wavelengths, namely 210, 254, 280, 300, 330 and 366 nm to obtain the best separation. Based on the respective chromatograms obtained, chromatogram established at the wavelength of 254 nm was found to show the best peaks separation , ethyl acetate and aqueous semi-purified fractions [The current work is a continuation of previous study by Abdul Rahim et al.  on the aractions , which w2 and PGF2), lipoxygenase (LOX) and cyclooxygenase (COX) in the peritoneal cavity that lead to indirect liberation/activation of various endogenous inflammatory mediators. These mediators stimulate the peripheral nociceptive neurons that are sensitive to opioid (i.e. MOR) and non-opioid (i.e. ASA) analgesics [Other than the acetic-acid-induced abdominal constriction test, PECN was found to attenuate nociceptive response when assessed using the hot plate test and formalin-induced paw licking test. The acetic acid–induced abdominal constriction test, which represents inflammatory–mediated peripheral nociceptive response, has been associated with increased production of prostaglandin (PG)  , involveBased on its ability to attenuate both the acetic acid- and thermal-induced nociceptive responses, PECN is suggested to have a potential to inhibit nociceptive response at the peripheral and central levels –22. ThisAlthough the three nociceptive assays used in the present study finally established that PECN possesses antinociceptive potential, it is necessary to determine the extract ability to modulate motor coordination of the mice upon consumption due to its centrally-acting effect since centrally-acting drugs are known to cause possible alterations in the motor coordination, balance and equilibrium ability of the animals . Accordil–arg/NO/cGMP pathway. The opportunity to study and identify any drugs/substances that modulate nociceptive response via mechanisms partly used by standard analgesic (MOR) (viz. opioid receptors and l–arg/NO/cGMP pathway) could be an added advantage in an attempt to find replacement to MOR, which has been associated with side effects such as dependence and tolerance.In an attempt to establish the antinociceptive profile of PECN, the mechanisms of antinociception modulated by PECN were also investigated by focusing on the role of opioid receptor systems  and Role of opioid receptor system in the modulation of antinociceptive action of drugs/substances can be determined via exploitation of certain pharmacological tools such as various antagonists of opioid receptor , 28. Thel–arg/NO/cGMP pathway in the regulation of analgesics action at peripheral and central levels haa been reported [l–arg to l–citrulline and NO by NO synthase, is known to play a complex part in the transmission of nociceptive signals peripherally and centrally [l–arg exerted insignificant increase in the nociceptive intensity when administered alone, it significantly reduced the antinociceptive intensity of PECN when pretreated together indicating the importance of NO presence in the modulation of PECN antinociceptive potential. On the other hand, increased presence of NO at the intracellular level stimulates the soluble guanylate cyclase (sGC), which augments the cGMP synthesis. The important of cGMP in the modulation of hyperalgesic response at peripheral sensory neurons and the involvement of NO/cGMP signaling pathway in the central and peripheral nociceptive processing have been established [l–arg/NO/cGMP pathway plays a pivotal role in the development of tolerance to MOR [C. nutans as an analgesic-based product with lack of unwanted side effects seen with MOR due to the difference in NO-mediated pathway modulated by the former.The involvement of reported . NO, derentrally . In factentrally . Althougablished . In the ablished . One exaablished , 38. Take to MOR , it is rC. nutans leaves demonstrated the presence of flavonoids, phenolic compounds, diterpenes, saponins, chlorophyll and its various derivatives, and steroids (i.e. β–sitosterol and stigmasterol) [Earlier investigation on the phytochemical content of asterol) , 5. Concasterol) , 41, triasterol) , 43, andasterol) , 45 exerGalanthus elwesii. Concurrent with this finding, there are reports on the potential of cinnamic acid derivatives [Further phytoconstituents analysis using HPLC revealed the presence of traces of only a few bioactive compounds , which upon comparison with several pure bioactive compounds revealed the presence of, at least, cinnamic acid. The lack of number of peaks and the presence of small peaks as detected in the chromatogram of PECN might be due to the lack of hydrophilic (water-soluble) bioactive compounds in the hydrophobic-based extract. The presence of cinnamic acid in PECN is parallel to the report of Tüzan and Özdemir  on the pivatives  to exertl–arg/NO-mediated, but cGMP-independent, pathway.In conclusion, PECN, partly through the presence of cinnamic acid, exerted antinociceptive activity at the peripheral and central levels via the non-selective activation of opioid receptors and modulation of the


