CRISPR/Cas9-mediated multiplex gene editing of gamma and omega gliadins, paving the way for gliadin-free wheat.

Wheat is a staple cereal in the human diet. Despite its significance, an increasing percentage of the population suffers adverse reactions to wheat, which are triggered by wheat gluten, particularly the gliadin fractions. In this study, we employed CRISPR/Cas multiplexing to introduce targeted mutations into γ- and ω-gliadin genes of wheat, to produce lines deficient in one or both immunogenic gliadin fractions simultaneously. For this work, eight single guide RNAs (sgRNAs) were designed and combined into four plasmids to produce 59 modified wheat lines, of which 20 exhibited mutations in the target genes. Characterization of these lines through Sanger or NGS sequencing revealed a complex pattern of InDels, including deletions spanning multiple sgRNAs. The mutations were transmitted to the offspring, and the analysis of homozygous derived lines by RP-HPLC and monoclonal antibodies showed a 97.7% reduction in gluten content. Crossing these lines with other CRISPR/Cas lines deficient in the α-gliadins allowed multiple mutations to be combined. This work represents an important step forward in the use of CRISPR/Cas to develop gluten-free wheat.

Importance of an Ongoing Nutritional Counselling Intervention on Eating Habits of Newly Diagnosed Children with Celiac Disease.

A strict lifelong gluten-free diet (GFD) is the current treatment for the management of celiac disease (CD). Several studies have demonstrated that without proper dietary assessment, this diet leads to nutritional deficiencies and/or imbalances. The present study aimed to improve the dietary habits of newly diagnosed children with CD through ongoing and face-to-face dietary counseling. Forty-three participants were followed during the first year after CD diagnosis. Dietary data were collected at diagnosis (Vt0), after 3 months on a GFD (Vt3), and after 1 year following a GFD (Vt12). Participants completed a 3-day 24-h food recall, a food frequency questionnaire, and the KIDMED index. After each data collection, participants received dietary assessment and nutritional education. Participants consumed more plant-origin foods after the intervention, with most of them reaching the daily recommendations. Fresh food intake increased and that of ultra-processed foods decreased. Compliance with the Mediterranean diet also improved. Personalized dietary assessment and ongoing follow-up improved the dietary patterns of children recently diagnosed with CD, highlighting the importance of dietitian involvement in the management of CD.

Knowledge Gaps in Gluten-Free Diet Awareness among Patients and Healthcare Professionals: A Call for Enhanced Nutritional Education.

Diet is the only treatment for celiac disease (CeD), and good adherence to a gluten-free diet (GFD) is the only way to ensure complete remission and to prevent complications. Limited education about the disease and a GFD is an attributing factor to inadequate adherence. Thus, our aim was to assess the current knowledge about a GFD and the clinical monitoring of adherence to the diet among CeD people and HCPs. Specific questionnaires were designed and distributed to assess the knowledge of CeD people (Q1 questionnaire) (n = 2437) and to analyze the follow-up of the disease from the perspective of patients (Q2 questionnaire) (n = 1294) and HCPs (Q3 questionnaire) (n = 346). Two-thirds of HCPs specialized in pediatric care, while one-third did so in adult care. In CeD people, general questions regarding food classification and cross-contamination are well understood. When patients have doubts, 51.4% reported using the Internet and social networks. Thus, it is crucial that resources like social media are reliable and provide valuable information. Q3 revealed the lack of time to follow up the diet after diagnosis (48% of HCPs allocate < 15 min), the interest in further training, and the need for a professional specialized in diets within the healthcare system. In conclusion, it is essential to enhance nutritional education to increase awareness of a GFD.

FODMAP Content Like-by-like Comparison in Spanish Gluten-free and Gluten-containing Cereal-based Products.

