This review presents the primary applications of various proteomic strategies to evaluate the impact of farming conditions on food quality and safety in aquaculture products. Aquaculture is a quickly growing sector that represents 47% of total fish production. Food quality, dietary management, fish welfare, the stress response, food safety, and antibiotic resistance, which are covered by this review, are among the primary topics in which proteomic techniques and strategies are being successfully applied. The review concludes by outlining future directions and potential perspectives. Aquaculture is the breeding of aquatic organisms under controlled conditions, involving both marine and freshwater fish along with algae, crustaceans, and mollusks. According to the Food and Agriculture Organization of the United Nations (FAO), this food sector represents a significant source of nutrients for the human diet and produces approximately 97.2 million tons of fish annually, which represents 47% of the global fish production . With niThe globalization of aquaculture markets presents important nutritional and economic benefits but also poses potential risks for food safety, such as the fraudulent substitution of fish species and the presence of food microorganisms, viruses, parasites, and vectors of their corresponding foodborne diseases . MoreoveProteomics is the high-throughput analysis of the proteins of a specific biological sample . ProteomProteomics has the potential to provide information useful to improve the production, welfare, health, nutritional value and wholesomeness of farmed fish. Discovery proteomics aims at identifying biological markers in a given proteome, frequently employing a bottom-up approach, in which the proteins of the sample are separated, proteolyzed with enzymes such as trypsin or Glu-C and the peptides obtained are subsequently analyzed by tandem mass spectrometry (MS/MS). Two-dimensional gel electrophoresis (2-DE) has traditionally been the technique selected for the separation of proteins samples . This ge18O) [https://blast.ncbi.nlm.nih.gov/), it is possible to select relevant peptide biomarkers to be used in the subsequent phase namely, targeted proteomics.In gel-free approaches, also known as shotgun proteomics, the proteins are directly digested in the extract with a selected enzyme, and the obtained mixture of peptides is subsequently analyzed by liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) ,16. It i18O) ; and lab18O) . After mm/z), represents a common analysis for detecting and identifying peptide biomarkers. These techniques are selective, sensitive, highly reproducible, with a high dynamic range and an excellent signal-to-noise (S/N) ratio [13C- or 15N-labeled absolute quantification peptide standards (AQUA) or concatemer of standard peptides (QCAT) can also be introduced to the sample as internal standards for absolute quantification of the proteins [Targeted proteomics refers to the monitoring of the relevant peptide biomarkers and it has become a recognized methodology to detect selected proteins with significant accuracy, reproducibility, and sensitivity . In targN) ratio . SRM/MRMN) ratio ,36,37. TN) ratio , can ideproteins . Programproteins ,41. The Fish farming environments and conditions are very different from the conditions in which fish live in nature. Farmed fish, for instance, do not need to actively swim to catch their prey or escape predators; therefore, they exercise less, which impacts on their muscle growth and phenotype. The heavily processed feed consumed by farmed fish differs considerably from their natural diet, which affects muscle metabolism and biochemical composition. In addition, the farming conditions in aquaculture may not be optimized regarding stocking densities, incidence of parasites and diseases, and establishment of hierarchies due to competition for space or feed, all of which have consequences for the wellbeing and development of abnormal behavior. All these variables exert a strong influence on the yield, quality and wholesomeness of farmed seafood. Moreover, development of analytical methods to ensure that fish was farmed minimizing stressful factors is also of high relevance to satisfy consumer demands and labeling on the welfare of fish to be used for food. To investigate all these topics, powerful proteomic methodologies (discovery and targeted proteomics) may have a considerable impact on the understanding of current aquaculture practices in several major areas: (i) dietary management, (ii) fish welfare and response to stress, (iii) food safety, and (iv) antibiotic resistance .Dietary management in aquaculture attempts to improve growth performance, health and immune status in living aquaculture organisms. Numerous works have shown that the composition of the feed influences the composition of fish fillet. Initial works using 2-DE revealed differences between the proteomes of skeletal muscle samples from wild and farmed fish. Carpene et al. , found dOptimization of fish diets has been a priority in the aquaculture sector for many years . One ingReduction of the use of fish meal and oil in aquaculture is a priority , which hUsing analytical techniques based on microfluidic electrophoresis and sequencing, some authors have consistently confirmed differences in the protein abundance and/or regulation in fish muscles depending on the production method. Monti et al. , using SThe effects of stress on growth have been studied extensively in animal production and aquaculture . DiffereFarming itself affects the levels of stress the fish suffer, their growth, and the biochemical composition of different tissues and organs, including the liver, brain, and muscle. For instance, exercising in salmonids lowers the levels of aggression and the building up of hierarchies, leading to increased fish welfare and growth ,59,60. IOne study on sea bream, however, was not able to show consistent differences between the 2-DE patterns of muscle from two natural repopulation lagoons and those of fish from four offshore mariculture plants in Italy by 2-DE, MALDI-MS and LC-ESI-Q-TOF-MS . The simDiscovery proteomics has been applied to improve our understanding of the mechanisms implicated in skeletal deformities . AnalysiThe Pacific geoduck clam is one of the species whose farming is seeing a successful bloom and, consequently, a species under study to improve its production . ProteomAquaculture has the capacity to provide food for millions of people over the world, however inappropriate facility management may severely damage aquatic ecosystems and affect health risks to consumers through contamination with environmental or human-made hazards. Moreover, several pathogenic microorganisms can be found in the aquatic environment with the potential to negatively affect, not only aquatic life, but also human health. In the last decade, the risk of dissemination of infectious or toxic agents and the occurrence of disease outbreaks has risen mainly due to increases in the following factors: (i) intake of raw or scarcely processed seafood; (ii) international trade of aquaculture products; (iii) suboptimal monitoring methodologies; (iv) alterations in ecological stability; and (v) contamination and climatic change . Food saTo make the reading of this review more comprehensible, the authors have decided to divide the hazards to which consumers of aquaculture products are exposed into two general groups: biotic hazards and abiotic hazards.Foodborne poisonings are a relevant cause of mortality and morbidity which result from drinking water or eating food contaminated with such pathogens as viruses, bacteria and parasites, and their toxins.Aeromonas spp., Clostribuim botulinum, Listeria monocytogenes, Vibrio cholerae, and Vibrio parahaemolyticus) and those derived from environmental contamination  [Staphylococcus aureus can infect aquaculture species during management due to inadequate hygiene conducts of operators in the processing factories [Regarding biotic hazards, there are two main groups of bacteria that affect food products of aquaculture: those naturally present in its habitat (la spp.) . Additioactories ,78.Vibrio spp., a Gram-negative bacteria causative of gastrointestinal diseases in humans after ingestion of poorly cooked infected seafood, such as seabream and mollusks [Ctenopharyngodon idella) against Vibrio mimicus [Proteomics has been applied to the detection and identification of bacterial species from aquaculture products both after their direct detection in fish products, or after their isolation and growing in different culture media. For instance, MALDI-TOF-MS techniques enabled us to achieve mass spectral fingerprints of mollusks ,80. The  mimicus . Vaccina mimicus . Recentl mimicus .Aeromonas veronii. This bacterium, a Gram-negative virulent pathogen associated with infections in freshwater fish species and mammals, is capable of adhering to biotic and abiotic surfaces surrounded by the extracellular matrix produced by the resident microorganisms. The study, which used an in vitro biofilm model [A. veronii to create biofilms and its increased dissemination.Targeted proteomics applications in the field of pathogenic bacteria have increased substantially in recent years. For example, both SRM and PRM have produced sensitive quantitative results about the proteins associated with bacterial infection, particularly in the fields of clinical diagnosis and antibiotic resistance . Thus, ilm model , showed Ichthyophthirius multifiliis, cause important economic losses to the aquaculture sector. Upon exposure to the parasite, LC-ESI-MS/MS revealed the differential regulation of some immune-related signal transduction proteins in the skin mucus of common carp [Protozoan parasites, such as mon carp . MultiplVirus infections can decimate the production in fish and shrimp farms. Among the latter, white spot syndrome virus is currently one of the most serious global hazards. A protein interactomics map for the white spot syndrome virus has been produced by means of a co-immunoprecipitation assay (co-IP) from a yeast two-hybrid approach .Alexandrium tamarense [Alexandrium minutum as described by Carrera et al. (2010) [Harmful algal blooms (HABs) generate shellfish poisoning toxins that affect aquaculture, particularly mussel farming. Gel-based proteomic approaches were used to distinguish and identify nontoxic dinoflagellates from the toxic dinoflagellate amarense . An alte. (2010) . Recent . (2010) . In that. (2010)  reviewedProteomic studies and systems biology analysis of allergenic proteins have also been critical determinants for the evaluation of the quality and safety of wild and cultivated fish and crustacean food