MSI was analysed by PCR using five microsatellite markers and classified as MSI‐H and MSI‐L. Frequencies of EBV(+), MSI‐H and MSI‐L in the biopsies before CTx were 4.2, 10.5 and 4.9% respectively. EBV(+) or MSI‐H did not correlate with response, but MSI‐L was associated with better response (p = 0.011). In the resected tumours, frequencies of EBV(+), MSI‐H and MSI‐L were 3.9, 9.6 and 4.5% respectively. Overall survival (OS) was significantly different in the non‐CTx group (p = 0.014). Patients with EBV(+) tumours showed the best OS, followed by MSI‐H. MSI‐L was significantly associated with worse OS . In the resected tumours after CTx, MSI‐H was also associated with increased OS . In multivariable analysis, molecular classification was an independent prognostic factor in the completely resected (R0) non‐CTx group (p = 0.035). In conclusion, MSI‐H and EBV(+) are not predictive of response to neoadjuvant platinum/5‐FU based CTx, but they are indicative of a good prognosis. In particular, MSI‐H indicates a favourable prognosis irrespective of treatment with CTx. MSI‐L predicts good response to CTx and its negative prognostic effect for patients treated with surgery alone suggests that MSI‐L might help to identify patients with potentially high‐benefit from preoperative CTx.Epstein–Barr virus positivity (EBV(+)) and high‐microsatellite instability (MSI‐H) have been identified as molecular subgroups in gastric carcinoma. The aim of our study was to determine the prognostic and predictive relevance of these subgroups in the context of platinum/5‐fluorouracil (5‐FU) based preoperative chemotherapy (CTx). Additionally, we investigated the clinical relevance of the low‐MSI (MSI‐L) phenotype. We analysed 760 adenocarcinomas of the stomach or the gastro‐oesophageal junction encompassing 143 biopsies before CTx and 617 resected tumours (291 without and 326 after CTx). EBV was determined by PCR and  Pre‐/peri‐operative chemotherapy (CTx) containing a platinum/5‐fluorouracil (5‐FU) combination is recommended for patients with advanced gastric carcinoma (GC) in western countries, but response rates are limited Recent studies suggest that molecular classification should be considered for optimal therapy planning. Molecular classification systems have been described by The Cancer Genome Atlas (TCGA) network and the Asian Cancer Research Group (ACRG). Both include tumours with microsatellite instability (MSI) as one subgroup The MSI‐L phenotype has been described in various tumours, including GC, but the biological and clinical significance is largely unclear and MSI‐L and MSS tumours are frequently combined in one group Another subgroup of the TCGA classification is formed by Epstein–Barr virus positive (EBV(+)) tumours, which represent about 4–10% of GC EBV positivity was shown to be associated with better prognosis in GC patients though there are studies that find no clear correlation between EBV status and survival and the relevance of EBV positivity to predict response to neoadjuvant CTx is unclear EBV positivity and MSI‐H were associated with good response in a clinical trial evaluating an immune check point inhibitor in metastatic GC In light of these new therapeutic options and against the background of still limited data available in the literature related to neoadjuvant CTx, further knowledge about the clinical relevance of MSI and EBV status in connection with classical treatment regimens is essential for the selection of the most appropriate treatment for each GC patient.Thus the goal of our study was to determine the prognostic and predictive significance of EBV positivity and MSI for carcinomas of the stomach and gastro‐oesophageal junction in the context of preoperative platinum/5‐FU based CTx. As various clinical aspects are relevant in this therapeutic setting, we performed a comprehensive analysis of overall 760 tumours encompassing three different patient cohorts each with specific characteristics.First, we analysed tumour biopsies before neoadjuvant CTx; this represents the most appropriate cohort to test for an association with therapy response as it allows inclusion of both non‐responding and completely or nearly completely responding patients. In addition, biopsies are the type of specimens that are available before the start of CTx in daily clinical practice. Second, we analysed the molecular subgroups in a relatively large cohort of resected tumours from patients treated with surgery alone and third, resected tumours from patients after neoadjuvant CTx, to determine if their prognostic role is comparable and might support the choice of subsequent treatment modalities. Finally, as the clinical significance of the MSI‐L phenotype in GC is poorly characterised, we aimed to fill this gap and analysed MSI in terms of MSI‐H and MSI‐L.Resected tumours from 704 patients with gastric adenocarcinomas including tumours of the gastro‐oesophageal junction : TRG1, TRG2 and TRG3, which corresponded to <10, 10–50 and >50% residual tumour cells/tumour bed respectively. The prognostic relevance of this classification system has been demonstrated in previous studies All three TRGs were present among the patients with tumour biopsies before CTx; patients with TRG1 were classified as responders, and those with TRG2 and TRG3 as non‐responders. Only tumours with TRG2 and TRG3 were present among the patients in the resected tumour cohort after CTx; these allow isolation of sufficient DNA from residual tumour cells.Follow‐up was performed as described The study was in accordance with the Declaration of Helsinki and was approved by the local Institutional Review Boards at the Technical University of Munich (reference: 502/15s) and at the University of Heidelberg (reference: 301/2001).DNA from formalin fixed paraffin embedded (FFPE) tissues was isolated after manual microdissection from 8 μm thick sections after deparaffinisation and proteinase K digestions using the Maxwell extraction system according to the instructions of the manufacturer . Details are described in supplementary material, Supplementary materials and methods. Only samples with a tumour cell content of at least 10% were included for MSI analysis according to the described detection limit for MSI of 2–10% tumour alleles MSI was determined by PCR analysing two mononucleotide repeats BAT25, BAT26 and three dinucleotide repeats D2S123, D5S346, D17S250 as recommended by the National Cancer Institute in situ hybridisation using the EBV early RNA Probe and the iViEW Blue detection kit  on an automated system  according to the instructions of the manufacturer. EBV