Gluten-free foods (GF) availability on supermarket shelves is growing and it is expected to continue expanding in the years ahead. These foods have been linked to a lower content of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs), molecules that trigger gastrointestinal symptoms in sensitive persons. In this study, the FODMAP content of 25 cereal-based GF foods in Spain (breakfast cereals, pasta, bread, biscuits, bakery products, and dough and puff pastry) and 25 gluten-containing equivalents (GC) available in the same supermarket were analysed and compared. Lactose, fructose, glucose, sorbitol, mannitol, raffinose, stachyose and fructans were quantified. In a like-by-like analysis, GF foods were found to generally contain fewer FODMAPs than their GC counterparts. The ingredients used in the manufacture of GF cereal-based foods may contribute to this fact. When the individually wrapped size was considered, the proportion of samples classified as high-FODMAPs in GC and GF foods showed a trend towards fewer samples in the GF. However, not all the GF samples were low-FODMAP. Altogether, our findings provide essential information for FODMAP content databases of GF products in Spain.

The Gluten-Free Diet for Celiac Disease: Critical Insights to Better Understand Clinical Outcomes.

The gluten-free diet (GFD) remains a complex paradigm in managing celiac disease (CeD) in children and adults, and there are many reasons why GFD adherence should be strict to improve outcomes. However, this is a challenging task for patients, since they need to have access to quality healthcare resources that facilitate optimal GFD adherence. Understanding the strengths and weaknesses of the GFD, tackling coexisting nutritional deficiencies, and dealing with complex situations, such as seronegative CeD or non-responsive CeD, all require the involvement of a multidisciplinary team. The short- and long-term follow-up of CeD patients should preferably be performed by a combined Gastroenterology and Nutrition service with well-defined quality standards and the multidisciplinary involvement of physicians, nurses, dietitians, and psychologists. Nutritional advice and counseling by an experienced dietitian can reduce the costs associated with long-term follow-up of CeD patients. Likewise, psychological interventions may be essential in specific scenarios where implementing and sustaining a lifelong GFD can cause a significant psychological burden for patients. This manuscript aims to provide guidelines to improve clinical practice in the follow-up and monitoring of CeD patients and provide information on the nutritional risks of an ill-advised GFD. Clinicians, biochemists, food technologists, dietitians, and psychologists with a global view of the disease have been involved in its writing.

Uncovering the Concerns and Needs of Individuals with Celiac Disease: A Cross-Sectional Study.

The treatment for celiac disease (CD) involves a strict gluten-free diet, which can be challenging and lead to questions for patients. Pinpointing these uncertainties can enable the creation of efficient educational resources. In this study, a questionnaire was specifically designed to gain insights into the knowledge, concerns, and needs of individuals with CD and their supporters. The questionnaire was distributed through the Instagram social network and received adequate responses from 300 participants, 258 (86%) being female and 152 (50.7%) falling in the age range of 25-44 years. The concerns of individuals with celiac disease and celiac supporters were rated on a 1-4 scale, with a mean score of 3.5 indicating significant concern. A total of 255 (85%) of all participants expressed that their principal concern was the social limitations they faced, such as difficulties in eating out and sharing food with others. Every participant evaluated their overall disease knowledge, averaging at 2.92 out of 4, indicating a reasonable level of awareness. When asked if they believed that improving general knowledge about CD in the general population would enhance their quality of life, the vast majority responded affirmatively. This finding underscores the importance of not only educating individuals with CD but also reaching out to the wider population, especially those who have a direct impact on the daily lives of individuals with CD, such as family members, friends, and food service providers.

Acute Stress-Induced Changes in the Lipid Composition of Cow's Milk in Healthy and Pathological Animals.

