Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 reveals three enhancer/silencer-rich regions and 38 spliceogenic variants.

Splicing is controlled by a large set of regulatory elements (SREs) including splicing enhancers and silencers, which are involved in exon recognition. Variants at these motifs may dysregulate splicing and trigger loss-of-function transcripts associated with disease. Our goal here was to study the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2. For this purpose, we used a previously published minigene with exons 6-10 that produced the expected minigene full-length transcript and replicated the naturally occurring events of exon 8 [Δ(E8)] and exon 10 [Δ(E10)] skipping. We then introduced 12 internal microdeletions of exons 8 and 10 by mutagenesis in order to map SRE-rich intervals by splicing assays in MCF-7 cells. We identified three minimal (10-, 11-, 15-nt) regions essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Then 87 variants found within these intervals were introduced into the wild-type minigene and tested functionally. Thirty-eight of them (44%) impaired splicing, four of which (c.883G>A, c.883G>T, c.884A>T, and c.1080G>T) induced negligible amounts (<5%) of the minigene full-length transcript. Another six variants (c.886G>A, c.886G>T, c.1075G>A, c.1075G>T, c.1076A>T, and c.1078G>T) showed significantly strong impacts (20-50% of the minigene full-length transcript). Thirty-three of the 38 spliceogenic variants were annotated as missense, three as nonsense, and two as synonymous, underlying the fact that any exonic change is capable of disrupting splicing. Moreover, c.883G>A, c.883G>T, and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based criteria. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants.

BACKGROUND: Disrupted pre-mRNA splicing is a frequent deleterious mechanism in hereditary cancer. We aimed to functionally analyze candidate spliceogenic variants of the breast cancer susceptibility gene CHEK2 by splicing reporter minigenes. METHODS: A total of 128 CHEK2 splice-site variants identified in the Breast Cancer After Diagnostic Gene Sequencing (BRIDGES) project (https://cordis.europa.eu/project/id/634935) were analyzed with MaxEntScan and subsetted to 52 variants predicted to impact splicing. Three CHEK2 minigenes, which span all 15 exons, were constructed and validated. The 52 selected variants were then genetically engineered into the minigenes and assayed in MCF-7 (human breast adenocarcinoma) cells. RESULTS: Of 52 variants, 46 (88.5%) impaired splicing. Some of them led to complex splicing patterns with up to 11 different transcripts. Thirty-four variants induced splicing anomalies without any trace or negligible amounts of the full-length transcript. A total of 89 different transcripts were annotated, which derived from different events: single- or multi-exon skipping, alternative site-usage, mutually exclusive exon inclusion, intron retention or combinations of the abovementioned events. Fifty-nine transcripts were predicted to introduce premature termination codons, 7 kept the original open-reading frame, 5 removed the translation start codon, 6 affected the 5'UTR (Untranslated Region), and 2 included missense variations. Analysis of variant c.684-2A > G revealed the activation of a non-canonical TG-acceptor site and exon 6 sequences critical for its recognition. CONCLUSIONS: Incorporation of minigene read-outs into an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme allowed us to classify 32 CHEK2 variants (27 pathogenic/likely pathogenic and 5 likely benign). However, 20 variants (38%) remained of uncertain significance, reflecting in part the complex splicing patterns of this gene.

Minigene-based splicing analysis and ACMG/AMP-based tentative classification of 56 ATM variants.

The ataxia telangiectasia-mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss-of-function variants are associated with 2-fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large-scale sequencing project BRIDGES. A total of 381 variants at the intron-exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4-9, 11-17, 25-29, and 49-52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF-7/HeLa cells. Forty-eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full-length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi-exon skipping. Twenty-seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL-transcript due to the use of a noncanonical GG-5'-splice-site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage-sensitive expression model in which variants producing ≥30% of FL-transcripts would be predicted benign, while variants producing ≤13% of FL-transcripts might be pathogenic. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Splicing predictions, minigene analyses, and ACMG-AMP clinical classification of 42 germline PALB2 splice-site variants.