positivity was defined when positive staining after in situ hybridisation was present in the nuclei of the tumour cells.Screening for EBV was performed by a PCR based assay using primers for amplification of EBV specific DNA in the BamHI‐W and BamHI‐K regions of the virus as described Chi‐squared tests or Fisher's exact tests were used for hypothesis testing of differences between the relative frequencies. Kaplan–Meier estimates of survival rates were compared by log rank tests. Relative risks were estimated by hazard ratios (HRs) from univariable Cox proportional hazard models or from Firth's corrected Cox‐regression. A multivariable Cox proportional hazards model was built by stepwise forward variable selection using likelihood‐ratio tests of pre‐therapeutically and post‐therapeutically available clinical factors.The pre‐therapeutically available factors were: sex, age (continuous variable), histological type according to Laurén , tumour localisation  and clinically determined tumour stage (cT2 versus cT3/cT4). The post‐therapeutic factors were: sex, age, histological type according to Laurén, tumour localisation, depth of tumour invasion (pT2 versus pT3/pT4), lymph node involvement (pN0 versus pN+), R‐category (R0 versus R+) and status of metastasis (M0 versus M+). Statistical analyses were performed using SPSS, Version 25 . Exploratory 5% significance levels (two‐tailed) were used for hypothesis testing.p < 0.01) and is shown in supplementary material, Figure Our study population consisted of different GC cohorts. The biopsy cohort encompassed patients with pre‐therapeutic tumour biopsies before CTx with inclusion of responding (TRG1) and non‐responding (TRG2/3) patients to accurately determine the predictive and prognostic value of the molecular subgroups for CTx treatment. Of 167 pre‐therapeutic biopsies, which were initially evaluated for the study, 24 were excluded. Among the 143 analysed biopsies, 45 of the patients showed TRG1, 34 showed TRG2 and 64 showed TRG3 in the resected specimens. The OS of patients in relation to TRG was significantly different  was detected in 6 (4.2%) of the 143 tumour biopsies and MSI‐H and MSI‐L were found in 15 (10.5%) and 7 (4.9%) of the samples respectively  of the 617 tumours were EBV positive, and 59 (9.6%) and 28 (4.5%) showed MSI‐H and MSI‐L, respectively .All MSI‐H tumours were negative for EBV. One biopsy and one resected tumour were positive for both MSI‐L and EBV. These two patients were excluded from further analyses and clinical parameters were compared for the four molecular subgroups, EBV(+), MSI‐H, MSI‐L and MSS/EBV(−), taking the latter as reference.p = 0.015), tumour localisation in the middle of the stomach (p = 0.033) and poor differentiation (p = 0.01). MSI‐H was associated with older age (p < 0.001), distal tumour localisation (p = 0.05) and absence of metastasis (p = 0.038). MSI‐L was more frequent in intestinal GC (p = 0.04)  was associated with male sex (p = 0.626 and p = 1.00 respectively). In contrast, MSI‐L demonstrated a significant association with better response (p = 0.011). Five (83%) of six MSI‐L biopsies were of responding patients with TRG1 in the resected specimens after CTx compared to 33 (28%) of 116 MSS/EBV(−) tumours  and MSI‐H were not associated with response to CTx in the pre‐therapeutic biopsies before CTx (p = 0.002)  : HR, 0.83, 95% CI, 0.26–2.64, p = 0.754). All survival data including the 1, 3 and 5 year OS rates are summarised in Table Comparison of OS of patients with biopsies before CTx regarding the four subgroups showed no statistically significant difference  Figure A. Essentp = 0.014) . Patients with MSI‐L tumours showed a statistically significantly worse OS  . In the non‐CTx group a statistically significant difference of OS regarding the four molecular groups was observed  Figure B. Patien1) Table .p = 0.038)  , age (p = 0.001) and the molecular classification  as significant prognostic factors. Interestingly, considering the molecular subgroups separately, MSI‐H emerged as an independent prognostic factor  revealed cT (or Table .p < 0.001), age (p = 0.004) and R‐category (p = 0.020) as independent prognostic factors (Table p < 0.001), age (p = 0.004), the molecular classification (p = 0.035) and pT (p = 0.023) as independent prognostic factors  . These results are included in Table In the CTx group, differences in OS were not statistically significant  Figure E. Howevep = 0.043) , but some experts endorse that patients with cT2 tumours can also be treated In contrast to the MSI‐H phenotype with a well‐known molecular background related to defects in one of the four DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2, the origin and biological significance of the MSI‐L phenotype is largely unclear and controversially discussed. MSI‐L has been related to elevated mutation rates, to defects in specific DNA repair genes and/or induction by DNA damaging agents Regarding MSI‐H, the majority of studies has demonstrated an association of this type of MSI with good prognosis in GC, which is essentially in line with our findings for patients in the resected cohorts EBV was detected in 4–5% of our tumours, which is similar to a recent report Regarding multivariable analysis for survival performed in the non‐CTx cohort, our results confirm the well‐known prognostic impact of lymph node involvement and completeness of tumour resection as independent prognostic factors Despite the comprehensive analysis of a very large cohort of patients comprising 760 tumour samples overall, our study has limitations which are mainly related to its retrospective nature. Regarding the analysis of the pre‐therapeutic biopsies, the availability of DNA or suitable tumour tissues presented the main limiting factor for inclusion of patients. Further limitations are that