Producers of milk and dairy products have been faced with the challenge of responding to European society's demand for guaranteed animal welfare production. In recent years, measures have been taken to improve animal welfare conditions on farms and evaluation systems have been developed to certify them, such as the Welfare Quality® protocol. Among the markers used for this purpose, acute phase proteins stand out, with haptoglobin being one of the most relevant. However, the diagnostic power of these tools is limited and more sensitive and specific technologies are required to monitor animal health status. Different factors such as diet, stress, and diseases modify the metabolism of the animals, altering the composition of the milk in terms of oligosaccharides, proteins, and lipids. Thus, in order to study oxidative-stress-associated lipids, a collection of well-characterized milk samples, both by veterinary diagnosis and by content of the acute stress biomarker haptoglobin, was analyzed by mass spectrometry and artificial intelligence. Two lipid species (sphingomyelin and phosphatidylcholine) were identified as potential biomarkers of health status in dairy cows. Both lipids allow for the discrimination of milk from sick animals and also milk from those with stress. Moreover, lipidomics revealed specific lipid profiles depending on the origin of the samples and the degree of freedom of the animals on the farm. These data provide evidence for specific lipid changes in stressed animals and open up the possibility that haptoglobin could also affect lipid metabolism in cow's milk.

Gluten-Free Products: Do We Need to Update Our Knowledge?

The gluten-free (GF) food market has been growing exponentially in recent years. However, GF products could contribute to imbalanced diets. The main objective of the present work was to perform a temporal nutritional comparison of GF foods over the last nine years. At the same time, the nutritional compositions of 104 GF products currently marketed in Spain were assessed and compared with their gluten-containing counterparts. Diets including GF products and the equivalent diets with homologous products with gluten were compared as well, the subjects being 25 adult celiac patients. A comparison of GF products (GFPs) in 2013 and 2022 showed nutritional differences in the groups of cookies, breakfast cereals, doughs/puff pastries/pizzas, and breads. The pasta group stands out from the rest due to an important decrease in energy, protein, simple carbohydrates, saturated lipids, dietary fiber, and salt. Comparing GF and gluten-containing homologous products in 2022, the major differences were found in protein and salt. Accordingly, GFPs lead to a diet lower in protein and higher in carbohydrates. Despite improvements in the formulation of GF products in recent years, their macronutrient profile maintains marked variation, and they cannot be considered nutritionally equivalent to their gluten-containing counterparts.

Gluten Assessment in Beers: Comparison by Different Commercial ELISA Kits and Evaluation of NIR Analysis as a Complementary Technique.

Traditionally, beers are made with gluten-containing cereals. It is crucial to have rapid analytical methodologies that allow gluten content control of the beers for celiac consumers. We assess the content of gluten in 65 conventional and 41 gluten-free labeled beers commercialized in Europe and compare the results in a subgroup of 71 beers with three ELISA kits. This research allows gathering information on the potential complementary utility of NIR analysis applied to gluten analysis of gluten-free beers in terms of time saving. Results obtained with the ELISA technique identified competitive R5 to be the most sensitive in detecting the prolamins, by eliciting a higher number of beers containing gluten above 20 mg/kg. The gluten content in conventional beers tested increased with the presence of wheat as raw material and with the use of ale-type yeasts. By using competitive R5, 3 out of the 41 gluten-free labeled beers appeared to contain gluten above 20 mg/kg, and conversely, 15 out of 65 of the conventional beers showed a gluten content below this threshold. According to our approaches, NIR did not achieve a suitable correlation with ELISA results, neither for gluten quantification nor for discrimination, and therefore, it cannot be proposed as a complementary technique.

Gluten and FODMAPs Relationship with Mental Disorders: Systematic Review.

Nowadays, gluten and FODMAP food components (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) are increasingly studied due to their possible relation with extraintestinal-associated conditions. In recent years, gluten-free diets (GFD) and low-FODMAP diets (LFD) are becoming more popular not only in order to avoid the food components that cause intolerances or allergies in some people, but also due to the direct influence of marketing movements or diet trends on feeding habits. Likewise, neurological and psychiatric diseases are currently of increasing importance in developed countries. For this reason, a bibliographic systematic review has been carried out to analyse whether there is a pathophysiological relationship between the dietary intake of gluten or FODMAPs with mental disorders. This review collects 13 clinical and randomized controlled trials, based on the PRISMA statement, which have been published in the last ten years. Based on these results, limiting or ruling out gluten or FODMAPs in the diet might be beneficial for symptoms such as depression, anxiety (7 out of 7 articles found any positive effect), or cognition deficiency (improvements in several cognition test measurements in one trial), and to a lesser extent for schizophrenia and the autism spectrum. Nevertheless, further studies are needed to obtain completely reliable conclusions.