PALB2 loss-of-function variants confer high risk of developing breast cancer. Here we present a systematic functional analysis of PALB2 splice-site variants detected in approximately 113,000 women in the large-scale sequencing project Breast Cancer After Diagnostic Gene Sequencing (BRIDGES; https://bridges-research.eu/). Eighty-two PALB2 variants at the intron-exon boundaries were analyzed with MaxEntScan. Forty-two variants were selected for the subsequent splicing functional assays. For this purpose, three splicing reporter minigenes comprising exons 1-12 were constructed. The 42 potential spliceogenic variants were introduced into the minigenes by site-directed mutagenesis and assayed in MCF-7/MDA-MB-231 cells. Splicing anomalies were observed in 35 variants, 23 of which showed no traces or minimal amounts of the expected full-length transcripts of each minigene. More than 30 different variant-induced transcripts were characterized, 23 of which were predicted to truncate the PALB2 protein. The pathogenicity of all variants was interpreted according to an in-house adaptation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) variant classification scheme. Up to 23 variants were classified as pathogenic/likely pathogenic. Remarkably, three ±1,2 variants (c.49-2A>T, c.108+2T>C, and c.211+1G>A) were classified as variants of unknown significance, as they produced significant amounts of either in-frame transcripts of unknown impact on the PALB2 protein function or the minigene full-length transcripts. In conclusion, we have significantly contributed to the ongoing effort of identifying spliceogenic variants in the clinically relevant PALB2 cancer susceptibility gene. Moreover, we suggest some approaches to classify the findings in accordance with the ACMG-AMP rationale. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Validity, reliability, and calibration of the physical activity unit 7 item screener (PAU-7S) at population scale.

BACKGROUND: Validation of self-reported tools, such as physical activity (PA) questionnaires, is crucial. The aim of this study was to determine test-retest reliability, internal consistency, and the concurrent, construct, and predictive validity of the short semi-quantitative Physical Activity Unit 7 item Screener (PAU-7S), using accelerometry as the reference measurement. The effect of linear calibration on PAU-7S validity was tested. METHODS: A randomized sample of 321 healthy children aged 8-16 years (149 boys, 172 girls) from the nationwide representative PASOS study completed the PAU-7S before and after wearing an accelerometer for at least 7 consecutive days. Weight, height, and waist circumference were measured. Cronbach alpha was calculated for internal consistency. Test-retest reliability was determined by intra-class correlation (ICC). Concurrent validity was assessed by ICC and Spearman correlation coefficient between moderate to vigorous PA (MVPA) derived by the PAU-7S and by accelerometer. Concordance between both methods was analyzed by absolute agreement, weighted kappa, and Bland-Altman statistics. Multiple linear regression models were fitted for construct validity and predictive validity was determined by leave-one-out cross-validation. RESULTS: The PAU-7S overestimated MVPA by 18%, compared to accelerometers (106.5 ± 77.0 vs 95.2 ± 33.2 min/day, respectively). A Cronbach alpha of 0.76 showed an acceptable internal consistency of the PAU-7S. Test-retest reliability was good (ICC 0.71 p < 0.001). Spearman correlation and ICC coefficients of MVPA derived by the PAU-7S and accelerometers increased from 0.31 to 0.62 and 0.20 to 0.62, respectively, after calibration of the PAU-7S. Between-methods concordance improved from a weighted kappa of 0.24 to 0.50 after calibration. A slight reduction in ICC, from 0.62 to 0.60, yielded good predictive validity. Multiple linear regression models showed an inverse association of MVPA with standardized body mass index (ß - 0.162; p < 0.077) and waist to height ratio (ß - 0.010; p < 0.014). All validity dimensions were somewhat stronger in boys compared to girls. CONCLUSION: The PAU-7S shows a good test-retest reliability and acceptable internal consistency. All dimensions of validity increased from poor/fair to moderate/good after calibration. The PAU-7S is a valid instrument for measuring MVPA in children and adolescents. TRIAL REGISTRATION: Trial registration number ISRCTN34251612 .

Usage of Plant Food Supplements (PFS) for weight control in six European countries: results from the PlantLIBRA PFS Consumer Survey 2011-2012.