our analysis was not performed in the context of a randomised clinical trial testing different treatment regimens but refers to a sample series from daily clinical practice of a local hospital with some variations regarding surgical approaches and treatment protocols. Thus, our study has to be considered an explorative analysis. Further prospective studies are needed to confirm our results and a comprehensive molecular analysis of MSI‐L tumours should be performed to clarify the biological background of this particular type of MSI.To conclude, in our study MSI‐H and EBV were not predictive of response to neoadjuvant platinum/5‐FU based CTx, but they were indicative of a good prognosis. In particular, considering MSI‐H, this was evident in principal regardless of the therapeutic approach chosen. MSI‐L, however, was predictive of good response to CTx. Furthermore, the negative prognostic effect of MSI‐L observed for patients treated with surgery alone, even in the group with clinically determined earlier tumour stages, indicates that MSI‐L might help to delineate patients with potentially high‐benefit from preoperative platinum/5‐FU based treatment. Clearly, additional studies are mandatory to confirm our results and the MSI‐L phenotype warrants further investigation to elucidate its role in chemosensitivity and tumour development.MKo, BG, WW and GK planned and conducted the study. MKo, BG, MKr, SB, AN, MR, TS, LI, DM, PM, MMG and LB collected the data. MKo, BG, MKr, JSH, MJ, AN, AH, KO, WW, GK interpreted the data. MKo, BG and GK drafted the manuscript. MJ, AN, AH, MMG, KO and WW reviewed the manuscript. All authors have approved the final drafts submitted.Supplementary Material and MethodsFigure S1. Discrimination of patient survival by tumour regression grade (TRG)Table S1. Chemotherapy regimens of the preoperatively treated patientsTable S2. Drug regimens and survival of the preoperatively treated patientsTable S3. EBV and MSI status of resected tumours without and after neoadjuvant CTx and association with patient`s characteristicsClick here for additional data file.


Three hypotheses were examined: (a) parents’ mind‐mindedness at 4 and 12 months predicts infants’ HRV at 12 months over and above infants’ initial HRV levels at 4 months, (b) mothers’ and fathers’ mind‐mindedness independently predict infant HRV, and (c) the effects of mind‐mindedness on infant HRV  operate via parenting behaviour. Infants’ HRV was assessed during rest and a stranger approach. Mind‐mindedness was assessed by calculating the proportions of appropriate and non‐attuned mind‐related comments during free‐play interactions, and parenting quality was observed at 4 and 12 months in the same interactions. Path analyses showed that mothers’ appropriate mind‐related comments at 4 and 12 months predicted higher baseline HRV at 12 months, whereas mothers’ non‐attuned comments predicted lower baseline HRV at 12 months. Similar, but concurrent, relations were found for fathers’ appropriate and non‐attuned mind‐related comments and infant baseline HRV at 12 months. In addition, fathers’ appropriate mind‐related comments showed an indirect association with infant baseline HRV at 12 months via fathers’ parenting quality. With regard to infant HRV reactivity during the stranger approach, mothers’ appropriate mind‐related comments at 4 months and fathers’ non‐attuned mind‐related comments at 12 months predicted a larger HRV decline during the stranger approach at 12 months. Infants’ HRV at 4 months did not predict parents’ later mind‐mindedness. The results indicate that mothers’ and fathers’ appropriate and non‐attuned mind‐related speech uniquely impacts the development of infants’ physiological emotion regulation.The main aim of this study was to test whether mothers’ ( This study examined the relations between parents’ mind‐mindedness and infants’ physiological emotion regulation capacity throughout the first year of life.Mothers’ and fathers’ appropriate and non‐attuned mind‐related speech independently predicted infants’ emotion regulation  at 12 months.Maternal mind‐mindedness at 4 and 12 months predicted infants’ HRV at 12 months, while paternal mind‐mindedness at 12 months concurrently associated with infants’ level of HRV.Mind‐mindedness of fathers showed a direct and indirect effect  on the infant's emotion regulation capacity at 12 months.1Most parents want their child to have emotions, but not be overpowered by them; to feel disappointed but not to cave in; to be thrilled but not get hysterical, to feel some anxiety (and therefore be careful), but not become overly worried or avoiding. In other words, parents hope that their child will find a balanced way to deal with the impact of emotions—to be able to regulate their emotions. For a substantial period of childhood—the first years—the child's regulation of emotions crucially depends on the parent's ability to manage the physical and emotional states of the child Taipale, . As infaIn behavioural science, emotion regulation refers to processes that affect the impact and/or time of an emotional response, either intentional or spontaneous, occurring at the physiological, behavioural, and cognitive levels Gross, . AlthougThe PNS is part of the autonomic nervous system and promotes functions associated with the restoration and conservation of bodily energy and the resting of vital organs. Activation of this system typically results in inhibitory effects on body functions, such as slowing down the heart rate Porges, . The othHeart rate variability is typically assessed during a resting (baseline) period or during an active situation, reflecting two different aspects of emotion regulation. The level of HRV during rest  is considered to represent an individual's general capacity and flexibility to regulate