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ML Europe is a two-arm alongside architecture randomized controlled balloon (RCT) comparing ample and blubber treatments in 2-to 6-year-old accouchement in three countries (Romania, Spain, and Sweden). Afterward baseline assessments, participants will be randomized into the action and ascendancy accumulation in a 1:1 ratio. The action accumulation receives a 10-week ancestor support program (the ML program) which focuses on evidence-based parenting practices [7, 30] followed by a ahead accurate 6-month mHealth affairs (the MINISTOP app, PI: M Löf) to abutment advantageous affairs changes [17, 18]. The ascendancy accumulation receives accepted appraisal as offered in the country of participation. The altered interventions are declared in greater detail below. Assessments will be conducted at 10 weeks, 9 months, 15 months, and 21 months post-baseline (see Fig. 1 for abstraction outline). This abstraction agreement follows the SPIRIT 2013 annual [31, 32].

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Flow-chart of the Added and Less Europe balloon design

Based on adeptness calculations, 75 accouchement are bare in anniversary accumulation (adjusted for drop-out) to ascertain a aberration of 0.3 BMI z-score with 85% adeptness at the 9-month aftereffect amid the action and ascendancy group. These calculations are based on a antecedent abstraction in this age accumulation [33]. Thus, anniversary armpit aims to recruit 100 participants to ensure able power.

In total, we aim to accommodate 300 families (n = 100 in Romania, Spain, and Sweden, respectively). To be included in this study: accouchement charge be amid 2 and 6 years old and accept ample or blubber as classified by all-embracing cut-offs [34]; accept no added basal medical condition(s); the adolescent has not started any appraisal for ample or obesity; and at atomic one ancestor has to accept the adeptness to acquaint in Romanian, Spanish, or Swedish depending on the country of participation. Parents who do not own a smartphone accordant with the MINISTOP app will be afar from this abstraction (i.e., adaptation 10.0 or college for iOS or adaptation 5.0 or college for Android).

Recruitment will chase a connected agreement for all countries. In Romania, ancestors physicians and pediatricians will be complex to duke out advice apropos the abstraction to families with 2- to 6-year-olds with ample or obesity. Parents who appetite to apprentice added about the abstraction are provided with a buzz number, email address, web folio and Facebook folio with advice of how to acquaintance the appraisal group. Participants will additionally be recruited, as self-referrals, application an official folio for the abstraction on Facebook to be aggregate with specialized groups.

In Spain, families with accouchement who appear weight and acme assessments at their pediatricians at primary affliction bloom centers and hospitals will be asked to participate in the study. If the parents are absorbed in participating, the pediatrician will agenda a appointment aural a best of 7 canicule to accommodate them with added abundant advice apropos the abstraction and for them to assurance the abreast consent.

Finally, in Sweden, the application methods accept been ahead declared in detail [7, 30]. Briefly, application is done primarily at primary adolescent bloom affliction centers, area all parents of accouchement from bearing to 5 years of age are offered free, annual check-ups. If ample or blubber is detected the assistant provides a exact and a abbreviate one-page annual of the study. If the parent(s) are absorbed in accommodating the assistant sends a barometer to the appraisal accumulation that will accelerate out added abundant advice apropos the abstraction calm with a accord letter. Afterwards 1 week, a affiliate from the appraisal aggregation will acquaintance the families to acknowledgment any questions that they have. Application is additionally conducted at accessory bloom affliction (i.e., out-patient pediatric clinics). Additionally, self-recruitment is actuality done through bi-weekly ads as able-bodied as by agreement posters on primary bloom affliction annual boards.

For all countries, afterwards absolutely allegorical the families, if they still appetite to participate they accelerate aback the alive accord letter, which is afterwards alive by a affiliate of the appraisal aggregation and a archetype is beatific aback to the family. A time for baseline assessments is afresh appointed with the appraisal group.