BACKGROUND: Obesity is increasing worldwide and weight-control strategies, including the consumption of plant food supplements (PFS), are proliferating. This article identifies the herbal ingredients in PFS consumed for weight control and by overweight/obese dieters in six European countries, and explores the relationship between their consumption and their self-reported BMI. METHODS: Data used were a subset from the PlantLIBRA PFS Consumer Survey 2011-2012, a retrospective survey of 2359 PFS consumers. The survey used a bespoke frequency-of-PFS-usage questionnaire. Analyses were performed in two consumer subsamples of 1) respondents taking the products for "body weight reasons", and 2) "dieters for overweight/obesity", to identify the herbal ingredients consumed for these reasons. The relationship between the 5 most consumed herbal ingredients and self-reported BMI in groups 1 and 2 is explored by comparing BMI proportions of consumers vs. non-consumers (using Chi-squared test). RESULTS: 252 PFS (8.8 %) were consumed for "body weight reasons" (by 240 PFS consumers); 112 PFS consumers (4.8 %) were "dieting for overweight/obesity". Spain is the country where consuming herbal ingredients for body weight control and dieting were most popular. Artichoke was the most consumed herbal ingredient. Considering only the 5 top products consumed by those who responded "body weight", when using the total survey sample, a greater proportion of BMI ≥ 25 was observed among consumers of PFS containing artichoke and green tea as compared to non-consumers (58.4 % vs. 49.1 % and 63.2 % vs. 49.7 % respectively). Considering only the 5 top products consumed by "dieters" and using only the "dieters" sample, a lower proportion of BMI ≥ 25 was observed among pineapple-containing PFS consumers (38.5 % vs. 81.5 %); however, when using the entire survey sample, a greater proportion of BMI ≥ 25 was observed among artichoke-containing PFS consumers (58.4 % vs. 49.1 %). CONCLUSIONS: A comparison of results among the scarce publications evaluating the use of weight-loss supplements at the population level is limited. Nevertheless every hint is important in finding out which are the self-treatment strategies used by overweight/obese individuals in European countries. Although limited by a small sample size, our study represents a first attempt at analysing such data in six EU countries. Our findings should encourage the conduction of further studies on this topic, long-term and large sample-sized studies, ideally conducted in the general population.

Splicing Analysis of 16 PALB2 ClinVar Variants by Minigene Assays: Identification of Six Likely Pathogenic Variants.

PALB2 loss-of-function variants are associated with significant increased risk of breast cancer as well as other types of tumors. Likewise, splicing disruptions are a common mechanism of disease susceptibility. Indeed, we previously showed, by minigene assays, that 35 out of 42 PALB2 variants impaired splicing. Taking advantage of one of these constructs (mgPALB2_ex1-3), we proceeded to analyze other variants at exons 1 to 3 reported at the ClinVar database. Thirty-one variants were bioinformatically analyzed with MaxEntScan and SpliceAI. Then, 16 variants were selected for subsequent RNA assays. We identified a total of 12 spliceogenic variants, 11 of which did not produce any trace of the expected minigene full-length transcript. Interestingly, variant c.49-1G > A mimicked previous outcomes in patient RNA (transcript ∆(E2p6)), supporting the reproducibility of the minigene approach. A total of eight variant-induced transcripts were characterized, three of which (∆(E1q17), ∆(E3p11), and ∆(E3)) were predicted to introduce a premature termination codon and to undergo nonsense-mediated decay, and five (▼(E1q9), ∆(E2p6), ∆(E2), ▼(E3q48)-a, and ▼(E3q48)-b) maintained the reading frame. According to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, which integrates mgPALB2 data, six PALB2 variants were classified as pathogenic/likely pathogenic, five as VUS, and five as likely benign. Furthermore, five ±1,2 variants were catalogued as VUS because they produced significant proportions of in-frame transcripts of unknown impact on protein function.

Minigene Splicing Assays Identify 20 Spliceogenic Variants of the Breast/Ovarian Cancer Susceptibility Gene RAD51C.

