Natural antimicrobials (NA) have stood out in the last decade due to the growing demand for reducing chemical preservatives in food. Once solubility, stability, and changes in sensory attributes could limit their applications in foods, several studies were published suggesting micro-/nanoencapsulation to overcome such challenges. Thus, for our systematic review the Science Direct, Web of Science, Scopus, and Pub Med databases were chosen to recover papers published from 2010 to 2020. After reviewing all titles/abstracts and keywords for the full-text papers, key data were extracted and synthesized. The systematic review proposed to compare the antimicrobial efficacy between nanoencapsulated NA (nNA) and its free form in vitro and in situ studies, since although in vitro studies are often used in studies, they present characteristics and properties that are different from those found in foods; providing a comprehensive understanding of primary mechanisms of action of the nNA in foods; and analyzing the effects on quality parameters of foods. Essential oils and nanoemulsions (10.9–100 nm) have received significant attention and showed higher antimicrobial efficacy without sensory impairments compared to free NA. Regarding nNA mechanisms: (i) nanoencapsulation provides a slow-prolonged release to promote antimicrobial action over time, and (ii) prevents interactions with food constituents that in turn impair antimicrobial action. Besides in vitro antifungal and antibacterial, nNA also demonstrated antioxidant activity—potential to shelf life extension in food. However, of the studies involving nanoencapsulated natural antimicrobials used in this review, little attention was placed on proximate composition, sensory, and rheological evaluation. We encourage further in situ studies once data differ from in vitro assay, suggesting food matrix greatly influences NA mechanisms. For many years, one of the concerns of the food industry was microbiological contaminants, which can cause damage to consumers’ health. Foodborne diseases can be related to the ingestion of pathogens or their toxins. These diseases involve public health issues and economic loss for the industry . In addiFurthermore, these antimicrobials can interact with food components such as proteins, fats, sugars, and salts, decreasing their antimicrobial activity, requiring large amounts to inhibit microbial growth, compromising the sensory properties of foods such as taste, color, and odor . MoreoveNanoencapsulation has been discussed with advantages over conventional microencapsulation due to the unique properties provided by the nanosized particle (1–100 nm) and its high surface/volume ratio, increasing the interaction with enzymes and microorganisms. However, there are still some challenges regarding the technology, and economic and regulatory concerns to its implementation in the food industry in relation to several factors, such as the influence of the wall material, the interaction with the food matrix, the behavior of the nanostructure during food processing, and the use of economic and straightforward strategies to obtain the nanostructures ,19,20,21In the face of those challenges and the relevance of the subject to food preservation, an increasing number of research papers and literature reviews on the micro-/nanoencapsulation of natural bioactive compounds for several purposes in the food industry have been published in the last five years. Some studies are concerned with the characterization of nanostructures and nNA used in foods , the phyAlthough several review articles focused on encapsulation techniques, materials characterization, or their application in food products, to the best of our knowledge no systematic review of the available literature was performed to compare the antimicrobial activity of free and nanoencapsulation form of NA into foods, addressing the differences between data of in vitro and in situ evaluation. The complex composition of foods  differs from simpler laboratory methods. This difference may influence the types of interaction that nNA can perform, and consequently its antimicrobial action. Differences in composition result in a difference in the viscosity of the medium and are consequential in the diffusion processor. Thus, the accessibility of microorganisms for nNA in food may differ from the lab environment. From the articles related to safety improvements, we also review the effects on quality parameters of foods products . Finally, we provide a comprehensive understanding of primary mechanisms of action of the NA nanoencapsulated into a food matrix. In this way, our work can address essential issues to help experts in the food industry improve the shelf life and quality of foods using nanoencapsulated natural compounds.Although several review articles focused on encapsulation techniques, materials characterization, or their application in food products, to the best of our knowledge no systematic review of the available literature was performed to (i) compare data of in vitro and in situ evaluation of the antimicrobial activity of free and