products . The majMytilus galloprovincialis by 2-DE and MS [Crassostrea angulata after HgCl2 contamination [Abiotic hazards in aquaculture have been extensively studied in mollusks exposed to contaminants in polluted areas. Discovery proteomics studies have been mainly performed for environmental assessment and marine pollution monitoring using the digestive glands of mussels E and MS . Bottom-E and MS . Moreovemination . The firmination .Gammarus fossarum) after in situ exposure to several aquatic environments [To assess complex field contamination, targeted proteomics using SRM methodologies was applied to the quantification of dozens of protein biomarkers in caged amphipods (ronments . The worronments .Pseudomonas spp. more strongly than did the nanoparticulate form of Ag in a bacterium isolated from waters in a region where fish is farmed for human consumption [+) have been predicted to reach EU freshwaters annually, mostly due to leaching of ionic AgNPs from biocidal plastics and textiles. Proteomic analysis using SWATH-MS allowed the identification of 166 proteins affected by exposure to the nanoparticulate form of Ag, which also impacted on the growth of Pseudomonas spp. The form of Ag induced different adaptive responses in the metabolic, stress, and energetic pathways in Pseudomonas spp., and proteins affected were transmembrane transporters, chaperones, and proteins related to the metabolism of carbohydrates and proteins, indicating their potential value as biomarkers of the stress induced by Ag+ and/or AgNPs. Among all the modified proteins, 59 had their content significantly changed by one or both forms of silver. In view of the evidences obtained in these studies, we believe that nanoparticle pollution should be considered an emergent hazard in waters with aquaculture production.Nanoparticle pollution is a recent issue of concern that has also been addressed by proteomic techniques. Thus, targeted proteomics has shown that the ionic form of Ag impacted on the growth of sumption . PossessAntibiotics are natural and synthetic compounds that kill bacteria and have been heavily used and abused in aquaculture for over 50 years  not onlyThe abuse in antibiotic treatments has provoked the development and spreading of bacterial resistance and the appearance and expansion of multidrug-resistant strains in such a way that the aquatic environment has become an important reservoir of antibiotic resistance genes/proteins (ARG/Ps) and a route for their dissemination and potential transmission to human pathogens. Until now, five main mechanisms of antibiotic resistance have been accurately recorded due to the related development of resistance to drugs that (a) deteriorate enzymes, (b) bypass target pathways, (c) change antibiotic focus sites, (d) alter the penetrability of porins, and/or (e) trigger flow systems .Proteomics techniques have the potential to significantly contribute to increase the knowledge about molecular mechanisms related to antibiotic resistance . In the Aeromonas spp., because this pathogen is responsible of hemorrhagic septicemia and hemolytic diseases in aquaculture, which cause large financial losses to farmers. Some of these studies are described below.Proteomic studies on antibiotic resistance of fish- and shellfish-borne bacteria have largely been performed on Aeromonas hydrophila  has been confirmed by different proteomics analyses. Comparison between the fitness and acquired resistance to CTC in an Aeromonas hydrophila biofilm by TMT-labeling-based quantitative proteomics indicated an increase in translation-related ribosomal proteins in both cases and an increase in proteins involved in fatty acid biosynthesis only in biofilm fitness, while proteins involved in other pathways were less abundant in acquired resistance biofilm. Targeting the up-regulation of fatty acid biosynthesis, the authors found that a mixture of CTC and triclosan (a fatty-acid-biosynthesis inhibitor) had a more powerful antimicrobial effect than either one of them alone. This information is highly relevant in the fight against this pathogen when forming biofilms, which are always a challenge in seafood farming and processing [Tetracyclines are commonly used antibiotics comprising the monocyclines group, doxycycline, and chlortetracycline (CTC), and they are very efficient against both gram-positive and gram-negative microbes. In aquaculture, tetracycline resistant ocessing .A. hydrophila [Two different quantitative proteomic studies, using dimethyl labeling and label-free methods, performed on the same year as the previous work, were conducted to examine the differential regulation of proteins in response to several doses of oxytetracycline (OXY) in drophila . The resA. hydrophila and in control strains [A. hydrophila. In addition, Elbehiry and colleagues described the application of MALDI-TOF MS for the discrimination of the Aeromonas genus from meat and water samples, with a spotlight on the antimicrobial resistance of A. hydrophila [In 2018, Li et al. published a quantitative proteomics experiment based on iTRAQ methodology, to compare proteins differentially regulated in CTC-resistant  strains . The majdrophila .A. hydrophila strains to find differences in the regulation of proteins [Edwardsiella, a gram-negative microorganism that also generates hemorrhagic septicemia in a broad number of cultivated fish species, including yellowtail carp and eels. As in the case of Aeromonas, the antibiotic resistance status of Edwardsiella tarda is of high relevance for seafood safety, particularly when the bacterium is forming biofilms. Sun and coworkers, used iTRAQ-based quantitative proteomics and high-resolution LC-MS/MS to analyze the differential protein regulation of E. tarda in response to OXY stress in biofilms [A. hydrophila in biofilms, many ribosomal proteins were upregulated in response to the stress in E. tarda, while treatment with OXY increased the amount of Uvr C, a member of the UvrABC system that plays an important role in multiple antibiotic resistance processes.Recently, another proteomic study using 2-DE and MALDI-TOF/TOF analysis was conducted by Zhu and coworkers with multidrug resistant (MDR) and sensitive proteins . The worbiofilms . Their wEdwardsiella piscicida and showed that a depressed P cycle seemed to be a characteristic of the differential proteome in the LTB4-RAMP [E. piscicida.iTRAQ and LC-MS/MS were used in conjunction to evidence the differential proteome of the ampicillin-resistant LTB4 (LTB4-RAMP) strain of the gram-negative facultative aerobic bacteria TB4-RAMP  strain, The above research works in aquatic organisms, using iTRAQ technologies, seem to indicate that the acquisition of antibiotic resistance involves chemotaxis, energy metabolism, biofilm characteristics and external membrane proteins, as well as networks of proteins associated to antibiotic resistance.Finally, “reprogramming proteomics” needs to be developed in a general manner to revert an antibiotic-resistance proteome to an antibiotic-sensitive proteome for the control of antibiotic-resistant pathogens .As presented in this review, proteomic approaches help to characterize some of the principal issues associated to farming conditions and to address some of the main challenges in aquaculture, such as dietary management, fish welfare, stress responses, food safety and antibiotic resistance.Proteomics helps to elucidate how dietary management in the aquaculture sector influences the production, growth, immunity and wellness/welfare of living aquaculture organisms and assists in the selection of optimal diets. A large number of publications have shown that the composition of the feed influences the fish muscle nutritional value and its proteome. Efforts have been made to discover protein markers for such quality traits. In addition, various proteomics investigations have been published on the identification of robust protein signatures for fish chronic stress. From this perspective, amelioration of aquaculture conditions to reduce chronic stress during farming and maintain fish welfare is one of the principal issues that can be addressed with discovery proteomics. Additionally, innovative fast targeted proteomics workflows have demonstrated the rapid detection of fish allergens, parasites and microorganisms in aquaculture. The characterization of species-specific peptides by MS/MS-based proteomics and their monitoring by targeted proteomics demonstrated the adequacy of these approaches for food safety control, enabling the differential detection of several hazards in the aquaculture sector. In this way, the utilization of rapid sample preparation methods, combined with sensitive and accurate MS for both the discovery and targeting of fish quality and safety biomarkers, may enhance quality control and safety in aquaculture. Moreover, proteomics offers a more holistic point of view on the molecular mechanisms of antibiotic resistance in the aquaculture sector and it can be directly linked to the metagenomic/metaproteomic approaches that are being applied to the study of a new concept known as the resistome, a current challenge of high relevance that needs an effective and rapid response and that may be elucidated through proteomics techniques.As the proteins are considered the principal functional macromolecules in all biological systems, we consider that proteomics strategies and their associated techniques can offer several advantages compared with other methodologies for the study of the impact of farming conditions on food quality and safety in aquaculture products. This is the case primarily because, with those methodologies, it is possible to identify and directly quantify protein/peptide signatures without the necessity of inferring conclusions based on other approaches such as genomics tools. Secondly, the benefits of proteomic analysis may be adapted for fish products with short shelf-life. Finally, the current advances in proteomic methodologies allow for the implementation of precise methods that may be useful for routine control test with a potentially lower cost and in a relatively short estimated time (<30 min).Lastly, the development and practical implementation of new advances based on protein arrays, microfluidics, and biosensors to the aquaculture sector offers a promising research area in which the results of proteomic studies can be established for the routine control test and diagnosis of fish products. We also assume that the digitalization of these new devices may be relevant to the aquaculture industry and control authorities in the next several years and may supply rapid monitoring information to effectively drive decision enforcing by the industry and authorities.