RAD51C loss-of-function variants are associated with an increased risk of breast and ovarian cancers. Likewise, splicing disruptions are a frequent mechanism of gene inactivation. Taking advantage of a previous splicing-reporter minigene with exons 2-8 (mgR51C_ex2-8), we proceeded to check its impact on the splicing of candidate ClinVar variants. A total of 141 RAD51C variants at the intron/exon boundaries were analyzed with MaxEntScan. Twenty variants were selected and genetically engineered into the wild-type minigene. All the variants disrupted splicing, and 18 induced major splicing anomalies without any trace or minimal amounts (<2.4%) of the minigene full-length (FL) transcript. Twenty-seven transcripts (including the wild-type and r.904A FL transcripts) were identified by fluorescent fragment electrophoresis; of these, 14 were predicted to truncate the RAD51C protein, 3 kept the reading frame, and 8 minor isoforms (1.1−4.7% of the overall expression) could not be characterized. Finally, we performed a tentative interpretation of the variants according to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, classifying 16 variants as likely pathogenic. Minigene assays have been proven as valuable tools for the initial characterization of potential spliceogenic variants. Hence, minigene mgR51C_ex2-8 provided useful splicing data for 40 RAD51C variants.

Improving upper-limb and trunk kinematics by interactive gaming in individuals with chronic stroke: A single-blinded RCT.

BACKGROUND: Commercial gaming systems are increasingly being used for stroke rehabilitation; however, their effect on upper-limb recovery versus compensation is unknown. OBJECTIVES: We aimed to compare the effect of upper-limb rehabilitation using interactive gaming (Nintendo Wii) with dose-matched conventional therapy on elbow extension (recovery) and forward trunk motion (compensation) in individuals with chronic stroke. Secondary aims were to compare the effect on (1) clinical tests of impairment and activity, pain and effort, and (2) trajectory kinematics. We also explored arm and trunk motion (acceleration) during Wii sessions to understand how participants performed movements during Wii gaming. METHODS: This single-centre, randomized controlled trial compared 12 hourly sessions over 4 weeks of upper-limb Wii therapy to conventional therapy. Outcomes were evaluated at baseline and 4 weeks. The change in elbow extension and trunk motion during a reaching task was evaluated by electromagnetic sensors. Secondary outcomes were change in Fugl-Meyer assessment, Box and Block test, Action Research Arm Test, Motor Activity Log, and Stroke Impact Scale scores. Arm and trunk acceleration during Wii therapy was evaluated by using inertial sensors. A healthy control group was included for reference data. RESULTS: Nineteen participants completed Wii therapy and 21 conventional therapy (mean [SD] time post-stroke 66.4 [57.2] months). The intervention and control groups did not differ in mean change in elbow extension angle (Wii: +4.5°, 95% confidence interval [CI] 0.1; 9.1; conventional therapy: +6.4°, 95%CI 0.6; 12.2) and forward trunk position (Wii: -3.3 cm, 95%CI -6.2;-0.4]; conventional therapy: -4.1 cm, 95%CI -6.6; -1.6) (effect size: elbow, d = 0.16, p = 0.61; trunk, d = 0.13, p = 0.65). Clinical scores improved similarly but to a small extent in both groups. The amount of arm but not trunk acceleration produced during Wii sessions increased with training. CONCLUSIONS: Supervised upper-limb gaming therapy induced similar recovery of elbow extension as conventional therapy and did not enhance the development of compensatory forward trunk movement in individuals with chronic stroke. More sessions may be necessary to induce greater improvements. CLINICALTRIALS: GOV: NCT01806883.

A randomized, placebo-controlled crossover trial of a decaffeinated energy drink shows no significant acute effect on mental energy.