emotions  is associated with more emotion regulation during a stressful or challenging situation  HRV change during social tasks  as well as the behavioural tendency to explicitly  communicate theories of the infant's mind during interactions Meins, . On the On the other hand, the mental component of mind‐mindedness  may enable parents to respond to and interact with their infant in an attuned manner, both on a conscious and an unconscious level . Hence, parents who are able to appropriately interpret their infant's state of mind seem better able to adjust themselves and the environment in a way that allows the infant to regulate arousal.This example suggests that mind‐mindedness may be a key predictor of infants’ developing emotion regulation capacity, and that the effect of mind‐mindedness may operate through parenting behaviour. This notion is supported by a recent meta‐analysis which showed that mind‐mindedness  predicted infant attachment security directly, but also indirectly via parents’ sensitive responsiveness  and a stronger decline in HRV levels (negative association) during a socially challenging task involving a male stranger at 12 months, taking into account infants’ HRV at 4 months. Conversely, we expected that parents’ non‐attuned mind‐related comments would predict lower levels of baseline HRV and less HRV decline during the stranger situation at 12 months.There is little research on the determinants of parents’ mind‐mindedness. The studies that are available suggest that the tendency to be mind‐minded is dependent on the familiarity with the interaction partner and therefore is an indicator of people's relationship quality  during interactions at 4 and 12 months, which, in turn, would positively affect infant regulation at 12 months. We further expected that the direct effect of mind‐mindedness on infant HRV would hold over and above the indirect effect via parenting quality.22.1Mage = 4.2, SD = 0.33), 135 fathers and mothers and their firstborns participated . At Time 2, when the child was 12 months old , 131 fathers (3% missing) and 130 mothers (4% missing) took part . Attrition was due to couples indicating that they did not have enough time to participate in the longitudinal study. Mean age of mothers at Time 1 was 31.30 years (SD = 4.41) and 33.87 years (SD = 4.77) for fathers. The mean educational level of parents was fairly high, M = 7.10, SD = 1.25 for mothers, and M = 6.53, SD = 1.55 for fathers . Parents were primarily Caucasian. All parents were living together at the time of the assessments.Families from the present study participated in an ongoing longitudinal study on social development from infancy to middle childhood  at 4 months, 40% of the infants were cared for by their mother, 11% were cared for by their father, and 49% were in non‐parental care. On an average weekday at 12 months (Monday to Friday), 34% of the infants were cared for by their mother, 11% were cared for by their father, and 55% were in non‐parental care. Fathers never took care of their child more than 2 days of the (work)week at 4 and 12 months. On an average weekend day at both 4 and 12 months, 11% of the infants were cared for by their mother, 5% by their father, and 82% of the couples reported that they both took care of the infant at the weekend.2.22.2.1At 4 months parental mind‐mindedness was assessed during a five‐minute free play period at the university research lab. The parent was asked to play with his or her child as naturally as possible. They were allowed to use all available space in the room. Each session began with the infant lying on a couch, with the parent sitting next to the infant. After two‐and‐a‐half minutes of play time without toys, the parent was provided with a box with five age‐appropriate toys to play with. At 12 months, mind‐mindedness was assessed during a 10‐minute free play session. The parent and child were seated on a play mat in the centre of the room with age‐appropriate toys and pillows. After five minutes the toys were replaced with animal‐printed pillows and the parent and child continued their play for another five minutes.Each comment made by the parent  was transcribed and coded by two trained observers using a translated version of the mind‐mindedness coding manual  the trained coder agreed with the parent's reading of the infant's internal state, (b) the internal state comment linked the infant's current activity with similar events in the past or future , or (c) the parent voiced (using the first person) what the child might say if he or she could speak. Comments were classified as non‐attuned when the independent coder believed (a) the parent misread the internal state of the child, or (b) the comment referred to a past or future event that had no obvious relation to the infant's current activity .father = .75, ICCmother = .88; at 12 months ICCfather = .84, ICCmother = .84. Next, we assessed the interrater agreement on the appropriateness of mind‐related comments by calculating Cohen's kappa. Interrater agreement was at 4 months кfather = .82; кmother = .85, at 12 months кfather = .92; кmother = .88. Disagreements were resolved by discussion.Interrater agreement was assessed on 90 out of 449 transcripts (20%). First, we assessed the interrater agreement on the number of mind‐related comments using intra‐class correlations . Inter‐rater agreement was at 4 months ICC2.2.2Physiological measures were obtained during baseline (two minutes) and the stranger approach task. The stranger task was designed on the basis of the Laboratory Temperament Assessment Battery .Parenting quality was assessed at 12 months by scoring parenting behaviour on four scales: responsiveness, intrusiveness (reversed), warmth/affectivity, and negativity (reversed). The scoring was based on the Meso Behavioural Rating