We determined the nutritional adequacy and overall quality of the diets of adult patients with eosinophilic esophagitis (EoE). Dietary intakes stratified by sex and age were compared to Dietary Reference Values (DRV). Overall diet quality was assessed by two independent Diet-Quality-Indices scores, the PANDiet and DHD-index, and compared to age- and gender-matched subjects from the general population. Lastly, food and nutrient intakes of EoE patients were compared to intakes of the general population. Saturated fat intake was significantly higher and dietary fiber intake significantly lower than the DRV in both males and females. In males, the DRV were not reached for potassium, magnesium, selenium, and vitamins A and D. In females, the DRV were not reached for iron, sodium, potassium, selenium, and vitamins A, B2, C and D. EoE patients had a significantly lower PANDiet and DHD-index compared to the general population, although the relative intake  of vegetables/fruits/olives was significantly higher (yet still up to 65% below the recommended daily amounts) and alcohol intake was significantly lower compared to the general Dutch population. In conclusion, the composition of the habitual diet of adult EoE patients has several pro-inflammatory and thus unfavorable immunomodulatory properties, just as the general Dutch population, and EoE patients had lower overall diet quality scores than the general population. Due to the observational character of this study, further research is needed to explore whether this contributes to the development and progression of EoE. Eosinophilic esophagitis (EoE) is a chronic, immune-mediated condition of the esophagus affecting both adults and children ,2. AlthoBesides the fact that food allergens can trigger an allergic reaction in EoE, in a previous study it was hypothesized that healthy and immune-enhancing nutrition may either be protective against the development of EoE or, in contrast, unhealthy nutrition may facilitate the development of food allergy through its impact on inflammation and esophageal mucosal integrity . HoweverTo further study the nutritional quality of the habitual diet in EoE, the present study aims were threefold: (1) to compare the nutrient intakes of Dutch adult patients with EoE with Dutch Dietary Reference Values (DRV) by age and sex; (2) to compare the overall diet quality of EoE patients with diet quality of the general Dutch population by using Diet Quality Indices (DQI), and (3) to compare the food and nutrient intakes of EoE patients with intakes of the general Dutch population. The study was performed prior to any elimination diets other than self-imposed dietary measures. DQI provide a single score for a combination of foods and/or nutrients, based on current nutrition knowledge established in food or nutrient based dietary guidelines ,17,18. SAdult patients diagnosed with active EoE (≥15 eosinophils/high power field (HPF) and symptoms of esophageal dysfunction), who participated in two trials  performed in the Academic Medical Center, Amsterdam, the Netherlands, were included between 2013 and 2015. Patients participating in Trial NTR4052 followed an allergen-microarray-guided dietary intervention as treatment of EoE . PatientAll patients completed a 3-day non-weighed food diary (2 working days and 1 weekend day) as described previously (online supporting information in ). In shoThe 3-day mean habitual intake of nutrients, stratified for age group and gender, was compared to the Dutch DRV for males and females ,25,26.For each patient, two DQIs were calculated from the dietary data, i.e., the Probability of Adequate Nutrient intake (PANDiet)  and the The PANDiet is a nutrient-based DQI which measures the adequacy of intake of 25 macro- and micronutrients in comparison to nutritional recommendations: protein, total fat, saturated fatty acids (SFA), linoleic acid (LA), α-linolenic acid (ALA), the sum of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), total carbohydrate, dietary fiber, vitamins A, B1, B2, B6, B12, C, D, E, calcium, copper, iodine, iron, magnesium, potassium, selenium, sodium, and zinc . FurtherThe DHD-index is a DQI that ranks participants according to their adherence to food based dietary guidelines, i.e., in the Netherlands, the Dutch Guidelines for a Healthy Diet of 2006 . These gn = 1114) and females 18 to 50 years ) ..23].In EoE is an emerging chronic disease, affecting individuals at any age with a predominance for Caucasian males under the age of 50 . To our In the current study, we showed that for several nutrients, the intake of adult EoE patients did not meet the DRV. Among the nutrients that were statistically significantly different from the DRV, there were several nutrients known for their beneficial or adverse effect on the microbiome, immune system, inflammation or mucosal integrity. For example, the high intake of (saturated) fat in EoE patients is likely to induce a shift in microbiome composition associated with inflammatory processes ,33. In ap = 0.009). However, higher vitamin D levels correlated with higher histologic eosinophil counts .Nutritional deficiencies might gain relevance in EoE. There are a few studies on nutritional deficiencies in EoE patients, predominantly in children . In thisp < 0.05). Moreover, EoE patients had lower intake of omega-3 fatty acids. These differences all point towards a diet which has higher pro-inflammatory, lower anti-inflammatory and unfavorable immunomodulatory properties, and overall seems less healthy. Although a significant percentage of EoE patients avoided fruit/vegetables due to pollen-food syndrome, food allergy or EoE symptoms, EoE patients still had a higher relative intake of well-tolerated fruits and vegetables  compared to the general Dutch population. However, fruit and vegetable consumption in both groups was far below (up to 65%) the recommended daily amounts. Remarkably, male EoE patients used less alcohol than male subjects from the general Dutch population. This may be due to the pain and discomfort caused by alcohol in EoE.When comparing the diet composition of our study population to intake levels of the general Dutch population, male EoE patients had significantly lower energy intakes. After correction for energy intake, we found that intakes of several of the nutrients were different between EoE patients and the general Dutch population. EoE patients  had higher total fat intakes and lower intakes of vitamins C and D . The difference in DHD-index score might be explained by differences in the intake of fatty acids. In EoE patients, the intake of total fat was higher, and the intake of omega-3 fatty acids was lower than in the general Dutch population, which led to a lower DHD-index in EoE patients. In contrast to fruits and vegetables that were avoided by half of the EoE patients due to pollen-food syndrome, food allergy or EoE, only few patients (9%) avoided the intake of fish , therebyThe differences in DQI scores between the total EoE patient population and the general Dutch population might also partly be explained by a difference in energy intake. Overall, the subjects in the general Dutch population had a higher mean energy intake than the EoE patients, which may have contributed to a better diet quality in the general population as individuals with a higher total energy consumption will meet the requirements for specific nutrients or food groups more easily .The strengths of this study include the use of a detailed, standardized way of recording diet history and the fact that, in addition to the assessment of individual nutrient and food intakes, we used DQI scores to assess the overall diet quality of EoE patients. DQI scores examine the effects of the overall dietary pattern and represent a broader picture of food and nutrient intakes as the combination of foods and nutrients in complex eating patterns and their potential synergistic effects are taken into account. The use of DQI scores may therefore be more predictive of disease risk/severity than individual foods or nutrients. The findings of this study are in line with our previous findings on the relationship between habitual diet and severity of disease in the same study population .Possible limitations of the present study are the small sample size of the EoE population , the fact that we did not take into account the potential influence of self-imposed dietary measures due to food allergy or EoE symptoms on food and nutrient intakes, the differences in timeframes between the dietary assessment of the general Dutch population (between 2007–2010) and the EoE patients (between 2013–2015) and the cross-sectional design of the study. The latter hampers the ability to draw conclusions about the causal relationship between diet quality and disease risk and severity. Large prospective cohort studies or intervention studies are needed to assess if there is a causal relationship between diet quality and EoE. Moreover, the dietary intake of the EoE patients was assessed by a 3-day food record. It is known that the dietary intake of individuals may vary from day to day, and hence the intake of infrequently consumed foods such as fish, may be underestimated.We did not correct for multiple comparisons in our analyses, due to the explorative character of this study. However, although after correction several significant comparisons will lose significance, the main conclusion of our findings remains intact, namely that the diet of EoE patients has several pro-inflammatory properties, just as the diet of the general Dutch population.Lastly, as in the DNFCS, we did not calculate the intake of supplements, because of the inconsistent use of amounts and types of supplements by patients, which may have influenced disease outcomes.In conclusion, intakes of dietary fiber and several micronutrients were below the DRV, while intakes of saturated fat were higher than the DRV in adult patients suffering from EoE. Total protein and total fat intakes were relatively high, yet within the range of the DRV, pointing towards a high intake of animal-based foods. Compared to the general Dutch population, the overall diet of EoE patients, as assessed by two independent diet quality scores, was generally less healthy than the diet of the Dutch population, with the exception of the relative intakes  of vegetables/fruits/olives which were significantly higher (yet still far below (up to 65%) the recommended daily amounts) and the relative intake of alcohol which was significantly lower. Thus, the habitual dietary intake of Dutch adult EoE patients has several pro-inflammatory and unfavorable immunomodulatory properties. These results support the hypothesis that an unhealthy diet is associated with development and progression of EoE. The results of this study are complementary to the results of our recently published cross-sectional study in this population .We are unable to determine whether the diet is changed because of the disease, or that an unhealthy diet precedes the development of EoE. Further prospective and interventional studies are needed to demonstrate causal relationships and effects of diet on the development of EoE. Once the relationship between nutrition and EoE has been established, anti-inflammatory nutrition advice about which foods and nutrients to include and which to avoid could be provided in order to prevent and maintain remission in EoE.