nanoencapsulation form of NA into foods; (ii) besides safety improvements, analyze the effects on the quality parameters of foods products rather than nutraceutical delivery ; and (iii) provide a comprehensive understanding of the primary mechanisms of action of the NA nanoencapsulated into a food matrix. In this way, our work can address essential issues to help experts in the food industry improve the shelf life and quality of foods by nanoencapsulated natural compounds.http://www.prisma-statement.org accessed on 21 September 2021). As support, we used a computational tool, State of the Art (Start) [This review was carried out according to A. P. A. de Carvalho & Conte Junior (2020). The issue in this review was “Application of nanoencapsulated natural antimicrobials in food”. For this, we followed the recommendations of the Preferred Report items for Systematic Review and Meta-Analysis (PRISMA) ( (Start) .Based on population, intervention, comparison, and outcome (PICO), the research questions focused on: (i) which NA and strategies have been used to apply nanoencapsulation in foods? (ii) How far has it been in vitro from in situ evaluation methods? (iii) What are the main modes of action of nanoencapsulated NA in the food matrix? (iv) What is the influence on the physicochemical quality of foods?To avoid possible sources of bias and based on the study’s objective, inclusion/exclusion criteria were used for eligibility.SCREENING—Title, keywords, and abstract:Inclusion: Studies published in English, at least one encapsulation strategy, at least one natural antimicrobial was encapsulated, applied in a food matrix.Exclusion: Studies that are not research articles, no encapsulation method has been studied, no natural antimicrobials have been learned, they have not been applied to food, no antimicrobial activity has been inspected, application to packaging, films, sachets, coating materials, and for cleaning.ELIGIBILITY—Full-text reading:Exclusion: Natural antimicrobials and the encapsulation process were not in line with the objective of the study.This research was carried out with the information acquired on 19 October 2020, in four electronic databases: Science Direct, Scopus, Web of Science, and PubMed. To search articles in the databases, we set up search strings, which were assembled based on keywords related to the research questions, synonyms related to encapsulation and antimicrobials, and the use of Boolean operators “AND”, “OR” and “NOT”. Search strategy: Science Direct: (encapsulation OR carrier OR delivery OR capsules) AND  AND (food) NOT (“food packaging”); Scopus: (*encapsulation OR *carrier OR *delivery OR *capsules OR *structures) AND  AND (food) AND NOT (“food packaging”); Web of Science: (*encapsulation OR *carrier OR *delivery OR *capsules OR *structures) AND  AND (food) NOT (“food packaging”); PubMed: (encapsulation OR nanoencapsulation OR microencapsulation OR nanocarrier OR microcarrier OR carrier OR nanodelivery OR delivery OR microcapsule OR nanocapsule OR capsule OR microstructure OR nanostructure OR structure) AND  AND (food) NOT (“food packaging”). English was used for the research between 2010 and 2020 . Due to the objective of applying antimicrobials directly to food, studies with focus and discussion of NA encapsulated in packaging, films, sachets, or as food coating materials were excluded. Despite not having a single particle size that can be used as a definitive cut-off point to distinguish nanoparticles, such as nanoemulsions, from conventional particles, according to the literature, one of the points to be considered is that the average particle radius of the system must be less than 100 nm ,29. Howe® tool. Duplicate/triplicate articles were automatically excluded, and the others went on to the stages of identification, selection, and extraction. Any doubts that arose during the articles’ identification were kept for further analysis based on the title, keywords, and abstract.After the selection process, the articles were imported into the StartThe systematic review results were presented on the PRISMA flow chart illustrated in As noted in The encapsulation by the emulsification method is based on the mixture of two immiscible and thermodynamically unstable liquids, one of which is dispersed in the other in the form of droplets. According to the nature of its dispersed phase, the emulsion is called water in oil (W/O), where the dispersed phase is water, and oil in water (O/W), in which the dispersed phase is oil. In the NA encapsulation analyzed in the studies, the emulsions used are O/W type, where the dispersed phase is oil. Most NA present hydrophobic characteristics and low water solubility, which justifies this type of emulsion (O/W). Formulating emulsions is thermodynamically unfavorable, requiring mechanical energy input or the supply of chemical energy obtained through low energy or high energy methods .L. delbrueckii and S. cerevisiae. This explains such an unexpected effect on the possible degradation of NA obtained by high-pressure homogenization [High energy methods use mechanical devices