BACKGROUND: "Energy drinks" are heavily marketed to the general public, across the age spectrum. The efficacy of decaffeinated energy drinks in enhancing subjective feelings of energy (s-energy) is controversial. OBJECTIVE: The authors sought to test the efficacy of the caffeine-free version of a popular energy drink compared with a placebo drink. METHODS: This study was a randomized, double-blind, placebo-controlled, crossover trial in 223 healthy men and women aged 18-70 y with intention-to-treat and completers analysis. Participants were randomly assigned to consumption of either the decaffeinated energy drink or a placebo drink on testing day 1, and the other drink a week later. A battery of computer-based mood and cognitive tests to assess s-energy was conducted at baseline and at 0.5, 2.5, and 5 h post-ingestion. The main outcome measures were 1) mood, which was assessed by using a General Status Check Scale and the Profile of Mood States 2nd edition brief form, and 2) cognitive measures, including the N-back task (reaction time and accuracy), Reaction Time test, Flanker task (distraction avoidance), and Rapid Visual Information Processing test. RESULTS: No statistically significant or meaningful benefits were observed for any outcome measure, including mood and cognitive measures. Analyses of mean differences, slopes, and median differences were consistent. CONCLUSIONS: No differences were detected across a range of mood/cognitive/behavioral/s-energy-level tests after consumption of the energy drink compared with a placebo drink in this diverse sample of adults. Thus, we found strong evidence that the energy drink is not efficacious in enhancing s-energy levels, nor any related cognitive or behavioral variables measured. In light of federal regulations, these findings suggest that labeling and marketing of some products which claim to provide these benefits may be unsubstantiated. This trial was registered at www.clinicaltrials.gov as NCT02727920.

Effects of dietary assessment methods on assessing risk of nutrient intake adequacy at the population level: from theory to practice.

The present study evaluated how applying different dietary methods affects risk assessment of inadequate intakes at the population level. A pooled analysis was conducted using data from two Spanish regional representative surveys both applying similar methodology with a total sample of 2615 individuals aged 12-80. Diet was assessed in the entire sample applying data from one 24 h recall (24HR), a mean of two non-consecutive 24HR, both crude and adjusted for intraindividual variability, and a FFQ. Intakes of vitamins A, C, E, thiamin, riboflavin, niacin, vitamin B6, vitamin B12, Fe, Mg, P and Zn were compared to the average nutrient requirement (ANR or estimated average requirement) in the entire sample and also excluding under-reporters applying the ANR cut-point method (and the probability approach for Fe). Higher percentages of intakes below the ANR were seen for 1-24HR and the mean of 2-24HR, except for nutrients with the highest rates of inadequacy (vitamins A, E, folate and Mg). For these micronutrients, higher percentages of inadequacy were obtained by adjusted 24HR data and the lowest with FFQ. For the remaining nutrients, adjusted data gave the lowest inadequacy percentages. The best concordance was seen between 2-24HR and 1-24HR as well as for adjusted 24HR, with the least observed between FFQ and the other methods. Exclusion of under-reporters considerably reduced inadequacy in both daily methods and FFQ. Crude daily data gave higher estimates of inadequate intakes than adjusted data or FFQ. Reproducibility of daily methods was also reasonably good. Results may differ depending on the micronutrient thus impeding reaching conclusions/recommendations common for all micronutrients.

How to find information on national food and nutrient consumption surveys across Europe: systematic literature review and questionnaires to selected country experts are both good strategies.

The present research was conducted within the framework of the EURopean micronutrient RECommendations Aligned project. In order to identify the best practice in assessing nutrient intakes, a search strategy for collecting data from national food consumption surveys/studies in Europe was developed. Systematic literature searches were carried out on twenty-eight European and the four European Free Trade Association countries. A questionnaire was also sent to two to five experts in each country. Systematic reviews using PubMed yielded 12 703 abstracts that were reduced to 200 studies using inclusion and exclusion criteria. Similarly, a search of ministry web sites yielded 3033 hits, and subsequently reduced to nine surveys. Belgium, France, Germany, Ireland, Sweden, Spain and the United Kingdom were the countries with most data and Slovenia and Liechtenstein were those with the least. Seventy-eight expert questionnaires were obtained from all countries except for Liechtenstein, Luxembourg and Slovakia. Detailed results and references are given. A systematic search and questionnaires are equally good at identifying national surveys across countries. Literature searching provides globally accessible and objective information albeit limited, whereas the questionnaire provides information that, depending upon responders, can be more complete. A combination of both strategies is recommended.