In December 2019, coronavirus disease 2019 (COVID-19) emerged in Wuhan, China, and caused a global pandemic. The disease has a systemic manifestation including the hematopoietic system. It is clear that the hematologic laboratory will play an essential role in this crisis, contributing to patient screening, diagnosis, and prognosis. To date, complete blood counts (CBCs) have been recommended as useful tools for patient monitoring. Quantitative hematologic abnormalities have been reported since the first published papers and all blood cells can be affected during COVID-19, mainly leukocytes and platelets [In Morocco, with a record of 6741 confirmed cases and 192 deaths, the situation is less critical than in Europe and the United States [Very few articles have been published on blood morphology during COVID-19 infection. Two recent publications described the blood smears of infected patients, where the morphology of the neutrophil and platelet lineages showed very frequent anomalies in terms of nuclear morphology, cytoplasmic granulation, and the presence of atypical and immature cells ,4. Like Our preliminary results remain limited and more investigation is required to study the reversibility of these abnormalities and their impact on severity. Compared to all inflammatory biomarkers, observation of blood cells can be a simple alternative for the first triage and early identification of the infection.


The most toxic NMs overall were the silver NMs. This paper describes the ecotoxicological effects of nanomaterials (NMs) as well as their testing methods. Standard ecotoxicity testing methods are applicable to nanomaterials as well but require some adaptation. We have taken into account methods that meet several conditions. They must be properly researched by a minimum of ten scientific articles where adaptation of the method to the NMs is also presented; use organisms suitable for simple and rapid ecotoxicity testing (SSRET); have a test period shorter than 30 days; require no special equipment; have low costs and have the possibility of optimization for high-throughput screening. From the standard assays described in guidelines developed by organizations such as Organization for Economic Cooperation and Development and United States Environmental Protection Agency, which meet the required conditions, we selected as methods adaptable for NMs, some methods based on algae, duckweed, amphipods, daphnids, chironomids, terrestrial plants, nematodes and earthworms. By analyzing the effects of NMs on a wide range of organisms, it has been observed that these effects can be of several categories, such as behavioral, morphological, cellular, molecular or genetic effects. By comparing the EC Both the study of nanomaterials and that of ecotoxicology are constantly increasing, as evidenced by the increasing number of scientific articles from Web of Science Core Collection  during 2010–2019 a,b. Due In the present work, a series of bibliometric investigations were carried out to observe and analyze the state of the research on the ecotoxicity, nanomaterials and the ecotoxicity of the nanomaterials .. These In this paper we address both the adaptation of the standard ecotoxicity testing methods for nanomaterials, as well as the mechanisms of the ecotoxicological effects of these nanomaterials described as their biological and biochemical effects.The term “ecotoxicology” was first used by Ernst Haeckel in 1866 in the sense of the “economy of nature” and the “science of all interactions between organisms and their environment”. In the following years the science of ecotoxicology was developed as the study, using the tools of toxicology, of the effects of radiation and chemical pollutants from the biosphere on organisms from the environment . In 1969The ecotoxicological research is under rapid development due to the pollution of the environment caused by the rapid industrial development and it is speeded up by severe industrial accidents. Since ecotoxicology became an important part in ecological and environmental risk assessment, ecotoxicity assessment policies were developed .The environment hazard assessment framework uses ecotoxicity tests which are tools used to answer questions about the dangers of chemical substances that may be released into the environment .The percentage of scientific articles by year in the Web of Science Core Collection  from the total of articles in 2010–2019 is increasing . This hiThe ecotoxicological assessment developed as aquatic and terrestrial, the terrestrial assessment lagging the aquatic one . AlthougThe ultimate concern of ecotoxicology is the establishment of the consequences of the effects at population level and on whole ecosystems, but in practice much work is done at individual level .○algae○plants○invertebrates○vertebratesaquatic organisms○algae○plants○invertebrates○vertebratesterrestrial organismsStandardized tests under the forms of guidelines were formulated by different organizations, such as Organization for Economic Cooperation and Development (OECD), United States Environmental Protection Agency (EPA), International Organization for Standardization (ISO), Government of Canada (GC) or American Society for Testing and Materials (ASTM). The standardized assay can be conducted on:These assays use a wide range of organisms depending on the type of test needed. A list of these organisms is provided in In Europe, there is a widespread tendency to reduce as much as possible the use of assays that involve the use of vertebrate organisms. Draft reports from EU REACH Implementation Project 3.3 recommend the use of vertebrates only if necessary. For example, in the aquatic ecotoxicity assessment, the sequence of testing involves the use of simple organisms for first test, and assays on fish only if necessary ..ParameciL-Cysteine-capped CdS NMs expressed a high uptake in the roots of Spirodela polyrhiza. This was confirmed by epifluorescence microscopy where the presence of NMs was observed inside the root tissues (as particles with different sizes in intracellular spaces), the NMs aggregates appearing as optically dense signals under fluorescence  caused abnormal foraging behavior  in elopment . The maielopment .Cobalt NMs are of great interest in both life-sciences and industry, due to their wide range of applications, such as in lithium-ion batteries, gas sensors or medicine .Microcystis and Oscillatoria, was observed at exposure to Co NMs. Other observed effects were the reduction of carotenoid, protein and carbohydrates contents and the decrease of SOD activity with increase of NMs concentration in the microalgae [Decline in the growth rate and reduction in biomass concentration of two cyanobacteria, croalgae .Allium cepa was used to investigate the effects of cobalt oxide NMs as an indicator organism. The observed phytotoxic effect at root level was the inhibition of root elongation due to the massive adsorption of NMs into the root system [t system .Due to their potential applications in diverse fields, such as biomedicine, electronics, and optics, copper NMs have been the focus of intensive study .When present in high concentrations, copper is supposed to be highly toxic in aquatic systems, causing irreversible damage . Due to Microcystis aeruginosa, was observed to be enhanced by dissolved organic carbon (DOC). The cell walls were crossed by the NMs through the cell wall pores and the cell plasma membrane was crossed via endocytosis, thus the NMs reaching the thylakoids and granules [L. minor, the CuO NMs alter the activity of antioxidative enzymes such as guaiacol peroxidase, glutathione reductase and ascorbate peroxidase, increasing necrosis and bleaching [The dissolution and adsorption of CuO NMs onto cell walls, of the prokaryotic alga granules . In L. mleaching .Hediste diversicolor and the clam Scrobicularia plana. In both organisms, the observed effects were oxidative stress defense system responses (affected oxidative stress markers were: glutathione S-transferase (GST) and catalase (CAT)) and induction of genotoxicity (comet assay was used to asses DNA damage) [A mesocosm that modeled tidal cycles was used for the assessment of CuO NMs toxicity on the worm  damage) .Vigna unguiculata; specifically, how NMs affect the ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) activities, lipid peroxidation, Cu uptake and bioaccumulation in roots, leaves and seeds [The effects of Cu NMs were assessed on cowpea, nd seeds .Due to their functions in medicine and therapeutics, in electronics, catalysis, cosmetic, and food industries, gold NMs are of special interest .Scrobicularia plana, Au NMs formed aggregates and gold was accumulated in the soft tissues of the clams. Biochemical effects of NMs were metallothionein induction, increase in catalase, superoxide dismutase and glutathione S-transferase activities (indicating defense against oxidative stress), while a behavioral effect was the impairment of the burrowing behavior [Mytilus edulis, Au NMs enhanced stress parameters in digestive glands, mantle and hematocytes, paradoxically protecting from the oxidative stress due to menadione [The exposure of marine bivalves to Au NMs was assessed. In behavior . In Mytienadione .Populus deltoides × nigra. The Au NMs were observed in the cytoplasm and various organelles of root and leaf cells, and these accumulated in the plasmodesma of the phloem complex in root cells suggesting that the transport between cells and translocation throughout the whole plant were done with ease, inferring the potential for entry and transfer in food webs [The vegetative uptake of gold NMs was assessed using as a model poplar plants ood webs .Iron NMs have a wide range of applications such as magnetic, electrical, catalytic and biomedical  . These NHeterocypris incongruens, an ostracod, and on Folsomia candida, a collembolan. The effects were observed after seven days and prolonged exposure led to the oxidation of nZVI, reducing its toxicity [Severe negative effects of nanosized zero valent iron (nZVI) were observed on toxicity .Pseudokirchneriella subcapitata, Chlamydomonas sp.), crustaceans (Daphnia magna), plants  and worms , was used for the assessment of the effects of nZVI. The testing of the iron NMs was difficult due to their turbidity, aggregation and sedimentation behavior in aqueous media, but nZVI proved to be toxic. The observed effects for plants were the inhibition of root elongation in Raphanus sativus and Lolium multiflorum [A test battery, composed from algae . The observed effects were counteraction of induction of oxidative stress by paraquat  and prolongation of life span of wild-type and short-living mutant mev-1 worms [Antioxidant effect of Pt NMs coated with polyvinylpyrrolidone was observed in larvae of -1 worms . The lif-1 worms .The major applications of Ag NMs include their use as optical sensors, catalysts, in engineering, optics, electronics, and, most importantly in the biomedical field, as a bactericidal and therapeutic agent .Dunaliella tertiolecta and Chlorella vulgaris. The observed effects were depletion of chlorophyll content, inhibition of photosystem II (PSII) electron transport, membrane damage via lipid peroxidation possibly via ROS-mediated processes [The effects of Ag NPs were tested on two microalgal species: rocesses .Mytilus edulis, polyamine (PAAm)-capped silver NPs caused the formation of a donut shaped microstructures on the nacreous layer of the bivalve, which can be explained by the disturbance of the shell calcification process. In the oyster Crassostrea virginica, Ag NPs caused the inhibition of embryo development and the destabilization of the lysosomal membrane of hepatopancreas cells of adults [In adult f adults .D. magna, silver NPs accumulated in the gut, under the carapace, in the brood chamber and on the antennae and body surface, affecting their swimming behavior [In behavior ,22.C. elegans were neurotoxicity, reduction of velocity, flex, amplitude, and wavelength of the body bend of exposed worms and reduction of survival and reproductivity [The effects of silver NMs on the nematode uctivity .Titanium dioxide NMs are frequently used in the production of paper, plastics, paints, cosmetics and welding rod coating material .2 NMs on the algae P. subcapitata were light shading, interference on nutrient uptake through adsorption onto the cell surfaces and production of ROS which cause lipid peroxidation of cell membrane, leading to leaching of DNA from the algal cells. In Chlamydomonas reinhardtii, TiO2 NMs caused up-regulation of genes associated with antioxidant activities, such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and plastid terminal oxidase (ptox2), but these did not alter the transcriptions of genes associated with photosynthesis and carotenoid biosynthesis [The effects of TiOynthesis .D. magna TiO2 NMs caused the reduction of brood size and body length, disruption of digestive enzymes, such as amylase and esterase, affecting nutrient assimilation and energy allocation. The increase of GST, GPX, and CAT activities demonstrate the induction of oxidative stress by titanium NMs [In nium NMs .C. elegans, it was found that proteins involved in oxidative stress protection and metal elimination, such as SOD isoforms, metallothioneins and heat shock proteins, have a key role in resistance to TiO2 NPs. These NPs led to a substantial decrease in both head thrash or body bend in nematode mutants  compared to the wild type [In ild type .Rhizobium leguminosarum bv. viciae 3841 on garden peas Pisum sativum was affected by TiO2 NMs by delaying root nodule formation and the onset of nitrogen fixation [The symbiosis of fixation .Zinc oxide NMs have been extensively used in products like coatings, paints and sunscreens due to their chemical stability and strong adsorption ability. These also have a high inherent risk of water contamination, being able to reach high concentrations in surface waters posing significant threat to aquatic ecosystems .Phaeodactylum tricornutum, was inhibited, as the contact between algal cells and NM particles increased [The ZnO NMs at lower concentration cause toxic effects, such as decrease of cell viability  due to cncreased ,163.Lymnaea stagnalis were suppressed by ZnO NMs, 86% of the Zn being retained inside the organism. The reproduction and survival of copepods was also altered by ZnO NMs, along with the impairment of their movement [The feeding and defecating rates of the snail movement ,22.Allium cepa to ZnO NPs. The mitotic index was inhibited in a concentration dependent way, indicating a mitodepressive effect of the NPs, which may prevent several cells from entering the prophase and blocking the mitotic cycle during interphase inhibiting DNA/protein synthesis  were identified and analyzed. The concentration values were taken into account only for the ecotoxicity tests selected as being adaptable for nanomaterials. In order to be able to compare the concentrations of different types of nanomaterials, one model species was selected for each test, because the concentrations show interspecific variations. The selected model species were:In order to compare the ecotoxicity of NMs and to classify these NMs into toxicity classes, from published data, the half maximal effective concentrations , for both the aquatic and terrestrial environments, only nanomaterials that had EC50 values were presented for multiple NM types based on the same substance, which might differ in shape and/or size, the mean value of all types of tested NMs was included in the comparison table. The average EC50 values were calculated as a mean of the concentration of each aquatic and terrestrial assay in order to compare the NMs, but only for those NMs that had available data for all assays, for the rest of NMs “not applicable” (N/A) was entered in the table. The data that was not an exact value but was represented as greater or smaller than a value, the value +1 and –1, respectively, was taken into calculation.Where ECThe aquatic ecotoxicity of substances during short term exposure (acute toxicity), for all organisms types  is divided into five toxicity categories , accordi50 values for all the selected assays and model test species. By comparing the average EC50 values for all assays, silver NMs are the most toxic (VHT/A1.1), followed by the highly toxic (HT/A1.2) copper NMs, the moderately toxic (MT/A2) cadmium-based NMs and the slightly toxic (ST/A3) iron and titanium NMs. The order of their toxicity is: Ag NMs > Cu NMs > Cd NMs > Ti NMs > Fe NMs.Only cadmium, copper, iron, silver and titanium-based NMs had available data regarding their ECRaphidocelis subcapitata), while the least sensitive is the duckweed assay (according to data for Lemna minor) as shown by the average EC50 values per assay.The most sensitive aquatic assay for NMs is the algae assay , for both plants and soil dwelling invertebrates, is divided into three toxicity categories , accordi50 values for terrestrial ecotoxicity assays suitable for NMs of some nanomaterials were compared , followed by the plant assay (according to data for Allium cepa) and the earthworm assay (according to data for Eisenia foetida).The most sensitive terrestrial assay, based on the scarce available data, was the nematode assay  organisms being considered and included in our study regarding the testing of nanomaterial ecotoxicity.Due to the special properties of nanomaterials with respect to their bulk material and their wide range of applications, it is essential to test the ecotoxicity of NMs. The use of standard tests for testing the ecotoxicity of nanomaterials is possible but requires some adaptation. We considered as adaptive tests only those that use only SSRET organisms, are suitable for nanomaterials , have a shorter duration than 30 days, do not require special equipment or training and have low costs. Also, the possibility of these tests being high throughput is an advantage.The tests considered to be adaptable for nanomaterials are the algal, duckweed, amphipod, daphnid and chironomid tests in the aquatic environment, and the terrestrial plants, nematodes and earthworm tests in the terrestrial environment. The adaptations of these tests to nanomaterials include the methods of preparing the material solutions, as well as different methods of preparing the culture media, quantifying the number of organisms, etc.Analyzing the different effects of nanomaterials on SSRET and other organisms, it can be observed that these effects are complex and are of different categories . NMs can50 values of some nanomaterials obtained in aquatic and terrestrial ecotoxicity tests, that were selected as conforming for nanomaterials and are conducted on selected model species, it can be observed that such values are available mainly for aquatic ecotoxicity. It can also be observed that from the three selected aquatic tests, the algae, duckweed and daphnids tests, the most sensitive was the algae test, while the least sensitive was the duckweed test. From the selected terrestrial tests, the plant, nematode and earthworm tests, the most sensitive was the nematode test, the least sensitive being the earthworm test.Comparing the EC50 values were described for all three selected tests. The toxicity order of these four NMs was Ag > Cu > Cd > Ti > Fe. The most toxic NMs to algae were silver, gold and iron, to duckweed and daphnids silver, while the least toxic to algae were graphene and carbon NTs, to duckweed iron, and to daphnids carbon NT.It can also be observed that in aquatic tests only for nanomaterials based on cadmium, copper, iron, silver and titanium EC50 values for all the selected assays. Comparing the concentration for the earthworm assay, the toxicity order of NMs was possible: Ag > Zn > Cu > Ce. In the terrestrial tests only silver NMs had ECIn conclusion, the assessment of the ecotoxicity of nanomaterials and its mechanism are essential, as well as the adaptation of the standard ecotoxicity testing methods for nanomaterials. There are still difficulties regarding the testing of NMs, but these could be resolved by further research on this subject. It is clear that the effects of nanomaterials on different types of organisms are complex, thus these must be further analyzed.