that generate intense, disruptive forces, causing a rupture and reducing the drops’ size. Thus, structures with reduced sizes, such as nanoemulsions, have unique properties and are more stable than emulsions. The high-intensity/frequency ultrasound method stands out in high-energy methods, which use sound waves to produce nanoemulsions. The technique made it possible to obtain emulsions with smaller diameters, high encapsulation efficiency (>80%), low polydispersity, low phase separation, and, consequently, high stability during storage ,31. In anization . AlthougAssociated with the effect of high energy methods used in nanostructures, such as nanoemulsions, the ratio of NA and surfactant also interferes with the size of the droplets ,31,32,48However, low-energy methods for forming emulsions containing NA have gained popularity, as it is unnecessary to use specialized and expensive equipment. The procedures allow the production of tiny droplets without or with gentle agitation using only internal energy from the system. Due to minimal energy generation, the method has the advantage of avoiding the degradation of nanoencapsulated compounds .Nevertheless, according to Chaudhari et al. (2021) the droplets formed by these methods are not stable at high temperatures. In this case, there is a need for a large amount of surfactant, in addition to being limited to a narrow range of compounds to be nanoencapsulated and surfactant type .The formation of emulsions can occur due to the phase inversion temperature (PIT), where nonionic surfactants are typically used, in which their solubility is altered due to temperature changes. Thus, nonionic surfactants are highly hydrated at low temperatures, and the formation of O/W type emulsions is favored. However, by increasing the temperature, the shape of W/O type emulsions is preferred. At a phase inversion temperature, the droplets exhibit a very low interfacial tension, and the mean spontaneous curvature of the surfactant molecules is zero, promoting emulsification. However, the formation of tiny droplets and consequently high curvature makes the emulsions at this temperature unstable, with a high coalescence rate. Therefore, a rapid departure from the phase inversion temperature is necessary to obtain, for example, kinetically stable nano-emulsions. If the temperature deviation is fast heating, W/O emulsions will be accepted, while the temperature deviation by cooling allows obtaining O/W emulsions. Changing the W/O to O/W emulsion enables the encapsulation of the NA. The amount of material encapsulated significantly influences the average diameter and polydispersity of the formed nano-emulsions . For theThe subsequent addition of water leads to a progressive increase in the surfactant’s hydration degree, promoting a change in the surfactant’s spontaneous curvature. This method made it possible to produce nanoemulsions containing orange essential oils (EO) with stability for at least three months under refrigeration . If we cAnother method that uses chemical energy to promote the encapsulation process by forming emulsions is self-emulsification. In self-emulsification, there is no change in the surfactant’s spontaneous curvature. As shown by Stratakos & Grant (2018), it occurs due to the dilution process in which carvacrol and thyme oil was encapsulated using triglyceride oil and tween 80 . AlreadyEmulsions are also used to encapsulate gel structures containing NA. Nanoemulsions containing gel structures formed by nisin and D-limonene have shown excellent stability stored at 28 °C for no less than three months, with no phase separation .As can be seen, emulsification technology is the most common for the nanoencapsulation of NA. However, phenomena such as flocculation, sedimentation, coalescence, phase separation, cremation, and Ostwald ripening are the main challenges related to the stability of emulsions .For better storage, emulsions can be subjected to additional processes such as lyophilization and drying .Liposomes, spherical structures, are based on hydrophobic-hydrophilic interactions between phospholipid compounds and hydrophilic agents, allowing the formation of lipid vesicles or bilayers capable of transporting hydrophobic and hydrophilic NA. Despite the ability to spontaneously form bilayers in aqueous solutions, one of the most used methods for producing liposomes is the thin film method. This method consists of evaporating an organic solution containing surfactant and NA and an aqueous phase and hydrophilic material, followed by the entry of a sufficient amount of thermal energy. Thus, liposomes create physical barriers due to their amphiphilic nature, protecting natural antimicrobials from external conditions ,52,53. TNanoliposomes of soybean and phosphatidylcholine (PC) containing lysozyme and nisin were coated with pectin and polygalacturonic acid. Although the coating does not differ significantly in size and polydispersity, the layer made the liposomes more negatively charged, providing greater electrostatic repulsion between the nanoparticles. Lysozyme’s nanoencapsulation efficiency (EE) decreased with the coating, mainly for pectin, which may be due to a rearrangement in the liposome after coating, releasing some lysozyme. The nanoliposomes encapsulating lysozyme-nisin did not differ from those without coating . One of Another series of natural antimicrobials such as monoterpene, limonene, menthol, linalool, and thymol were nanoencapsulated using dripping ionic gelation. The authors were able to produce particles with diameters smaller than 55 nm. Despite the small size that helps in the stability of nanoemulsions, low values (−0.1690 mV) in the module of the zeta potential were observed . We beliOther methods for NA encapsulation are available in the literature, such as (i) a spray dryer consisting of solubilizing, homogenizing, and spraying the NA’s material in the drying chamber, in a stream of hot water air, instantly producing powder particles. The conversion occurs due to heat transfer from the air to the atomized droplets and the mass transfer of the atomized droplets to the air. Therefore, due to the rapid evaporation of the solvent, the natural antimicrobial is trapped. The starting material that feeds the sprayer can be a solution, emulsion, or suspension ,58. DespDespite the potential these techniques demonstrated in encapsulating NA, they could not produce structures with a diameter < 100 nm, which is the focus of our review ,67,68,69Through the data obtained from the articles, it is not possible to determine which is the best method to obtain structures with a diameter < 100 nm, since factors such as the type of wall material, the ratio surfactant/NA, and the oil used (in the case of emulsions) demonstrate the influence of the size of the formed structure. However, the formation of nanoemulsions stood out the most for the nanoencapsulation of NA.We believe that studies aimed at the development of new encapsulation methods and analyses that propose novel wall materials, studies trying to improve existing processes, the best conditions for the formation of nanostructures, and the choice of the best wall material for each technique is interesting in order to achieve industrial application faster and more efficiently.When analyzing Escherichia coli (E. coli) was changed when the NA was encapsulated. In addition, differences were observed among studies. Limonene, linalool, menthol, and thymol have been shown to reduce MIC from 48% to 72% in nanoencapsulated form, compared to free NA. A mixture of terpenes -trans, cyclohexanol, terpinolene, and δ-terpinene) reduced the MIC for E. coli. It showed without significant effect for the minimum bactericidal concentration (MBC). Thymol also confirmed the MIC reduction by 20%, and thyme reduced by 50%. As observed for MIC, thyme EO was more effective in lowering MBC (66.6%) for E. coli after nanoencapsulation compared to the effect of nanoencapsulation for thymol (20%) [E. coli activity may be associated with the synergistic effect between the components (mainly thymol and carvacrol) present in the essential oil, different from the isolated form (only thymol) [E. coli. At pH 4 the nanoencapsulated sweet orange EO reduced by 66% E. coli compared to free form. However, at pH 7 this effect was not observed [Salmonella Typhimurium was 30% to 72% lower in the nanoencapsulated form than in the free form [S. Typhimurium in the study by Badawy et al. (2020) [S. Typhimurium by 87.5% [The minimum inhibitory concentration (MIC) for ol (20%) ,34,43,44ol (20%) ,71. We s thymol) . Furtherobserved . We suggobserved . Hexanalobserved . Therefoobserved . The MIC. (2020) . In addi. (2020) . More prby 87.5% .Staphylococcus aureus from 50% to 100% compared to free eugenol [S. aureus were decreased by 87%, in a concentration of 0.78 mg/mL (MIC) and 3.13 mg/mL (MBC) [S. aureus by up to 40% in a 5% concentration of EO oregano in the nanoemulsion [S. aureus than nanoemulsified eugenol. This can be explained by the different wall materials and oils used in the NA nanoencapsulation process and the different sizes of the nanoemulsions obtained. The smaller size of the nanoemulsion, the greater the area of contact with the external environment. Intermolecular interactions between the core material (NA) and the wall material can occur during nanoencapsulation. These interactions may have different magnitudes depending on the other molecules used, influencing NA diffusion from the nanoemulsion to the external environment. The wall material will also influence the solubility and stability of the hydrophobic compounds, directly reflecting on their antimicrobial activity. A 0.2% concentration of nanoemulsion containing thyme EO reduced the MIC and MBC for S. aureus by 33.3% compared to free thyme EO, supporting those above [S. aureus. Trans 2-hexanal increased the MIC and MBC for S. aureus by up to 150%, and both compared to their free forms [Nanoemulsions containing 0.003%, 0.03%, 0.3% eugenol have been shown to reduce  eugenol . After bmL (MBC) . In thismL (MBC) . Nanoemuemulsion . When cose above . ContradListeria monocytogenes were not so promising. Nanoemulsion containing thymol showed less