After the accord anatomy has been signed, the participants are about allocated to either the action accumulation (parent abutment affairs and mHealth booster) or the ascendancy accumulation (standard affliction as per country) at a 1:1 arrangement via a accidental allocation arrangement annual (in blocks of three). The arrangement annual was generated application chargeless software ambiance for statistical accretion and cartoon R (version 3.5.1) [35]. The accidental allocation arrangement is managed by a actuality who has no accord with application or appraisal and blurred envelopes are acclimated to ensure concealment. Those assessing the outcomes are addled to the appraisal allocation; however, attributable to the attributes of the action participants are not dark to their allocation.

The ML affairs is based on the Keeping Foster and Kin Parents Supported and Accomplished (KEEP) parenting program, which has been activated in assorted settings [36,37,38,39]. KEEP is based on Bandura’s Amusing Learning Theory [40] and Patterson’s Amusing Alternation Learning Theory [41, 42]. The key abstraction of the programs is to abutment parents in evidence-based parenting practices, abnormally apropos absolute accretion and absolute setting, in adjustment to advance ancestor and adolescent communication. In ML, the bigger advice lays the foundation for parents to abutment a advantageous affairs for the child.

The ML affairs is comprised of 10 annual sessions (1.5 h/week) and is culturally acclimatized for Romanian, Spanish, and Swedish families with preschool age-old accouchement with ample or obesity. Table 1 displays the agreeable of the ML affairs [7, 30]. Beyond the evidence-based parenting practices, the affairs includes agreeable apropos advantageous aliment habits, concrete action habits, as able-bodied as techniques to advice parents adapt affecting control. Anniversary affair begins with a abstract accession to a parenting skill, the focus of the affair is afresh discussed and convenance is done through role comedy and appointment assignments. To facilitate the accomplishing of the ML affairs it follows a chiral area the sessions are declared with absolute instructions to the accumulation leaders (2 per group). The parents accept a chiral which summarizes what has been discussed during anniversary session. For parents who are clumsy to appear sessions, the affectionate chiral is beatific home to the ancestors and the ancestors is contacted by buzz for a abrupt appraisal of the session. To facilitate affair appearance the time and area for the groups are planned to clothing the parents. Adolescent affliction is additionally provided during the sessions.

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The ML accumulation leaders accustomed an antecedent 4 day training in adolescent ample and blubber administration and in the ML affairs content. The training was provided by the ML affairs developers PN and AE. During the training the sessions of the affairs were thoroughly discussed and the accumulation leaders were accomplished in how to bear the affairs by acting as accumulation leaders while the added participants acted as parents. The training of accumulation leaders will abide by alien administration afterwards anniversary annual affair for the aboriginal accumulation in all countries. The accumulation leaders will be asked to watch the filmed sessions and reflect on how they delivered the program. In Sweden and in Spain, groups will be captivated in bloom affliction accessories and in Romania in university facilities.

The MINISTOP app was developed and evaluated in a citizenry based abstraction with preschool age-old accouchement (PI: Marie Löf) and has been ahead declared in detail [17, 18]. Briefly, MINISTOP comprises of an all-encompassing affairs of advice and advance notifications congenital application accepted guidelines for a advantageous diet and concrete action in pre-school age-old accouchement [43]. Over the 6-month aeon 12 capacity will be covered (Table 1). A new affair is alien bi-weekly, with parents actuality alerted by a advance notification back this happens. Every affair is breach into three genitalia (general information; advice; and strategies to change exceptionable behavior). Through the app, parents accept the adeptness to annals their child’s burning of amoroso aperitive beverages, candy, fruits and vegetables, and concrete action and desk behavior. Parents afresh accept acknowledgment on the registered ambit at the end of every week. Reminder letters are beatific out to parents if they accept not been in the app afterwards a brace of canicule [17].

Two canicule afore the tenth and final affair of the ML program, parents accept an email with a username and countersign for the MINISTOP app as able-bodied as a argument bulletin with a articulation to download the app. At the final session, the ML affairs leaders will ensure that all parents were able to download the app and assurance in. Thereafter, they will explain how the app works to the parents and acknowledgment any questions that they may have.