System for Families with young children . We also coded whether infants cried during the stranger approach, and checked whether the infants who cried were at risk of having a noisy signal due to movement artefacts (chi‐square test of independence). Infants who cried at 4 and 12 months were not more likely to have a noisy signal, 4 months: χ2 (1) = 0.07, p = .795, and 12 months: χ2 (1) = 0.15, p = .700.From the participating families, data on mind‐mindedness at 4 months was available for 114 mothers and 110 fathers. At 12 months, data on mind‐mindedness and parenting behaviour was available for 112 mothers and 113 fathers. The scores on mind‐mindedness were missing due to no lab visit at one of the time points, parents speaking a foreign language during the interaction that we did not master , and technical problems. Valid data on baseline HRV were available for 111 and 99 infants at 4 and 12 months, respectively. For HRV decline valid data were available for 97 and 84 infants at 4 and 12 months, respectively. Missing data were due to families not visiting the lab at one of the time points or disturbance due to movement artefacts or technical problems. We conducted 2.3.2Path models were assessed using structural equation modelling . Missing data were handled using the full information maximum likelihood (FIML) method. The FIML method obtains maximum use of the data, without substituting an actual value for missing data points or deleting cases with missing data points Kline, . InsteadThe first hypothesis concerns the effects of parents’ mind‐mindedness on the infant's development of HRV from 4 to 12 months. To examine the hypothesis, we tested a path model with all transactional relations between parental mind‐mindedness and infant HRV at 4 and 12 months for mothers and fathers separately (Models 1 and 2). These models enabled us to study whether parental mind‐mindedness predicted infant HRV at 12 months, over and above the effects of infants’ initial HRV status at 4 months. We tested a mother and father model because a path model with all variables  included in a single model would be too comprehensive in terms of parameter estimates, considering the amount of participants in this study Kline, . Model 1The second hypothesis involved the independent contributions of mothers and fathers in predicting infant HRV. We tested two models (Models 3 and 4). In Model 3 we examined the direct longitudinal effects of mothers’ and fathers’ mind‐mindedness at 4 months on infant HRV at 12 months. In Model 4 we tested the direct concurrent effects of mothers’ and fathers’ mind‐mindedness at 12 months on infant HRV at 12 months. As both mothers and fathers were included in the same model, we were able to examine whether parents uniquely contribute to infant HRV.The third hypothesis focused on whether parents’ mind‐mindedness predicts infant HRV via the quality of parenting behaviour. To examine this hypothesis, we added measures of mothers’ and fathers’ parenting quality at 4 and 12 months to the above‐mentioned models (in Models 5 and 6). In Model 5 we tested the indirect effects of mothers’ and fathers’ mind‐mindedness at 4 months via parenting quality at 4 months on infant HRV at 12 months. In Model 6 we tested the indirect effects of mothers’ and fathers’ mind‐mindedness at 12 months via parenting quality at 12 months on infant HRV at 12 months. For both models we first tested whether a full mediation model would fit the data. Subsequently we examined whether the model fit improved after adding the direct effects of mind‐mindedness to the model .2.3.3Paternal appropriate and non‐attuned mind‐mindedness at 4 months, maternal non‐attuned mind‐mindedness at 12 months and baseline HRV at 4 months had outliers (z > 3.29), which may exert a disproportionate influence on the results. We assigned the outliers with scores one unit larger than the next most extreme score  and 12 months . The mean proportions of appropriate mind‐related comments at 4 months produced by mothers and fathers were 7% and 8%, respectively. The mean proportion of appropriate mind‐related comments at 12 months decreased slightly to 6% for both mothers and fathers. Non‐attuned mind‐related comments were made less often: at both time points means were 1% for both mothers and fathers.Descriptive statistics for the infant and parent variables are reported in Table rmothers(105) = .33, p = .001; rfathers(102) = .24, p = .016. Non‐attuned mind‐related comments showed stability only for fathers, rfathers(102) = .42, p < .001; rmothers(102) = .03, p = .79. There was concordance in mothers’ and fathers’ appropriate mind‐related comments at 12 months, r(109) = .22, p = .018. Table Correlations between the mind‐mindedness variables and parenting quality, as well as the concordance within couples are presented in Table 3.13.1.12(3) = 1.35, p = .718, CFI = 1, RMSEA = .00. Mothers’ appropriate, but not non‐attuned mind‐related, comments were moderately stable over time, βappropriate = .32, SE = .09, p < .001; βnon‐attuned = .05, SE = .10, p = .642. Mothers’ appropriate mind‐related comments at 4 months predicted higher levels of baseline HRV at 12 months, β = .29, SE = .10, p = .004, as well as greater HRV decline during the stranger task, β = −.25, SE = .11, p = .021. Non‐attuned comments at 4 months predicted lower levels of baseline HRV at 12 months, β = −.24, SE = .09, p = .011. Infants’ baseline HRV at 4 months did not predict mind‐mindedness at 12 months. Mothers’ mind‐mindedness at 12 months was unrelated to infant HRV at 12 months. Infants with higher HRV baseline levels showed a stronger decline in HRV levels during the stranger approach at 4 months, β = −.60, SE = .11, p < .001 and at 12 months β = −.57, SE = .11, p < .001, than infants with lower HRV baseline. Infants’ baseline HRV