effect for MIC than free thymol and increased MBC by 14.2% after nanoencapsulation [L. monocytogenes) [L. monocytogenes at 30 °C. However, at 7 °C, it had no effect compared to free bacteriocins. We believe that at a temperature of 30 °C, more significant agitation of the molecules, compared to 7 °C, facilitated the diffusion of bacteriocin outside the nanostructure and in the food, making it more effective as an antimicrobial. L. monocytogenes is a pathogenic microorganism frequently associated with food contamination, representing risks to consumer health, making new studies with nNA highly relevant for its inhibition.The results found for sulation . The MICtogenes) . Malheirtogenes) , when usPseudomonas aeuroginosa was also improved after nanoencapsulation, reducing the MIC and MBC by 50% compared to the free form [Saccharomyces cerevisiae [S. cerevisiae compared to its free forms, showing that the mixture of nanoencapsulated terpenes was more promising against S. cerevisiae than hexanal and trans-2-hexenal nanoencapsulated [The activity of trans-cinnamic acid against ree form . Nanoemurevisiae . Nanoemupsulated ,34.E. coli, Gram-negative microorganism, and S. aureus, Gram-positive microorganism, there was greater effectiveness of Origanum majorana EO and thyme EO against E. coli [E. coli compared to S. aureus, demonstrating that, despite the Gram-negatives, microorganisms are more susceptible to the antimicrobial action of EO, and that this cannot be generalized and extrapolated to the nanoencapsulation process.When comparing the effects of the same nanoencapsulated NA against  E. coli ,36. The The association of NA can be used to improve antimicrobial activity, as demonstrated by Bei et al. (2015) , who fouOriganum majorana EO nanoencapsulated more effectively against Aspergillus flavus (reduced the MIC by 60%) and aflatoxin B1 (AFB1) (reduced the MIC by 33.3%) compared to the free forms [Coriandrum sativum EO reduced the MIC of a mixture of 14 fungi by 44.4% and the MIC for AFB1 by 50% compared to free forms [Effects against fungi and their toxins were also evaluated. ee forms . Coriandee forms , demonstThrough the demonstrated results we can conclude that nanoencapsulation, in general, improves the effect of NA on several microorganisms. The nanoencapsulation process can enhance the solubility of NA in aqueous media and strengthen its stability in temperature, pH, and light, which provides improvements for application. However, it is essential to emphasize that when considering nanoencapsulation of the NA, it must be taken into account that the NA must cross the protective barrier to act on the microorganisms, which can hinder its immediate action. Possible intermolecular interactions between NA and the materials used to form the nanostructures will also make it difficult for the NA to exit to act on microorganisms.The food matrix is complex and diverse, formed by water, proteins, lipids, carbohydrates, vitamins, fibers, and ions. In addition, these molecules are arranged in different forms, forming different structures. The molecules can be soluble, dispersed, forming clots, structured networks (gels), and interacting with each other and with other molecules. These characteristics make food different from the culture media used in laboratories. Therefore, in situ studies (food) are relevant to an accurate explanation of the nNA. S. Typhimurium in fresh-cut pineapple [E. coli in zucchini was reduced after nanoemulsification of carvacrol [Trans-cinnamic acid, when nanoencapsulated, demonstrated effectiveness against aerobic mesophilic bacteria and aerobic psychrophilic bacteria present in fresh-cut lettuce, reducing 50% of the growth of the microorganism compared to free antimicrobial . Linalooineapple , and 1 larvacrol . We founE. coli. However, this effect was not proportional. Limonene, menthol, linalool, and thymol nanoencapsulated reduced the growth of E. coli in minced meat by approximately 76%, 66%, 72%, and 75%, respectively, compared to free form. The 2.5-fold increase in concentration reduced 3%, 6%, 14%, and 9% more E. coli growth [E. coli and S. aureus was obtained using oregano EO nanoencapsulated in chicken pate. However, in this study, the control was chicken pate without the addition of antimicrobial [E. coli in beef by 20% and 30%, respectively, compared to free forms [Donsì et al. (2014)  also anai growth . A reducicrobial . Both caee forms . These sOriganum majorana), nanoencapsulated did not show any effect against aflatoxin B1 (AFB1) in maize compared to its free form [Coriandrum sativum EO nanoencapsulated also showed no impact on AFB1 in rice compared to its free form. However, it increased the protection against fungal infestation by 25.6%. When using 2xMIC (1.0 μL/mL), the effect was less (21.7%) than the MIC (0.5 μL/mL) (25.6%) [At 2.5 times lower concentration of oregano, EO (ree form . Coriand (25.6%) , demonstLactobacillus delbrueckii in juice (orange and pear) was reduced by 37.5% using 1.0% of nanoencapsulated mixture terpenes. The reduction was total in 5% and 10% concentrations compared to the