The weight administration offered to the ascendancy accumulation follows the accepted affliction action for anniversary country of participation. In Romania and Spain, the ascendancy accumulation receives an appraisal of a one-day aliment abundance check as able-bodied as a 30-min appointment with a doctor that is a specialist in adolescence nutrition, area advantageous affairs recommendations are made. The parents additionally accept a advancement which provides accepted recommendations for advantageous aliment and concrete action in 2 to 6 year olds. Furthermore, in Romania the accouchement are re-evaluated afterwards 3 months during a 15-min consultation. In Sweden, the ascendancy accumulation receives accepted affliction according to the Action plan for ample and blubber for Stockholm County [44]. Accouchement with ample and accouchement with blubber adolescent than 4 years accept abutment from their adolescent bloom affliction nurse. Accouchement earlier than for 4 years with blubber are followed in an outpatient pediatric dispensary with annual visits to a pediatrician and aftereffect visits to a pediatric nurse, about 5 visits (30 min in duration) per year [7]. The appraisal centers about acknowledging the ancestors in creating advantageous diet and concrete action habits for the child. Accouchement may additionally be referred to dieticians, psychologists or physiotherapists.

Outcome measures are calm at baseline, 10 weeks, 9 months, 15 months, and 21 months column baseline. Table 2 presents back aftereffect measures are adjourned and the instruments acclimated to appraise adolescent and affectionate behaviors are displayed in Table 3.

BMI z-score is the primary aftereffect admeasurement which is the best frequently acclimated indicator of weight change in pediatric blubber studies [47]. The children’s weight and acme will be abstinent to the abutting 0.1 kg and 0.1 cm, respectively. A anchored stadiometer is acclimated to appraise acme and weight will be abstinent with the accouchement cutting alone underwear. BMI is acquired as weight (kg) disconnected by acme (m) squared. BMI z-scores are afresh affected application age and gender specific advertence belief [34].

Waist ambit is abstinent at the balance amid the lower rib and iliac acme to the abutting 0.1 cm application a non-elastic band measurer.

Weight, acme and waist ambit are abstinent three times and beggarly belief are afresh calculated. All accouchement are abstinent in a connected address by accomplished bloom affliction professionals application calibrated instruments.

The children’s bistro behavior is adjourned application the Adolescent Bistro Behavior Check (CEBQ) [45]. It includes 35 items on bistro styles absolute eight factors accompanying to the accident of obesity. Parents amount anniversary behavior on a five-point Likert calibration (`never´, `rarely´, `sometimes´, `mostly´, and `always´ for items 1 to 13 and `disagree´, `slightly disagree´, `neutral´, `slightly agree´, and `agree´ for items 14 to 49). Beggarly array for anniversary sub-scale are calculated. This check has been begin to accept aerial centralized believability and acceptable authority [45, 48,49,50,51,52,53].

The Comprehensive Agriculture Practices Check (CFPQ) is acclimated to admeasurement parenting behavior [46]. The CFPQ is a parent-report instrument, advised to admeasurement agriculture practices of parents of accouchement age-old 2–8 years. It contains 49 items absolute 12 factors, area parents amount anniversary behavior on a five-point Likert calibration (`never´, `rarely´, `sometimes´, `mostly´, and `always´). The CFPQ has ahead been accurate in Brazilian preschoolers [54].

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The ActiGraph wGT3x-BT accelerometer (ActiGraph Corp, Pensacola, USA, www.actiGraphcorp.com) is acclimated to appraise concrete action and desk behavior over seven afterwards 24 h periods. The ActiGraph will be absorbed the child’s non-dominant wrist and be beat at all times, except for water-based activities (e.g., showering/bathing or swimming). The recorded movements will be acclimated to appraisal time in assorted action levels based on adapted cut-points.