How does the quality of surveys for nutrient intake adequacy assessment compare across Europe? A scoring system to rate the quality of data in such surveys.

Research was conducted within the EURopean micronutrient RECommendations Aligned (EURRECA) Network of Excellence, to find the best practice in assessing nutrient intakes. Objectives include: to search for and use data on individual nutrient intake adequacy (NIA) assessment collected in twenty-eight European countries and the four European Free Trade Association countries; to design and test innovative tools for data quality analysis. The information was obtained using the method described by Blanquer et al. in the present issue. The best-practice criteria were devised to select the most appropriate survey in each country. Then a survey quality scoring system was developed in consultation with experts and tested on these surveys. Weights were allocated according to a variable priority order agreed by consultation. The thirty-two countries yielded twenty-four national surveys (eight countries excluded). Data collection techniques: eleven countries/surveys used personal interviews only; six used combinations of techniques. Dietary assessment methods: two used repeated 24 h recalls only; eleven used combinations. NIA assessment methods: two used probabilistic approach and sd/Z-scores only; eleven used comparison with estimated average requirements/RDA only. Countries were ranked according to the survey quality scoring, but careful interpretation is needed because of incomplete data from some surveys; bearing this in mind, the information quality is high in 37.5 % countries, medium in 50.0 % and low in 12.5 %. Although there is room for improvement and caution should be taken when drawing conclusions and recommendations from these results, the lessons learned and tools developed at this first attempt form the basis for future work within the EURRECA framework for aligning European micronutrient recommendations.

How is the adequacy of micronutrient intake assessed across Europe? A systematic literature review.

EURopean micronutrient RECommendations Aligned is a network of excellence funded by the European commission, and established to address the problem of differences between countries in micronutrient recommendations as well as to understand how nutritional information including requirements and adequacy of intake is processed among different population groups. The aims of the present paper were to review the methods used for the adequacy assessment of the intake of six micronutrients of public health concern (vitamin A, folate, vitamin B12, Fe, Zn and iodine) in non-European and European nutrition surveys carried out on the apparently healthy population and to compare in particular the adequacy across surveys for folate intake. A systematic literature review was conducted to identify nutrition surveys that assessed micronutrient intake adequacy. The search yielded 9049 records, out of which 337 were eligible for the selected micronutrients. The majority (83.9 %) of the European surveys compared the adequacy of the nutrient intake against the Recommended Dietary Allowances (RDA); only a few surveys (8.0 %) used the estimated average requirement cut-point method, while none of them used the probability approach. The comparison of folate inadequacy across eight countries revealed that about 25 % of the adult female population had inadequate intakes when judged against the different recommendations used by the respective investigators, but nearly 75 % had inadequate intakes when compared against the estimated average requirement cut-point value of 320 microg/d. The present review showed that different methods were applied across Europe to estimate the adequacy of micronutrient intake, which led to different prevalence estimates of micronutrient inadequacy.

Overview of methods used to evaluate the adequacy of nutrient intakes for individuals and populations.

The objective of the present paper is to review the methods of measuring micronutrient intake adequacy for individuals and for populations in order to ascertain best practice. A systematic review was conducted to locate studies on the methodological aspects of measuring nutrient adequacy. The results showed that for individuals, qualitative methods (to find probability of adequacy) and quantitative methods (to find confidence of adequacy) have been proposed for micronutrients where there is enough data to set an average nutrient requirement (ANR). If micronutrients do not have ANR, an adequate intake (AI) is often defined and can be used to assess adequacy, provided the distribution of daily intake over a number of days is known. The probability of an individual's intake being excessive can also be compared with the upper level of safe intake and the confidence of this estimate determined in a similar way. At the population level, adequacy can be judged from the ANR using the probability approach or its short cut - the estimated average requirement cut-point method. If the micronutrient does not have an ANR, adequacy cannot be determined from the average intake and must be expressed differently. The upper level of safe intake can be used for populations in a similar way to that of individuals. All of the methodological studies reviewed were from the American continent and all used the methodology described in the Institute of Medicine publications. The present methodology should now be adapted for use in Europe.