control, without antimicrobial [S. aureus, the trans-2-hexanal and hexanal mixture after nanoencapsulation reduced 1 log cycle on the 2nd day and 1.5 log cycles on the 8th day at 10 °C. For L. monocytogenes and E. coli, there was an increase of 1 log cycle and 0.5 log cycle on the 2nd day, respectively, and a reduction of 1 log cycle and 0.5 log cycles on the 8th day, at 10 °C. For S. cerevisiae, a decrease of 2 log cycles was observed on the 14th day, and on the 22nd day showed without effect, both at 10 °C [L. monocytogenes and E. coli in cantaloupe juice, below the detection limit in 2 h, compared to the free form that presented 5 log cycles [E. coli during 30 min of exposure [icrobial . Nanoencicrobial . For S. at 10 °C . Nanoencg cycles . No effeL. monocytogenes was close (~20%) for skim milk and full-fat milk. The concentration used in full-fat milk was 4.5 times higher. Greater effectiveness against E. coli was observed in full-fat milk (72.2%) compared to skim milk (47.3%). However, the concentration used in full-fat milk was higher (4.5 times). Regardless of the difference observed as a function of the fat content in milk, in both situations nNA was more effective than its free form against E. coli and L. monocytogenes [L. monocytogenes was more significant in whole milk than in skim milk, both at 37 °C and 7 °C of storage. In skim milk, an increase of up to 3.5 log cycles was observed over 10 h. Nisin and lysozyme nanoencapsulated showed no effect on S. enteritidis and mixtures of Listeria strains in milk (whole milk and skim milk) at 37 °C and 7 °C. No effect was observed against L. monocytogenes in UHT goat milk at 37 °C and 7 °C of storage when bacteriocins were nanoencapsulated [E. coli, 50% by MIC, and 35.5% by MBC for S. aureus in milk, compared to free NA [The effectiveness of unencapsulated thymol varied according to the fat content present in the milk. Comparing the full-fat milk and 2% reduced-fat milk that used the same concentration of the nanoemulsion containing thymol (4.5 g/L), greater effectiveness was observed in the 2% reduced-fat milk. The effect against ytogenes . The infytogenes . Howeverytogenes . In the ytogenes , the efffat milk .2% compapsulated . Thymol  free NA .We can verify that nanoencapsulation makes NA more promising to be applied in food. The effect of nNAs is variable depending on the different types of food and possible variations in terms of composition that they may present. Therefore, further studies of nNA in food matrices are necessary to understand better and clarify its effect.L. monocytogenes. As for the storage temperature of 7 °C, no difference was observed in the in vitro study of the UHT goat milk [E. coli [Coriandrum sativum EO was less effective in protecting against fungal infestation and AFB1 in rice than in vitro study [S. aureus, S. typhimurium, and P. aeuroginosa [Some studies have demonstrated greater effectiveness in minced meat and milk. However, a higher concentration of nanoencapsulated antimicrobials was used in the food than in the laboratory environment ,40,43,44oat milk . Chaudha[E. coli . The nanro study . Trans-croginosa .The laboratory methods used in microbial growth are simpler than food in terms of composition. Some foods are more complex than others in composition and structure, explaining the differences found in different foods. In addition to composition, differences in viscosity can influence the diffusion of the natural antimicrobial and its action. Therefore, it is important to study different foods since their composition and rheological characteristics vary, influencing different nNA. Although in vitro studies are very relevant, they cannot be extrapolated to food, demonstrating the importance of a more excellent linkage between NA research in vitro and in situ.+, Mg2+, and K), nucleic acids, and proteins, in addition to inhibiting methylglyoxal. A reduction in carbohydrate catabolism in the presence of EO is also one reason for the inhibitory action of mycotoxins’ occurrence [Of the articles selected in our studies, some analyzed the mechanisms of action of nNA. In these studies, the primary mechanism of action involves damage to the cell membrane with cell constituents and cell lysis release. Such damage to the cell membrane can be irreversible, and components such as proteins and nucleic acids can be released. In the case of fungi, damage to the plasma membrane also occurs, inhibiting the content of ergosterol, causing cell leakage of ions . However, when applying cinnamon oil, it has been shown to reduce the TVB-N formation, being that the nanoencapsulated form decreased the TVB-N from 2.5% to 15% compared to the free form . These dThe results so far are promising, showing that natural antimicrobials can also increase antioxidant activity in food and act on food preservation. However, sensory and rheological characteristics are also essential to be analyzed so that the insertion of natural antimicrobials does not reduce the acceptance of food by the consumer. Orange juice added with sweet orange EO nanoemulsions showed a 14% reduction on the hedonic