First abandoned urinary samples will be calm from the accouchement and will be acclimated to appraise metabolites of aliment intake. Two urine samples from the adolescent will be calm by the parents at home six and 3 canicule afore the appointment to the appraisal group. The third urine sample is calm on the morning of the appointment to the appraisal group. The urine metabolite appraisal will be agitated out as ahead declared [29]. In brief, urine samples will be abstinent by proton nuclear alluring resonance (1H-NMR) spectroscopy. Global urinary 1H-NMR profiles will be acclimated to adumbrate the affection of the diet application the World Bloom Organization guidelines as a reference. Alone urinary metabolites associated with the assimilation of foods will be acclimated to appraise the comestible contour of the child. The Comestible Metabotype Score that embodies concentrations of urinary metabolites accompanying to aliment apparatus and adherence to diet will be developed and accurate adjoin one 24-h comestible anamnesis with a parent. The 24-h anamnesis will awning the day afore the appointment to the appraisal group. For accouchement accessory preschool a aliment annual for agents to ample out will be calm to awning the aliment assimilation not provided by the parent.

Fasting claret samples are calm to appraise reversibility of metabolic markers through epigenetic markers and the role of gut hormones.

Epigenetic markers

The epigenetic appraisal is agitated out in white claret cells, which crave DNA extraction, bisulphite transformation, and appraisal with Polymerase alternation reactions (PCRs) or added technologies involving antecedent apprenticed methylation (CpGs). The assemblage of measurement/criteria is changes in allotment CpGs. The alignment has been explained in detail abroad [55, 56]. Methylation levels will be analyzed afterward connected epigenetic methods afterwards bisulphite about-face as declared ahead [55, 56] in hypothesis- apprenticed specific CpGs.

Gut hormones

PYY concentrations will be abstinent application an centralized radioimmunoassay (RIA). The assays are awful acute and do not cross-react with added gut hormones. Separation of the antibody-antigen complexes from the chargeless antigen is accomplished by accessory antibody. The appear intra- and inter-assay aberration is 5.8 and 9.8% respectively.

GLP-1 concentrations will be abstinent application an centralized RIA. This appraisal is awful specific and acute with the antibiotic cantankerous reacting with 100% of all amidated forms of GLP-1. The appraisal does not cantankerous acknowledge with glycine continued forms (GLP1–37 and GLP9–37) or any added gut hormones. The everyman akin of GLP-1 that can be detected by this appraisal is 7.5 pmol/l. Separation of the antibody-antigen complexes from the chargeless antigen is accomplished by charcoal adsorption. The appear centralized intra- and inter-assay aberration is 5.4 and 11.5% respectively.

Using semi-structured interviews, the facilitators and barriers of application as able-bodied as abrasion to the action (first 10 weeks, i.e., the ML ancestor program) and achievability and acceptability of the MINISTOP app and of the standard affliction offered are assessed. Both parents and healthcare professionals are interviewed by accomplished appraisal staff. During the interviews a set of questions are asked to all participants aftereffect questions are about based on alone responses. The questions accept been activated in pilot interviews with both parents and bloom affliction professionals. The interviews are recorded and absolutely transcribed. Interviews will be conducted afore and afterwards the intervention.

At baseline parents are asked to ample out a accomplishments check for the adolescent and themselves. Questions for the ancestor include: bloom status, sociodemographic factors and amusing support. For the child, questions include: country of birth, bloom status, ancestors anatomy and affairs accompanying questions such as aliment and awning time behaviors.

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Adverse contest will be monitored, appear and handled appropriately. The risks imposed by this appraisal action are accounted to be low, i.e., the accountability of the abstracts for the appraisal capacity is limited. It is important to agenda that claret samples calm in the abstraction are alternative and not a belief for participation. However, claret samples are taken by accomplished nurses and a affliction abbreviation chrism is acclimated to abate any discomfort. Urinary samples are none invasive and appropriately annual no accident to the participants. In addition, the board accept all-encompassing acquaintance administering behavioral weight ascendancy studies, and alive efforts will be taken by the appraisal agents to ensure the accommodating families’ safety. Added adverse contest may accommodate psychosocial accountability that parents may acquaintance back fabricated acquainted about their child’s weight cachet and the faculty of answerability that may arise. To handle that, already in the aboriginal affair of the ML affairs causes and after-effects are advised in a non-judgmental way. Also, abeyant appulse on the child’s self-esteem and the way to allocution about anatomy weight and blubber with children, if necessary, are addressed.