Relationship Between Weekly Patterns of Caloric Intake and Reported Weight Loss Outcomes: Retrospective Cohort Study.

BACKGROUND: Although millions of overweight and obese adults use mobile phone apps for weight loss, little is known about the predictors of success. OBJECTIVE: The objective of this study was to understand the relationship between weight loss outcomes and weekly patterns of caloric intake among overweight and obese adults using a mobile phone app for weight loss. METHODS: We examined the relationship between weekly patterns of caloric intake and weight loss outcomes among adults who began using a weight loss app in January 2016 and continued consistent use for at least 5 months (N=7007). Unadjusted and adjusted linear regression analyses were used to evaluate the predictors of percentage of bodyweight lost for women and men separately, including age, body mass index category, weight loss plan, and difference in daily calories consumed on weekend days (Saturday and Sunday) versus Monday. RESULTS: In adjusted linear regression, percentage of bodyweight lost was significantly associated with age (for women), body mass index (for men), weight loss plan, and differences in daily caloric intake on Mondays versus weekend days. Compared with women consuming at least 500 calories more on weekend days than on Mondays, those who consumed 50 to 250 calories more on weekend days or those with balanced consumption (±50 calories) lost 1.64% more and 1.82% more bodyweight, respectively. Women consuming 250 to 500 calories or more than 500 calories more on Mondays than on weekend days lost 1.35% more and 3.58% more bodyweight, respectively. Compared with men consuming at least 500 calories more on weekend days than on Mondays, those consuming 250 to 500 calories or more than 500 calories more on Mondays than on weekend days lost 2.27% and 3.42% less bodyweight, respectively. CONCLUSIONS: Consistent caloric intake on weekend days and Mondays or consuming slightly fewer calories per day on Mondays versus weekend days was associated with more successful weight loss. TRIAL REGISTRATION: ClinicalTrials.gov NCT03136692; https://clinicaltrials.gov/ct2/show/NCT03136692 (Archived by WebCite at http://www.webcitation.org/6y9JvHya4).

The PlantLIBRA consumer survey: Findings on the use of plant food supplements in Italy.

BACKGROUND: Food supplements, and in particular those containing botanicals (plant food supplements, PFS), have in recent decades been of great interest both to consumers and to food/pharmaceutical industries. OBJECTIVES: The aim of this paper is to examine replies by Italian consumers to the PlantLIBRA consumers' survey in order to: 1) assess the behaviour of an Italian population with respect to the use of PFS, and to compare it with that of other 5 countries involved in the whole survey; 2) identify different habits in the 4 Italian cities selected according to their geographical distribution; 3) collect independent information on the actual intake of PFS and consumers' behaviour. SUBJECTS/SETTING: 397 Italian consumers enrolled, 187 males (49.5%) and 191 female (50.5%). The distribution of subjects among the 4 cities included was: Milan 99; Venice 90; Rome 96 and Catania 96. RESULTS: The interest in PFS in Italy is high, the prevalence of "regular" consumers being 22.7%. Some differences were observed between the 4 cities involved: the pattern of use during the year was specific to each city; consumers in Milan reported reasons to use PFS significantly different from those in the whole Italian sample and did not indicate supermarkets as an important place of purchase; respondents from Rome and Catania more frequently used family doctors and pharmacists as a source of recommendation. Some significant difference among cities, sex and age groups were observed when the most frequently used botanicals were ranked. CONCLUSIONS: The results provide new insights on the socio-economic characteristics and lifestyle of Italian PFS consumers, on their reasons for and pattern of use, and on their behaviour and expectations. The value of this information is not restricted to the specific country (Italy) but allows for a more general evaluation of the pattern of use, according to habits and geographical area.

[Trends in the nutritional status of the Spanish population: results from the Catalan nutrition monitoring system (1992-2003)]. / Tendencias del estado nutricional de la población española: resultados del sistema de monitorización nutricional de Cataluña (1992-2003).