scale than the control . No significant difference was observed for apple juice added from the same nanoemulsion . HoweverThe addition of nanoemulsion containing cinnamon oil in sea bass fillets reduced the values of b*, increased the value of a*, and the effect on L* depended on the concentration used . DespiteOnly one study analyzed the rheological parameters of the food after the addition of NA. The nanoencapsulated form of cinnamon oil, an increase in hardness (12.57% to 52.27%) and reduction in cohesiveness (1.56% to 2.85%), and springiness (3.53% to 3.65%) in sea bass fillets were observed regardless of the concentration used. For adhesiveness, a reduction of 15.5% was observed for the concentration of 1429 mg/L and an increase of 6.81% in the concentration of 11,429 mg/L. When NA is added in the free form, lower hardness values can be attributed to interactions between cinnamon oil and sea bass fillet proteins, decreasing the muscle’s water-binding capacity. The hardness values decreased with the storage time for the control treatment (without NA) due to bacterial action and enzymatic autolysis. However, the samples added of NA maintained their hardness which corroborates the antibacterial activity of NA .Therefore, it was observed that sensory, rheological, and composition parameters can be altered with NA, and this alteration is different when NA is nanoencapsulated. In general, nanoencapsulation prevents negative changes in the sensory characteristics of food, which is highly relevant for consumer acceptance. Further studies are needed to understand better the possible effects of free and nanoencapsulated NA on foods’ different characteristics and properties.We verified that the review’s principal method used in the nanoencapsulation of NA applied in food is emulsification. We believe that this fact is related: (i) to the mainly hydrophobic characteristic of NA, especially the EO, (ii) to the different ways (high and low energy) for obtaining emulsions and, (iii) to the fact that they demonstrate a certain ease in penetrating the bacterial cell membrane. At the end of the nanoencapsulation process, factors such as EE, particle size and homogeneous distribution, stability, and resulting charge are essential for the nanoencapsulated NA’s antimicrobial activity. They are often not analyzed in the studies.Lipids, proteins, polysaccharides, and carbohydrates are provided to be the most common food-grade materials used as wall material in an NA nanoencapsulation system. These materials are inexpensive, non-toxic, and compatible with food formulations. However, there is still no consensus as to which wall material is best, as it will depend on the nanoencapsulation process, the core material, and the food to be inserted.In large part, the NA does not act with a single mechanism but a set of mechanisms inhibiting and even destroying the microbial cell. Although the action of free NA can be faster, immediate, it is precisely the slow and controlled release of NA from nanostructures that makes it able to act throughout the food shelf life.E. coli, L. monocytogenes, Salmonella enterica, and S. aureus are frequently evaluated in food. For fruits and cereals, fungal deterioration stands out due to low pH and water content.Among the analyzed microorganisms, The NA’s efficiency, even nanoencapsulated, was different in vitro (the laboratory environment) and in situ (the food matrix). Possible interactions between NA and food constituents can be difficult in diffusion and the action against the microorganisms. Food matrices may differ in composition and characteristics, which may influence the activity of NA in different ways. Moreover, the repair of microbial cells may vary in the food compared to the simple medium.Although nanoemulsion is promising for NA nanoencapsulation, further analysis related to the variation in the composition of foods, especially the fat content, is relevant. A study with low-fat food should also be carried out to verify whether nanoemulsions can increase the fat/lipid content in low-fat foods.A more significant explanation of the antioxidant activity after the insertion of nNA is relevant based on the data found in this review.Studies improving nanoencapsulation methods are relevant to quickly obtaining effective, cheap, and industrially applicable methods. Studies with direct food application are necessary since laboratory means have not shown results compatible with the food matrix’s results. A more significant explanation of the antioxidant activity after the insertion of nNA suggests that more prolonged studies in the food matrix should be attractive due to the slow and controlled release of encapsulated NA. The low-concentration combination of free and encapsulated NA in foods can be attractive due to an immediate action of free NA and a prolonged action of encapsulated NA, without affecting the sensory characteristics of food. Due to possible intermolecular interactions between the natural antimicrobials and components present in the food matrix, we believe that studies should be carried out concerning potential compounds formed from these interactions, ensuring the quality and safety of food. We agree with Lopes & Brandelli, (2018)  that fut