FODMAP Intake in Spanish Population: Open Approach for Risk Assessment.

Fermentable oligo-, di- and monosaccharides and polyols' (FODMAP) were related with intestinal complications. The present study aimed to determine the FODMAP consumption of Spanish children, adolescents and adults, analyzing the real FODMAP risk of foods, and to set an open methodology for the measurement of this intake in other regions as well as nutrient intake assurance. Total fructan analysis was performed analytically in eighty-seven food samples. Daily intake of FODMAPs, fiber and micronutrients was calculated by combining the food composition for selected fermentable carbohydrates with the national food consumption stratified by age in an open software. Spanish child and adolescent total FODMAP consumption was settled as 33.4 ± 92.4 and 27.3 ± 69.0 g/day, respectively. Both intakes were higher than that of the adult population (21.4 ± 56.7 g/day). The most important food sources of lactose, excess of fructose and total fructan, considering their content and dietary intake were different between age groups. The contribution of these foods to dietary calcium and fiber and the consequent risk of deficiency if they are withdrawn was highlighted. We demonstrated the relevance of stratifying the total FODMAP intake by age. An open approach for FODMAP intake quantification and nutrient control was provided.

Effect of analytically measured fiber and resistant starch from gluten-free products on the diets of individuals with celiac disease.

OBJECTIVE: The aim of the present work was to assess the content of fiber and resistant starch (RS) in several gluten-free products (GFPs), compare them with their gluten-containing analogs (GCP) analytically and with label data, and measure the contribution of these foodstuffs to the fiber content of a gluten-free diet. METHODS: Total dietary fiber and RS content in a large amount of GFP (n = 55) and their counterparts (n = 55) were measured by analytical techniques based on AOAC methods. Dietary assessment was carried out with a sample of 141 individuals (pediatric and adult patients with celiac disease) submitted to a food frequency questionnaire and a 24-h recall survey (three times on different days). RESULTS: In general terms, GFP and GCP showed similar fiber and RS content, with the exception of breads, which revealed a higher percentage of both in GFPs. This food group was the main contributor to fiber intake in both groups of patients. Even if GFPs were to be blamed for being nutritionally poorer than GCPs, this was not the case for fiber. CONCLUSIONS: The fiber and RS content were not different in the studied samples, except for the bread. It is noteworthy that label information can underestimate fiber consumption among those with celiac disease, and this must be considered for future database revision. Additionally, we demonstrated a significant effect on the fiber intake of children and adults due to GFPs, and especially to bread, a widely consumed basic product.

New Software for Gluten-Free Diet Evaluation and Nutritional Education.

Following a gluten-free diet (GFD) is the only treatment for celiac disease. This diet must ensure the absence of gluten but also needs to be nutritionally balanced. Dietitians working in this field cannot properly evaluate energy and nutrient intake of celiac people because dietary programs available on the market do not contain the nutritional composition of gluten-free products (GFP). Here we present a new GFD evaluation software that contains more than 700 gluten-free rendered foodstuffs and their macronutrient composition. Apart from diet evaluation and design, the software represents a tool for nutritional education as well, since it shows diet appropriacy and indicates how to promote balanced self-care. Moreover, anthropometric and biochemical data or symptoms presence and diet adherence can be recorded and evaluated. This open free software, can be downloaded in its app format for mobiles and tablets. Software evaluation indicated its correct functionality and the importance of assessing a GFD with GFP instead of with their gluten-containing analogues. Thus, this software represents an essential e-Health tool, not only for proper GFD evaluation, but also for improving life quality of celiac and gluten sensitive people.