at 4 months was positively related to baseline HRV at 12 months, β = .25, SE = .12, p = .040.Figure 3.1.22(3) = 4.16, p = .527, CFI = 1, RMSEA = .00. Fathers’ appropriate and non‐attuned mind‐related comments were stable over time, βappropriate = .25, SE = .10, p = .010; βnon‐attuned = .39, SE = .09, p < .001. Fathers’ appropriate mind‐related comments at 12 months were related to higher infant baseline HRV at 12 months, β = .29, SE = .10, p = .002, whereas fathers’ non‐attuned comments at 12 months were related to lower baseline HRV at 12 months, β = −.23, SE = .10, p = .022. Infant HRV at 4 months did not predict paternal mind‐mindedness at 12 months.Figure 3.1.3SE = .09, p < .001, whereas mothers’ non‐attuned mind‐related comments predicted lower baseline HRV at 12 months, β = .19, SE = .09, p = .048. Maternal appropriate comments no longer predicted greater HRV decline, β = .20, SE = .11, p = .070.Figure 3.1.4SE = .10, p = .031. Fathers’ appropriate mind‐related comments at 12 months predicted higher levels of baseline HRV at 12 months, β = .22, SE = .10, p = .025, whereas fathers’ non‐attuned mind‐related comments predicted lower baseline HRV at 12 months, β = −.24, SE = .10, p = .011.Figure 3.1.52(12) = 40.67, p < .001, CFI = .37, RMSEA = .14. We then added the direct effects of mind‐mindedness (4 months) on infant HRV at 12 months, χ2(4) = 8.66, p = .070, CFI = .89, RMSEA = .09. A chi‐square difference test of the full mediation model against the partial mediation model was significant, χ2(8) = 32.01, p < .001, indicating that the partial mediation model showed a better fit to the observed data than the full mediation model . This model showed a poor fit to the observed data, χl Kline, . The est3.1.62(12) = 18.60, p < .001, CFI = .85, RMSEA = .07. We added to the model the direct effects of mind‐mindedness (12 months) on infant HRV at 12 months, χ2(4) = 2.14, p = .710, CFI = 1, RMSEA = .00. A chi‐square difference test of the full mediation model against the partial mediation model was significant, χ2(8) = 16.46, p = .036. This indicates that the partial mediation model showed a better fit to the observed data than the full mediation model . This model showed a satisfactory fit to the observed data, χl Kline, . The estSE = .09, p = .009. Mothers’ appropriate mind‐related comments positively related to infant baseline HRV at 12 months, β = .21, SE = .10, p = .031. Parenting quality of mothers was unrelated to maternal mind‐mindedness and infant HRV.Paternal appropriate mind‐related comments were positively related to fathers’ parenting quality. In turn, fathers who showed higher levels of parenting quality at 12 months were more likely to have infants with higher baseline levels. The indirect effect of paternal appropriate mind‐related comments on infant HRV via parenting quality was significant, β = .08, CI 95% . The direct effect of fathers’ appropriate mind‐related comments on infant HRV was no longer significant in this model. Fathers who made non‐attuned mind‐related comments were more likely to have infants with lower HRV baseline levels, β = .24, 3.1.7r(109) = −.20, p = .036, we reran the two maternal path analyses including mothers’ educational level as a covariate. The outcomes of these analyses were essentially similar to the outcomes presented in Figures Prior to the analyses, we checked whether any of the predictor variables were related to infant gender and the level of education of the parents. Since mothers with a higher education level made fewer non‐attuned comments at 12 months, 4The present study tested three hypotheses: (a) parents’ mind‐mindedness at 4 and 12 months predicts infant HRV levels at 12 months over and above infants’ initial HRV levels at 4 months, (b) mothers’ and fathers’ mind‐mindedness independently predict infant HRV, and (c) the effects of mind‐mindedness on infant HRV  operate through parenting behaviour. With regard to the first hypothesis, we found that mothers’ higher proportions of appropriate mind‐related comments during interactions with their infants at 4 and 12 months predicted infants’ higher baseline HRV at 12 months, whereas higher proportions of non‐attuned comments at 4 months predicted lower baseline HRV at 12 months. Furthermore, we observed a larger HRV decline in infants during the stranger task at 12 months when mothers made more frequent appropriate mind‐related comments at 4 months. For fathers, higher proportions of appropriate mind‐related comments at 12 months were associated with infants’ higher baseline HRV at 12 months, whereas higher proportions of non‐attuned comments at 12 months were associated with lower baseline HRV at 12 months. Paternal non‐attuned comments at 12 months were related to less HRV decline during the stranger task. Infants’ baseline HRV and HRV decline at 4 months did not predict mothers’ and fathers’ mind‐mindedness at 12 months.With regard to the second hypothesis, we found concordance within couples regarding appropriate mind‐related comments at 12 months (but not at 4 months). Parents thus start to become more alike in terms of their mind‐mindedness. It may be that parents influence each other's mentalizing stance and mind‐related speech throughout the first year of the infant's life. It could also be that the child's individual characteristics increasingly evoke higher or lower mind‐mindedness in both parents, and thus couples’ mind‐mindedness starts to correlate at a later age (at least after 4 months). Despite the correlation between paternal and maternal appropriate mind‐related speech at 12 months, mothers’ and fathers’ mind‐mindedness independently predicted infant HRV. With regard to the third hypothesis, paternal, but not maternal, mind‐mindedness showed an indirect effect on infant HRV via parenting quality. Fathers’ appropriate