BACKGROUND: The purpose of this study was to evaluate the changes in the nutricional habits and nutricional status of the Catalan population over 10 years interval of the two Catalan Nutricional Surveys conducted in 1992-93 and 2002-03. METHODS: 2641 individuals in 1992-93 and 2060 individuals in 2002-03 aged 10 to 75 years participated in the surveys. Two 24 hour recall, a food frequency questionnaire, and a general questionnaire with information on physical activity, knowledge and opinions on nutrition and supplements use were administered. Weight (Kg), height (cm) and waist circumference (WC) (cm) were measured. A subsample of the population underwent a biochemical evaluation. Total cholesterol, HDL cholesterol, LDL cholesterol, tryglicerides and beta-carotene, alpha-tocopherol and retinol were measured. RESULTS: A decrease in the consumption of fruit, vegetables, potatoes, meat and fish and an increase in the consumption of dairy products and fast food (in young individuals) were reported. An increase in the mean value of Body Mass Index (BMI) was observed among males, and an increase in WC mean value was observed in males and females. BMI value decreased in females (except among the younger ones). The prevalence of obesity increased among males (from 9.9% to 16.6%), but not in females. Both total cholesterol and HDL cholesterol decreased. A decrease was observed in the percentage of population with sedentary habits during leisure time. CONCLUSIONS: There is a need for an effective nutrition policy promoting healthy nutrition in accordance with the ongoing dietary guidelines.

Functional classification of grasp strategies used by hemiplegic patients.

This study aimed to identify and qualify grasp-types used by patients with stroke and determine the clinical parameters that could explain the use of each grasp. Thirty-eight patients with chronic stroke-related hemiparesis and a range of motor and functional capacities (17 females and 21 males, aged 25-78), and 10 healthy subjects were included. Four objects were used (tissue packet, teaspoon, bottle and tennis ball). Participants were instructed to "grasp the object as if you are going to use it". Three trials were video-recorded for each object. A total of 456 grasps were analysed and rated using a custom-designed Functional Grasp Scale. Eight grasp-types were identified from the analysis: healthy subjects used Multi-pulpar, Pluri-digital, Lateral-pinch and Palmar grasps (Standard Grasps). Patients used the same grasps with in addition Digito-palmar, Raking, Ulnar and Interdigital grasps (Alternative Grasps). Only patients with a moderate or relatively good functional ability used Standard grasps. The correlation and regression analyses showed this was conditioned by sufficient finger and elbow extensor strength (Pluri-digital grasp); thumb extensor and wrist flexor strength (Lateral pinch) or in forearm supinator strength (Palmar grasp). By contrast, the patients who had severe impairment used Alternative grasps that did not involve the thumb. These strategies likely compensate specific impairments. Regression and correlation analyses suggested that weakness had a greater influence over grasp strategy than spasticity. This would imply that treatment should focus on improving hand strength and control although reducing spasticity may be useful in some cases.

Trends in the association between smoking history and general/central obesity in Catalonia, Spain (1992-2003).

OBJECTIVES: To examine trends in the relationship between smoking history and both general and central fatness in adults from a Mediterranean setting. Materials and methods: The ENCAT 1992-1993 and 2002-2003 surveys were used; samples consisted of 482 men, 589 women from 1992-1993, and 515 men, 613 women from 2002-2003, aged 25-60 years. Measured anthropometry and self-reported data on smoking habits, diet, lifestyle and SES were collected. General fatness was defined as WHO's BMI overweight and obesity, and central fatness was defined as WHO's Increased-Risk-for-metabolic-complications Waist Circumference (IR WC) and Substantially-Increased-Risk WC (SIR WC). Simple logistic regression was used to estimate multivariate-adjusted associations between general/central fatness and smoking history. RESULTS: By 2002-2003, most associations observed in 1992-1993 had been strongly attenuated: only male current-heavy-smoking remained associated with IR/SIR WC (three-fold) and female current-moderate-smokers were 0.57 times less likely to have an IR/SIR WC (p < 0.10). CONCLUSIONS: Although causality cannot be established, results suggest a positive association between heavy smoking and central fatness among men, but no association between former smoking and general/central fatness; findings strengthen arguments for promoting smoking cessation to reduce smoking -and obesity- associated morbidity and mortality.