To carry out a systematic review on the effects of phytosterolsupplementation on the treatment of dyslipidemia in children andadolescents. Review in the SciELO, Lilacs, Bireme, PubMed and Web of Science databases,with no time limit. Descriptors: phytosterols or plant sterols anddyslipidemias, hypercholesterolemia, cholesterol, children, adolescent, inEnglish and Portuguese. The articles included were published in Portuguese,English or Spanish and evaluated the effect of phytosterol supplementationin pediatric patients with dyslipidemia. Documents that involved adults oranimals, review papers, case studies and abstracts were excluded. Twoauthors performed independent extraction of articles. Of 113 abstracts, 19were read in full and 12 were used in this manuscript. Phytosterol supplementation to reduce cholesterol levels has been shown tobe effective in reducing LDL-cholesterol levels by approximately 10%, withreductions above 10% in LDL-cholesterol levels observed after 8 to 12 weeksof intervention. Studies have not shown significant changes inHDL-cholesterol and triglyceride levels. Based on the absence of adverseeffects, its use seems to be safe and of good tolerance in children andadolescents. Phytosterol supplementation seems to be of great therapeutic aid for thetreatment of hypercholesterolemia in children and adolescents. Furtherstudies assessing the long-term effect of phytosterol supplementation arenecessary. Dyslipidemia in children and adolescents represents a determinant risk factor foratherosclerosis and can contribute with coronary disease in adulthood.Dyslipidemia is a metabolic disorder characterized by abnormal concentrations oflipids and/or lipoproteins in the blood.Lipid disorders may have primary (of genetic origin) or secondary  causes.The prevalence of dyslipidemia in the juvenile age group in Brazil ranges from 24 to40%.The I Guideline for preventing atherosclerosis in childhood and adolescencerecommends the pharmacological lipid lowering treatment for children aged more than10 years, and proposes changes in dietary pattern and in the lifestyle of childrenolder than two years of age, which include a diet with reduced levels of saturatedfat and cholesterol, and practice of physical activity.Plant sterols and stanols are also known as phytosterols, which are bioactivecomponents whose structure is similar to that of cholesterol; sterols are theunsaturated forms (sitosterol and campesterol), and stanols are the saturatedderivatives (sitostanol and campestanol).,,,The inverse correlation between the frequent intake of phytosterols in the diet andserum levels of cholesterol or LDL-cholesterolphytosterols or plant sterols,dyslipidemias, hypercholesterolemia,cholesterol, children,adolescent, in English and in Portuguese. The bibliographicresearch was based on the question: “What are the effects of phytosterolsupplementation on the treatment of dyslipidemia in pediatric patients?”, which wasbased on the Population, Intervention, Comparison, Outcome model.This review was carried out based on the recommendations in the Preferred ReportingItems for Systematic Reviews and Meta-AnalysesInclusion criteria were clinical trials in English, Spanish and Portuguese, whichassessed the effect of phytosterol supplementation in pediatric patients withdyslipidemia. We also considered the studies referred to by selected articles whichmet the inclusion criteria. Documents that did not concern the purpose of thisstudy, involving adults or animals, case reviews, case studies, abstracts andanalyses that did not include samples with dyslipidemia were excluded. There was nolimitation as to the year of publication of the articles.The identification of the articles was first carried out through the analysis of thetitle to exclude repeated articles, or the ones that did not contemplate thepredefined criteria. Afterwards, the remaining abstracts were evaluated regardingtheir adaptation to the inclusion and exclusion criteria. Studies presenting thepredetermined criteria were fully acquired for a detailed analysis and dataextraction. The selected articles were read by two evaluators who decided,independently, about their inclusion based on the eligibility criteria. Anydivergence about the selection of articles was decided consensually.The selected studies had the following characteristics:Characteristics of the study participants , and inclusion and exclusioncriteria of the study.Type of intervention .Main results .Then, a synthesis and a critical analysis were carried out considering thestudies.To verify the validity of the clinical trials, the following were determined: samplesize, loss extension in the follow-up of participants in the studies and blindedpatients, health professionals, data collectors and evaluators of results.,,,,,,,,,,,Initially, 311 studies were identified in the electronic databases. After refinementby title, 113 abstracts were selected for analysis. The potentially relevant studieswere fully acquired for a detailed analysis of the eligibility criteria.Additionally, seven studies identified in other reference lists of the selectedarticles, and related to the theme, were included. Finally, the descriptivesynthesis was composed of 12 references, whose periods of publication were between1993 and 2017.,,,,,,,,,,,,,,,,,,,,,,,,All of the selected studies are randomized,,,,,,,,,,,,,,,,,,,,In total, 370 children aged between 2 and 19 years were included, and the sample sizevariation of the studies was from 9 to 59 participants. As to the type ofdyslipidemia, nine studies included children/adolescents with FH,,In two clinical trials,,,,,,,,,,,During the study period, the children and adolescents’ diet contained low levels ofsaturated fat and cholesterol, established by the respective authors.The intervention in the studies regarding the type of phytosterol, dose, duration andfood vehicles used is represented in The studies did not show significant changes in levels of HDL-cholesterol and TG,except for the results found in a single clinical trial, Only two studies included the analysis of the effect of phytosterol supplementationafter the intake period. The study by Matsuyama et al.,,,,,,After the intervention, some of the clinical trials observed reduction in theplasmatic concentration of hydrocarbon carotenoids  in children aged between 2 and 19 years. However, the BrazilianSociety of Cardiology suggests that the use of phytosterols should be part of thechanges in lifestyle, and is indicated for children aged more than 5 years with FH,as approved for use in Brazil.,The selected clinical trials used the administration of plant sterols or stanols, orboth, for intervention. The main phytosterols used were sitosterol and sitostanol.About the different subclasses of phytosterols, the studies that compared theefficacy of plant stanols and plant sterols verified that even though both have ledto reduction in LDL-cholesterol, a lower dose of plant stanol seems to be able toreduce LDL more effectively.,,The administration of plant sterols/stanols occurred during meals, especiallybreakfast, lunch and dinner. The studies that used margarine and spread as foodmatrix or supplementation vehicle oriented its consumption with bread,,,,The vehicles used for supplementation were: tablets, margarine, yogurt, milk, bread,spread, emulsions. Even though all of them significantly reduce LDL-cholesterol, themost significant reduction seems to have occurred with the intake of tablets,whereas bread and emulsions represented inferior reduction. According to a study byClifton et al.,,,,,,,,,It is also possible that the efficacy of phytosterols may decrease with time ofsupplementation, as observed in the selected clinical trials in whichsupplementation was superior than 12 weeks.,Regarding the beneficial effect of phytosterol supplementation on cholesterolemia, apotential negative effect is phytosterolemia, also known as sitosterolemia, a rareinherited autosomal recessive disorder which is related to mutation in the genes ofthe cotransporters of phytosterols/cholesterol, ABCG5 and ABCG8. These mutationspromote an increase 50 times higher in the circulating concentration of plantsterols, and are associated with early onset atherosclerosis.,,,,,,,,,,Another possible side effect is related to the absorption of fat-soluble vitamins andantioxidants. Phytosterols can interfere with the absorption of fat-solublevitamins,,,,,,,,,,,In the pediatric age group, few studies evaluated the effect of phytosterolsupplementation regarding lipid profile. However, the studies published until thismoment recommend the use of phytosterols as a treatment only for children withhypercholesterolemia who could not reach the ideal levels of LDL-cholesterol afterchanges in lifestyle.,,,,,The clinical trials included in this review presented some limitations, even thoughthey answered the initial question. Some debatable limiting factors were samplesize, the short period of follow-up, and the possible confounding variables.Besides, five studies did not report their financing sources,Despite its limited capacity of reducing the LDL-cholesterol fraction, especiallywhen administered in enriched foods, phytosterol supplementation seems to be ofgreat therapeutic help to reduce cholesterol among children and adolescents withfamilial hypercholesterolemia, of undetermined etiology or without genetic cause,after the age of 5 years, as established by the Brazilian Society of Cardiology.It is worth to mention that phytosterols should be used as a form of treatment,instead of prevention, in children/adolescents with hypercholesterolemia who couldnot achieve the ideal levels of LDL-cholesterol after changes in lifestyle. Theadministration in meals (lunch and dinner), for a period of 8 to 12 weeks, seems toreach significant reduction in LDL-cholesterol, higher than 10%, together with anadequate diet.Besides, based on the absence of adverse effects in experimental trials, their useseems to be safe and of good tolerance. Further studies are necessary to analyze theeffect of phytosterol supplementation in the long term, in order to verify if itshypocholesterolemic effect is sustained after the intake period.