Micronutrient Analysis of Gluten-Free Products: Their Low Content Is Not Involved in Gluten-Free Diet Imbalance in a Cohort of Celiac Children and Adolescent.

Data about the nutritional composition of gluten-free products (GFP) are still limited. Most studies are based on ingredient and nutrition information described on the food label. However, analytical determination is considered the gold standard for compositional analysis of food. Micronutrient analytical content differences were observed in a selection of GF breads, flakes and pasta, when compared with their respective gluten-containing counterparts. In general terms, lower iron, piridoxin, riboflavin, thiamin, niacin, folate, manganese and vitamin B5 can be underlined. Variations in biotin and vitamin E content differed among groups. In order to clarify the potential contribution of the GFP to the gluten-free diet's (GFD) micronutrient shortages, analytical data were used to evaluate GFD in a cohort of celiac children and adolescent. Participants did not reach recommendations for vitamin A, vitamin E, folic acid, vitamin D, biotin, iodine, and copper. It does not seem that the lower micronutrient content of the analyzed GFP groups contributed to the micronutrient deficits detected in GFD in this cohort, whose diet was not balanced. Nevertheless, GFP fortification for folate and biotin is proposed to prevent the deficiencies observed in GFD, at least in the case of pediatric celiac disease.

Consideraciones analíticas en la determinación de gluten en alimentos / No disponible

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Celiac Male's Gluten-Free Diet Profile: Comparison to that of the Control Population and Celiac Women.

The aim of the present work was to analyze the body composition and dietary profile of Spanish celiac men and to compare them to control men and celiac women from our previous studies. Forty-two celiac men (31.5 ± 11.9 years) were recruited and anthropometric measurements were taken. Analysis of energy consumption, macro- and micronutrient intake and food frequency consumption was carried out. Celiac men were more overweight and obese than celiac women, but less than the control population, reporting the same energy intake and macronutrient distribution. Most micronutrient deficiencies in celiac men were not directly related to a gluten free diet; these were also observed for the entire population. The least adherence to Dietary Reference Intakes in women was reported for iron, iodine, potassium and selenium, whereas magnesium intake was higher than in men. Among celiac participants (both genders), cereal, vegetable and legume consumption was poor and meat intake was contrastingly excessive. In conclusion, the dietary profile of celiac men is as unbalanced as that of control men but slightly more than that of celiac women. General nutritional education should be given to both general and celiac populations, and specific advices to celiac men, in order to decrease the risk of celiac disease-related pathologies.

Evolution of Gluten Content in Cereal-Based Gluten-Free Products: An Overview from 1998 to 2016.

The treatment of Celiac disease consists in a strict lifelong gluten-free (GF) diet. As the ingestion of small amounts can have damaging complications, there has been an ongoing discussion regarding the safe threshold for dietary residual gluten. The aim was to analyze the evolution of gluten content in cereal-based GF foodstuffs (n = 3141) from 1998 to 2016 measured by the enzyme-linked immunosorbent assay (ELISA) technique. Eight categories were defined: flours, breakfast cereals/bars, bakery, pasta, breads, dough, snacks, and yeasts, and these were divided into GF labeled-foods (GF-L) or reportedly GF foodstuffs, but not certified (GF-NC). Gluten-detection was decreased over time in line with the evolving European regulations about food information and gluten content claims. This decline started sooner in GF-L products than in GF-NC. As a whole, gluten was detected in 371 samples, with breakfast cereals/bars being the most contaminated group. Snacks and yeasts changed from being high gluten-detected samples to being totally GF over the years. The downside is that, of contaminated samples, those in the low levels of gluten detection range have decreased while flour samples containing over 100 mg/kg gluten have risen in the 2013-2016 period. Obtained data confirm that GF cereal-based foods are becoming safer but gluten control must be maintained.

Analysis of Body Composition and Food Habits of Spanish Celiac Women.