mind‐related comments at 12 months were positively related to parenting quality, which in turn positively related to infant baseline HRV.We tested physiological emotion regulation capacity in two situations, during a baseline period and a potentially stressful situation. Infants’ baseline HRV was found to be somewhat stable over time, which is in line with the majority of the studies conducted previously  effects of mind‐mindedness for mothers and fathers. First, fathers’ appropriate and non‐attuned mind‐related speech related only concurrently to infant HRV at 12 months, as opposed to the concurrent and predictive effect of mothers’ mind‐mindedness. Given the fact that there was considerable stability in fathers’ mind‐related comments, it was surprising that only the 12‐month variable was related to infant HRV. Because the path model simultaneously estimated the path coefficients for all relations in the model, we checked the raw correlations. These also showed no association between fathers’ mind‐mindedness at 4 months and infants’ later HRV levels. The magnitude of the paternal and maternal path coefficients were very similar, with appropriate comments showing larger effects than non‐attuned comments.Second, the effects of parents’ mind‐mindedness on HRV decline during the stranger approach also differed between mothers and fathers. Only for fathers, non‐attuned mind‐related comments at 12 months were associated with less HRV decline during the stranger task. Thus, when fathers tended to misinterpret the infant's state of mind, or project their own state of mind onto that of the child, their infants showed less active emotion regulation during the stranger situation. On the other hand, only for mothers, appropriate mind‐related comments at 4 months predicted stronger HRV decline. Mothers’ appropriate mindreading early in life may thus stimulate infants’ parasympathetic system to become more actively involved in regulation arousal during stressful and unfamiliar social situations. Third, only fathers’ mind‐mindedness at 12 months was related to infants’ HRV directly and indirectly via parenting quality at 12 months. Fathers’ tendency to form representations of the infant's mind thus seemed to connect to responsive, non‐intrusive, affective parenting behaviour, which in turn explained part of the variation in infants’ emotion regulation.Altogether the findings described above are in line with proposed paternal and maternal differences regarding child development and the proposed unique role of fathers in the prediction of children's emotional development . Getting to understand and respond appropriately to the emotional arousal of an infant may require time to develop, which mothers in the present sample may have had more than fathers. Furthermore, nursing mothers engage in an interaction that is critical in soothing arousal during the first months. Differences in the nature and quantity of infant–father and infant–mother interactions during the infant's first months may thus also explain the earlier onset of the maternal effects.Parenting quality at 4 months did not relate to appropriate mind‐related comments at 4 months, whereas fathers’ parenting quality at 12 months did correlate with appropriate mind‐related comments at 12 months. Thus, at 4 months, parents’ mind‐minded stance is not (yet) guiding their parenting behaviours during interaction. One explanation is that at 4 months first‐time parents are very new to parenting in general and they are just learning about the mind of their infant. For instance, parents could just be beginning to understand that their 4‐month‐old is highly sensitive to external stimuli and easily over‐aroused. Their behaviour may not yet be guided by this kind of early “mental sensitivity”. Another explanation is that at 4 months, parenting is more non‐verbal than at 12 months , and not yet guided by verbal comments about their mental states . At 12 months, fathers who tend to be attuned to their infant's state of mind also tend to be more responsive and warm, and less intrusive and negative. This suggests that the fathers representational mindset and their actual behaviour become synchronized throughout the first year of the infant's life.r = .23, p = .016. For mothers, the correlation was in the positive direction, but did not reach significance, r = .15, p = .115. Although meta‐analytic evidence shows that the pooled correlation between appropriate mind‐related comments and sensitivity is significant, the range of correlations among studies varies greatly, from r =. 14  mind‐mindedness and the infant's development of emotion regulation. The body‐based interactive processes between the parent and the infant play an important role in understanding the effects of parents’ mentalizing stance, especially in the case of very young infants. Parental Embodied Mentalization (PEM) is a construct developed to assess parental mentalization at the nonverbal and primarily unconscious level  in combination with a father who is not inclined to interpret the infant's mind  may result in more regulation problems compared to an infant that has either one of these parents in combination with a highly attuned parent . This type of research would help to gain insight into familial risk factors.4.2Both parents’ appropriate and non‐attuned mind‐related commenting linked to the developing status of infants’ HRV levels, although the impact of mothers’ mind‐mindedness may show an earlier onset. The effects of parental mind‐mindedness held over and above the effects of parenting quality. The present study provides initial evidence that mothers’ and fathers’ tendency to make sense of their infant's mind in an attuned manner influences the development of infants’ emotion regulation capacity throughout the first year of life.