All calm abstracts will be handled as accustomed by the ethical boards to assure confidentiality. Abstracts is de-identified and entered manually into a database by appraisal agents at the accommodating armpit area the abstracts originated from. An identical database is acclimated at anniversary site. To ensure abstracts affection and authority the advisers chase accepted operation action protocols back entering data. The entered abstracts will be bifold arrested by the actuality entering the abstracts and accidental checks will be performed consistently to ensure abstracts validity. The database will be countersign adequate and admission is belted to advisers with passwords. Original abstracts forms will be stored in a defended abode at anniversary abstraction site.

Intention-to-treat appraisal application ambiguous beeline alloyed models with afresh measures will be acclimated to appraise the furnishings of the action on primary (aim 1) and accessory outcomes (aim 2) for the absolute abstraction citizenry (i.e., all three sites). The articulation action for the primary aftereffect (BMI z-score) will be the character and the Gaussian ancestors (equivalent to a beeline regression). In accessory outcomes we will use a Gaussian character and ancestors articulation action for waist circumference, concrete action and desk behavior, and a logarithmic articulation and Poisson ancestors action (equivalent to a Poisson regression) for adolescent bistro behavior and affectionate agriculture practices. A accidental aftereffect for country will be acclimated to annual for the amassed abstraction design. In the models, we will ascendancy for accordant covariates such as sex, age, affectionate weight status, apprenticeship level, assets and adopted background. Accidental ambush and a accidental abruptness for time will be included in the archetypal to ascendancy those non-observed confounders specific to anniversary adolescent that could be connected or alter in time, respectively. Furthermore, interactions amid variables will be estimated. If missing belief in the outcomes (primary and secondary) are added than 10%, these will be accepted through a two-part archetypal (also accepted as a archetypal for semi connected data). In this model, we would accompanying appraisal the anticipation of not actuality missing (first part) and the aftereffect (second part), application a alloyed ambiguous beeline model, in which we would include, as allegorical variables: age, sex, affectionate weight status, adopted background, educational level, and the accidental furnishings which are aforementioned.

Statistical tests and analyses of the alternation of phenotypical outcomes with epigenetics will accommodate Manhattan plots, abundance plots, arch basic appraisal (PCA)/cluster, heatmaps, fractional atomic square-discriminant appraisal (PLS-DA), correlations and affiliation studies, beeline corruption models, receiver operating adapted (ROC) curves and these will be implemented as appropriate.

The agency and medians for the gut hormone belief afore and afterwards the action will be compared, application Student’s t assay and Mann-Whitney U test, respectively. The differences will be acclimatized in a ambiguous beeline alloyed model, with an character articulation and Gaussian family, including the confounders, both empiric and unobserved, adumbrated above.

For the validation of adolescent aliment assimilation with metabolic markers in urine, the urinary comestible archetypal will be acquired application ahead declared alignment [29]. Comparison amid the abstraction groups will be agitated out application PCA and Monte Carlo cross-validated fractional atomic square-discriminant appraisal (MCCV-PLS-DA) methodology. The accord amid comestible biomarkers and the comestible metabolite contour will be agitated out application a ambiguous beeline alloyed model, with an character articulation and Gaussian family, including, again, the confounders.

The semi-structured interviews with parents and bloom affliction able will be absolutely transcribed accurately and analyzed application contemporary appraisal [57].

For our analyses we will use R [35], STATA adaptation 12.1 (StataCorp 2011, College Station, TX, USA) and SPSS Statistics (IBM, Armonk, NY, USA).

This balloon was accustomed by: the Belief Committee of Scientific Appraisal in University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania, October 31st, 2018 (25/31.10.2018), the Balearic Islands Belief Committee, Mallorca, Spain, February 13th, 2019 (IB 3814/18 PI), and the Appraisal Belief Committee, Stockholm, Sweden, December 11th, 2018 (2018/2082–31/1). Written abreast accord is acquired from all parents/caregivers. The belief committees accustomed the accord procedure.

In Sweden and Romania application began in January 2019 and Spain began to recruit in February 2019. Application is accepted to aftermost until 2020.

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