The capability of some polymers, such as chitosan, to form low cost gels under mild conditions is of great application interest. Ionotropic gelation of chitosan has been used predominantly for the preparation of gel beads for biomedical application. Only in the last few years has the use of this method been extended to the fabrication of chitosan-based flat structures. Herein, after an initial analysis of the major applications of chitosan flat membranes and films and their usual methods of synthesis, the process of ionotropic gelation of chitosan and some recently proposed novel procedures for the synthesis of flat structures are presented. Chitosan (Chit) flat structures have not been yet classified in literature and thus, reviews on their methods of synthesis, properties and applications are still lacking. Chit flat structures are a subset of Chit membranes and films and their applications range from wastewater treatments, food packaging and biomedical uses to novel energy conversion and storage devices. Every method of synthesis possesses advantages and disadvantages depending on the physico-chemical, structural and shape/size features of the chitosan-based flat structures required for a specific application.Since the 1990s, ionotropic gelation has been used in the synthesis of polymeric micro and nanoparticles for biomedical applications , interfeThis review aims to shed light to the ionotropic gelation method mechanism for the synthesis of chitosan flat structures. After a brief introduction to the properties of chitosan, the methods of synthesis and application of chitosan flat structures are reviewed with a detailed discussion of recent research studies. Novel procedures introduced in the last years based on chitosan ionotropic gelation methods to synthesize chitosan flat structures for both biomedical  and ener2, C-O-C)  . From a nstrated . These pnstrated ,18,19.The increasing use of Chit combines its excellent properties with those of other materials to obtain composites that exhibit superior performance for specific applications. The incorporation of Chit to polymeric blends, multilayer coatings, scaffolds and nanostructures, is being actively studied nowadays . In thisUses in the biomedical area include Chit nanocomposites with metal and graphene oxides for hyperthermia therapy , combinaThe cationic behavior of chitosan in acidic solutions and the induction of gelation reaction mediate interactions with other materials and the formation of composites. The importance of chitosan and chitosan-based structures in many fields of daily life is largely due to their multifunctionality and the advantages related to the properties of the composites obtained, which will be evidenced in subsequent sections.3+ because of the protonation of its amine groups and breaking of hydrogen bonds. At neutral pH , biocomChitosan flat structures (membranes or films) can be synthesized by many methods, the most representative of which are solvent evaporation, crosslinking, layer-by-layer assembly, electrodeposition, and ionotropic gelation. These techniques can be combined, and different follow-up treatments are also performed.The most used technique for the synthesis of flat chitosan films is the solvent evaporation or solution-cast method ,37,38,39® and, thus novel studies exploring the possibility to use low-cost chitosan have been proposed  chitosan chloride and they cross-linked it with glutaraldehyde. The cross-linked polymer was blended with chitosan to improve the performance of alkaline fuel cells in terms of mechanical strength, compactness and ionic transport within the electrolyte. Wan et al. studied the ionic properties of various chitosan membranes such as pure chitosan, chitosan in different deacetylation form, di-o-butyryl chitosan, glutaraldehyde cross-linked chitosan and they found that all of studied membranes were suitable for use as electrolyte in alkaline fuel cells /[chitosan]r.u.) ranges from 5.2 to 3.8 at the beginning of the process .,36.13,36The mechanism underlying chitosan macroscopic gelation is supposed to include three consecutive steps: (i) the formation of small primary complexes as already known in the synthesis process of Chit micro-gel ; (ii) thw/v). The presence of NaCl is necessary at low chitosan concentration - hence low viscosity of solution -to shield strong electrostatic interactions between the polymer and TPP, hence reducing binding kinetics. The overall effect is to limit chain diffusion toward the TPP-gel interface and, consequently, formation of a polymer gradient. If the latter occurs , chitosan progressively accumulates forming a gelling zone, resulting into an inhomogeneous network with the highest concentration of polymer at the boundary and a liquid-like core [Supporting salts like NaCl can be eventually added in the process in order to improve the gel homogeneity when low chitosan amounts are tested . Recently, this set of biomaterials have been studied as cell substrates for tissue engineering applications and, more broadly speaking, for the investigation of cell-to-extracellular matrix interplay ,209 AlonLastly, chitosan gels can be freeze-dried to generate pliable membranes incorporated with silver nanoparticles . These cOver the last years the ionotropic gelation of chitosan has been widely exploited for the preparation of nano- and micro-gels especially employed in the biomedical sector. Chitosan-based flat structures assembled through ionotropic gelation are gaining significant interest among scientists, since potential applications thereof encompass ones ranging from wastewater treatment to energy conversion and storage to mention but a few. Here, we have provided the actual state of the art on this hot matter explored in a very limited way hitherto. From this perspective, ionotropic gelled chitosan-based flat structures emerge as intriguing networks that deserve to be further investigated allowing simplicity, low cost and physico-chemical tunability as a function of the required application. The numerous references discussed in the paper provide a comprehensive and unprecedent focus on the topic that should be useful to researchers interested in scientific developments of ionotropic-gelled chitosan flat structures.


Mesoamerica is a historically and culturally defined geographic area comprising current central and south Mexico, Belize, Guatemala, El Salvador, and border regions of Honduras, western Nicaragua, and northwestern Costa Rica. The permanent settling of Mesoamerica was accompanied by the development of agriculture and pottery manufacturing (2500 BCE–150 CE), which led to the rise of several cultures connected by commerce and farming. Hence, Mesoamericans probably carried an invaluable genetic diversity partly lost during the Spanish conquest and the subsequent colonial period. Mesoamerican ancient DNA (aDNA) research has mainly focused on the study of mitochondrial DNA in the Basin of Mexico and the Yucatán Peninsula and its nearby territories, particularly during the Postclassic period (900–1519 CE). Despite limitations associated with the poor preservation of samples in tropical areas, recent methodological improvements pave the way for a deeper analysis of Mesoamerica. Here, we review how aDNA research has helped discern population dynamics patterns in the pre-Columbian Mesoamerican context, how it supports archaeological, linguistic, and anthropological conclusions, and finally, how it offers new working hypotheses. Genomics, namely the study of the complete set of genetic information carried by an individual, has become a powerful tool to map the peopling of the world in detail. Ancient DNA (aDNA) adds a crucial layer of information by providing a snapshot of genetic diversity in past populations, most of which is often hardly accessible in modern-day populations due to recent demographic events. Therefore, aDNA provides evidence in addition to archaeology, linguistics, and anthropology to better comprehend human expansions and demographic events. Within the Americas, the genetic history of the Arctic, northern North America, and South America have been recently studied using aDNA ,2,3,4. HMesoamerica is a historically and culturally defined geographic area comprising current central and south Mexico, Belize, Guatemala, El Salvador, and border regions of Honduras, western Nicaragua, and northwestern Costa Rica  Figure . MesoameArchaeological evidence indicates a continuous occupation of Mesoamerica since the end of the Late Pleistocene, specifically the Lithic period (~13000–5000 BCE)  11]. Ho. Ho11]. The permanent settling of Mesoamerica took place in the Preclassic (or Formative) period 2500 BCE–150 CE), with the development of agriculture and pottery manufacturing. Agricultural practices intensified and improved during the Classic and Epiclassic periods (150–900 CE). The number of large population centres rapidly increased and cultural differences incremented between regions. During the Postclassic period (900–1519 CE), Mesoamerica was inhabited by large intermingled cultural groups connected by trade and agriculture, which fostered migrations that intensified admixture and built up the diversity patterns found across Mesoamerica. The Postclassic period ended with the start of the Spanish conquest (1519–1521 CE) and the subsequent colonial period (1521–1821 CE) ,15. The 0 CE, witMesoamerica was home to different human cultures connected by commerce and farming, and whose varied genetic backgrounds created an invaluable source of genetic diversity in the continent. Previous studies have attempted to uncover the peopling process of Mesoamerica using genetic approaches on modern-day Indigenous and cosmopolitan populations, mostly from Mexico and Guatemala. These analyses mainly focused on studying the so-called uniparental markers: the mitochondrial DNA (mtDNA) ,25,26,27Most of these genetic studies proposed a single origin for Mesoamerican populations ,28,29,33Inferring the population history of Mesoamerica through genetic studies that solely incorporate modern-day populations can be challenging due to recent historical events, such as population admixture with European, African, or Asian populations, that can distort pre-colonisation genetic signals. For instance, present-day Mexicans have, in general, three different ancestries: Native American, European (closely related to Iberians), and African. While the African component is generally minimal, the European component has a much lower proportion in Native Mexicans than what is found in cosmopolitan Mexicans. Notably, the Native American ancestry component can be divided into six separate ancestries . Three oIn spite of the extensive archaeological, linguistic, and anthropological research on pre-Columbian Mesoamerica, there have been surprisingly few aDNA studies. Therefore, there is a current lack of genetic knowledge about the genetic history of the region, probably because of the highly degraded nature of genetic material from tropical environments. Thus, currently available ancient genetic data rely mostly on mtDNA studies. The human mtDNA is a circular double-stranded DNA molecule that is maternally inherited, does not recombine ,38, and Interestingly, existing aDNA data come from the Central and Southeast areas of Mesoamerica, corresponding to the Basin of Mexico and the Yucatán Peninsula and its nearby