The purpose of the present work was both to analyze composition of Spanish celiac women and to study the food habits and gluten-free diet of these celiac patients, in order to determine whether they achieve a balanced and healthy diet as well as to highlight nutritional qualitative and/or quantitative differences. 54 adult celiac women (34 ± 13 years) took part in the six-month study. Height, weight and body composition were measured. An analysis of energy consumption and of the macronutrient distribution of their diet was carried out. Their fulfillment of micronutrient intake recommendations was verified. Participants showed a Body Mass Index of 21.6 ± 2.4 kg/m2. Energy Intake was slightly lower than the Dietary Reference Intakes. Excessive protein apart from over-consumption of fat was observed. More than three quarters of participants consumed meat in excess. Carbohydrate consumption along with that of fiber was below recommended levels. Vitamin D, iron, and iodine had a low percentage of recommendation compliance. In general, participants followed the recommendations of dairy products and fruit intake whereas vegetable consumption was not enough for the vast majority. We conclude that although the diet of celiac women does not differ much from the diet of general population, some considerations, such as reducing fat and protein consumption and increasing fiber intake, must be taken into account.

Resveratrol regulates lipolysis via adipose triglyceride lipase.

Resveratrol has been reported to increase adrenaline-induced lipolysis in 3T3-L1 adipocytes. The general aim of the present work was to gain more insight concerning the effects of trans-resveratrol on lipid mobilization. The specific purpose was to assess the involvement of the two main lipases: adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in the activation of lipolysis induced by this molecule. For lipolysis experiments, 3T3-L1 and human SGBS adipocytes as well as adipose tissue from wild-type, ATGL knockout and HSL knockout mice were used. Moreover, gene and protein expressions of these lipases were analyzed. Resveratrol-induced free fatty acids release but not glycerol release in 3T3-L1 under basal and isoproterenol-stimulating conditions and under isoproterenol-stimulating conditions in SGBS adipocytes. When HSL was blocked by compound 76-0079, free fatty acid release was still induced by resveratrol. By contrast, in the presence of the compound C, an inhibitor of adenosine monophosphate-activated protein kinase, resveratrol effect was totally blunted. Resveratrol increased ATGL gene and protein expressions, an effect that was not observed for HSL. Resveratrol increased fatty acids release in epididymal adipose tissue from wild-type and HSL knockout mice but not in that adipose tissue from ATGL knockout mice. Taking as a whole, the present results provide novel evidence that resveratrol regulates lipolytic activity in human and murine adipocytes, as well as in white adipose tissue from mice, acting mainly on ATGL at transcriptional and posttranscriptional levels. Enzyme activation seems to be induced via adenosine monophosphate-activated protein kinase.

The effect of birth weight on low-energy diet-induced changes in body composition and substrate-energy metabolism in obese women.

OBJECTIVE: The objective of this study was to examine the association of birth weight (BW) with body composition, resting metabolic rate (RMR), nonprotein respiratory quotient (NPRQ), and insulin sensitivity changes after a 12-week diet intervention program. METHODS: A total of 78 obese (body mass index [BMI] 34.0 ± 2.8) women aged 36.7 ± 7 years volunteered to participate in a 12-week diet intervention program. We assessed body fat mass (FM), lean mass (LM), and bone mass (BM; measured by dual energy x-ray absorptiometry), RMR (measured by indirect calorimetry), fasting plasma glucose, and insulin before and after the intervention. We calculated BMI, FM to LM ratio (FM/LM), and HOMA-IR. BW and gestational age were self-reported, and the BW Z-score was calculated. RESULTS: At baseline, the BW Z-score was positively associated with LM (p < 0.01) and RMR (p < 0.05). The BW Z-score was significantly associated with diet-induced FM (p < 0.05) and FM/LM ratio changes (p < 0.01) independently of potential confounders including weight loss. The BW Z-score was not associated with diet-induced RMR or insulin resistance changes. CONCLUSIONS: Lower birth weight is associated with lower LM and RMR in obese women and could program a lower FM loss achievement after an energy-restriction diet intervention.