territories, respectively. Therefore, this review will focus on the research that has been performed in these two geographical areas. Nonetheless, it should be noted that Mesoamerica contains other regions with extensive archaeological and anthropological records that have not been investigated in aDNA studies yet. These regions include the Gulf Coast and Oaxaca. The Gulf Coast was initially peopled by the Olmecs (the earliest known major Mesoamerican civilisation) and was later home to the city of El Tajín. Oaxaca comprises the current Mexican state of Oaxaca as well as the border regions of Guerrero, Puebla, and Veracruz. It was home to the Mixtec and Zapotec civilisations, the Preclassic–Classic city of Monte Albán being the most important archaeological site. In contrast, the northern and western regions of Mesoamerica are largely understudied from an archaeological and anthropological perspective and have been severely looted. The North was the transition area between Mesoamerica and Aridoamerica inhabited by both hunter-gatherers and farmers and it was nearly abandoned during the Postclassic period. The West was a geographically diverse area that led to the rise of highly diverse societies characterised by the shaft tomb tradition in the Preclassic and Classic periods and the Tarascan state during the Postclassic .The Basin of Mexico is a highland plateau that presently includes Mexico City and parts of the states of Mexico, Hidalgo, Tlaxcala, Morelos, and Puebla. The Basin of Mexico previously contained five lakes drained by Spanish colonists in order to expand Mexico City and prevent flooding . This arMesoamerica’s settling took place during the Early Preclassic period with the emergence of agrarian villages with egalitarian societies. The Middle Preclassic was defined by the development of agriculture, the increase in population sizes in settlements with hierarchical societies, and a strong influence from the Gulf Coast. This influence later disappeared during the Late Preclassic, when some settlements  became capital towns with strong economic and political power. At the end of this period, Teotihuacan rose as the undisputed power in Mesoamerica . Teotihuacan was a powerful political and military centre that controlled trade routes and attracted large masses of people from distant regions, coordinating them into an effective multi-ethnic system. The metropolis grew into the largest city of Mesoamerica and one of the world’s largest and most urbanised population centres of the first millennium, reaching 80,000–100,000 inhabitants by approximately 150 CE . TeotihuTeotihuacan was first occupied ~500 BCE and consisted of villages of local people as the valley offered several advantages: an abundance of freshwater springs, volcanic and raw materials for construction and technology, and a crucial location along the route from the Gulf Coast to the Basin of Mexico, skirting the Trans-Mexican Volcanic Belt also known as Sierra Nevada .During the first century CE, Teotihuacan was the destination of numerous migrations from Puebla and Tlaxcala, probably due to the Popocatépetl volcano eruption . It has Around 350 CE, a new episode of population displacement from Cuicuilco and surrounding regions likely took place when the Xitle volcano erupted . This miFollowing the political crisis, Teotihuacan became a multi-ethnic city inhabited by locals, returnee migrants, people from the Tlaxcala–Hidalgo–Puebla corridor, and individuals from distant regions, such as the Gulf Coast, Oaxaca, and Chiapas. This pattern has been sustained by archaeological studies , trace eAround 550 CE, Teotihuacan’s population size declined by half, and the city collapsed without any clear explanation. Although some of the earliest theories postulated that Teotihuacan was sacked by outside invaders , the mosThe Epiclassic, also known as Late Classic in other parts of Mesoamerica, was marked by the decadence of Teotihuacan and extensive population movements. Various ephemeral centres of power emerged throughout the Basin of Mexico, such as Xochicalco, Teotenango, Cacaxtla, and Cholula . The latThe study of the Postclassic includes not only archaeology and anthropology, but also linguistic information from accounts written in Spanish and different Indigenous languages. While the Classic period was a peaceful epoch of cultural climax, the Postclassic period was marked by political instability and war. This period emerges with the apogee of Tula , the capital of the Toltec Empire. Even though the political system and area of influence of the Toltec culture remain a point of discussion among scholars, especially in the city of Chichen Itza, inhabited by the Mayas in the Yucatán Peninsula, it has been proposed that its influence was widespread and persisted through time in central Mesoamerica . After the collapse of the Toltec Empire, numerous city-states emerged and fought one another for the control of political power. Even though Azcapotzalco  initially ruled most of the Basin of Mexico, the Triple Alliance of the city-states of Tenochtitlan, Texcoco, and Tlacopan reduced Tepanec influence and allowed the rise of the Aztec Empire. The Aztecs conquered a large number of cities across Mesoamerica over the following years and established a large Empire that lasted until the Spanish conquest . For exaIn contrast, various studies have focused on the archaeological site of Tlatelolco, which is buried beneath modern-day Mexico City and was founded around 1337 CE. Even though Tlatelolco was a sister city to Tenochtitlan , religious and cultural practices were distinct between the two cities. Tlatelolco was the biggest and most diversified marketplace in Mesoamerica during the Late Postclassic . The mosThe Yucatán Peninsula is located in southeastern Mexico and separates the Caribbean Sea from the Gulf of Mexico. Currently, it comprises the Mexican states of Yucatán, Campeche, and Quintana Roo, as well as part of Guatemala and almost the whole of Belize. The first humans inhabiting this area were hunter-gatherers. In fact, the oldest individual (~10500 BCE) with published genetic data in Mesoamerica comes from this region. This individual was found in Hoyo Negro , a submeHaplogroup D1 was actually found in two genetically related individuals ~5300 BCE) from the archaeological site of Saki Tzul in the Maya Mountains (Belize) 00 BCE fr that expThe entire Yucatán Peninsula together with the Mexican states of Tabasco and Chiapas, the rest of Guatemala and Belize, as well as the border regions of Honduras and El Salvador, were the home to the Maya civilisation , one of The geographical diversity of the Yucatán Peninsula was one of the most important traits for the Mayan settling in the Preclassic period, allowing the emergence of different cultural focuses that led to the rise of various linguistic families. Despite the existence of a cultural influence from the Gulf Coast during the Middle Preclassic, it disappeared in the Late Preclassic when cities started to urbanise with architectural gigantism . The ClaRegarding genetic studies of the region, Ochoa-Lugo and colleagues  performeAdditionally, other aDNA studies just focused on the analysis of mtDNA haplogroups in one archaeological site during a specific period of time. For example, the largest mtDNA collection from Mesoamerica to date comes from the Classic–Epiclassic site of Midnight Terror Cave (Belize), which is believed to have been a site of human sacrifice . A totalFinally, a further study focused on the Epiclassic–Postclassic site of Xcaret (Mexico), one of the most active ports in the eastern Mayan coast . This reThe Greater Nicoya was an ancient Central American cultural area currently located in the Pacific Nicaragua and northwestern Costa Rica. It was situated between southern Mesoamerica and northern Isthmo-Colombia . This reRecent improvements in the field of aDNA have revolutionised the study of the history of past populations, providing additional information highly valuable for archaeologists, linguists, and anthropologists. Given that Mesoamerica is the geographical link between the North and South American continents, aDNA studies from this region may be essential to understand the migration patterns of populations across the region, as well as their territorial expansions and interactions. For example, the aDNA study performed by Moreno-Mayar and colleagues  describeArchaeological evidence strongly suggests that after the settling of Mesoamerica, most cultures remained connected by trade, agriculture, culture, politics, empires, and conquests. Therefore, it is essential to include genetic analysis to examine the patterns of population structure, biological adaptation, cultural impacts, and natural phenomena  on the evolutionary and demographic history of Mesoamerican populations. Crucially, aDNA can help to discern these patterns in pre-Columbian Mesoamerica and provide further support to archaeological studies as well as offer new insights and working hypotheses. This is particularly important for the highly understudied Preclassic period, which could benefit from interdisciplinary approaches that help to unravel the demographic and genetic architecture of the settling process of Mesoamerica. Regarding the Basin of Mexico, a detailed study focused on samples from the Classic city of Teotihuacan, concretely in multi-ethnic neighbourhoods such as Teopancazco, could not only help to determine aspects of population demography of Teotihuacan, but also be useful to infer and contextualize the population history of Mesoamerica during the Classic period. This should be aided by similar studies focusing on the understudied Epiclassic and the Postclassic periods, which could help to explain how people were connected after the fall of Teotihuacan and during the empires that emerged before the Spanish conquest, respectively. Although Mayan culture remained in the Yucatán peninsula throughout all these cultural changes, it was never isolated and was also affected by the Classic collapse. A genetic study focusing on the Mayas could further expand our knowledge of this important civilisation and reveal connections with other regions of Mesoamerica. In fact, a knowledge gap currently exists in several Mesoamerican areas that could help to define population interactions in the region. These include the Gulf Coast, the North, the West, and Oaxaca regions, as well as the cultural and historical areas of Isthmo-Colombia, Aridoamerica, and Oasisamerica.Mesoamerica represents a critical tropical transition in the southward journey of the First Peoples of the Americas. A thorough description of genetic variation in ancient Mesoamerican populations through space and time may enable us to identify mechanisms of adaptation to changing environments, climates, lifestyles, and social structures, with potential consequence for present-day people’s health. For instance, this kind of research could target the Mesoamerican Classic collapse and investigate a possible genetic link to severe droughts or epidemic diseases, and potential consequences to the genetic make-up of contemporary Mesoamerican populations. Thus, it is highly important to reconstruct the genetic past of Mesoamerica before and after European arrival and estimate the genetic impact of European colonisation in the demographic and adaptive history of these populations, which currently remains unknown.