The aim of the present work was to characterize effect and susceptibility biomarkers in non-smokers from those hospitality venues occupationally exposed to ETS comparatively to non-exposed ones. A group of smokers was also included for comparison. The sister chromatid exchange (SCE), micronucleus (MN) and comet assays in whole peripheral blood lymphocytes (PBLs) and the micronucleus assay in exfoliated buccal cells, were used as biomarkers of genotoxicity. Furthermore, a comet assay after ex vivo challenge of leukocytes with an alkylating agent, ethyl methanesulfonate (EMS), was used to analyze the repair capacity of those cells. Genetic polymorphisms in genes associated with metabolism and DNA repair were also included. The results showed no clear association between occupational exposure to ETS and the induction of genotoxicity. Interestingly, the leukocytes from non-smoking ETS-exposed individuals displayed lower DNA damage levels in response to the ex vivo EMS challenge, in comparison to those from non-exposed workers, suggesting a possible adaptive response. The contribution of individual susceptibility to the effect biomarkers studied was unclear, deserving further investigation.Environmental tobacco smoke (ETS) has been recognized as a major health hazard by environmental and public health authorities worldwide. In Portugal, smoke-free laws are in force for some years, banning smoking in most indoor public spaces. However, in hospitality venues such as restaurants and bars, owners can still choose between a total smoke-free policy or a partial smoking restriction with designated smoking areas, if adequate reinforced ventilation systems are implemented. Despite that, a previous study showed that workers remained continuously exposed to higher ETS pollution in Lisbon restaurants and bars where smoking was still allowed, comparatively to total smoke-free venues. This was assessed by measurements of indoor PM In addition, the measurement of a biomarker of tobacco smoke exposure, i.e., cotinine, a metabolite of nicotine, confirmed the employees' exposure to ETS. Although all workers exhibited normal lung function, a proteomics approach identified differentially expressed proteins in the plasma of those ETS-exposed non-smoking workers, suggestive of alterations that may precede the first symptoms of tobacco-related diseases , also known as second-hand smoke (SHS), passive smoke or involuntary smoke is a widespread indoor pollutant of significant public health concern, and a major risk factor for lung cancer and other diseases –5. The mdiseases .in-vitro, in-vivo and biomonitoring studies  and DNA repair  genes.The aim of the present work was to characterize the local and systemic genotoxic effects induced by occupational exposure to ETS in non-smoking workers from Lisbon restaurants and bars and to assess whether the genetic susceptibility could influence the observed effects. The sister chromatid exchange, micronucleus and comet assays in PBLs and the micronucleus assay in exfoliated buccal cells, were used to assess DNA and chromosome damage in ETS-exposed workers comparatively to non-exposed workers from the previously characterized hospitality venues , 19. A gposition , 44–50, 2.5), CO and CO2 in all venues and a full description of the methods and results obtained can be found elsewhere , smoking (Sre) and mixed restaurants and bars with both smoking (Sro) and non-smoking rooms (NSro), as previously described by Pacheco et al. . In addilsewhere , 19.p-value of 0.05 and a difference between the exposed and control groups that corresponds to a 25% higher mean level of MN among the exposed, a minimum of 49 subjects would be needed in each group; if the difference increases to 30%, 33 subjects would be needed. Despite the estimates made and the invitation to participate to a larger number of employees, only 97 accepted to provide blood and buccal cells samples for genotoxic assessment.To estimate the adequate sample size of the study groups, a power analysis was performed based on the frequency of micronuclei (MN) in lymphocytes, a sensitive biomarker of an early biological effect. Based on published and our own data from control groups, the mean frequency of MN was expected to be 7.0/1,000 cells and the SD = 3.0. An 80% power is generally considered as acceptable . To obtaEthics approval for this study was secured from Instituto Nacional de Saúde Dr. Ricardo Jorge (INSA) ethics committee, Lisbon. Each potential participant was informed about the procedures and the objectives of the study and those who accepted to participate provided written informed consent for the collection and utilization of biological specimens. During the medical surveillance phase of the study, each subject was interviewed to evaluate clinical history, demographic and lifestyle information, particularly about smoking habits  or self-reported exposure to ETS at home. According to the inclusion criteria, healthy subjects with more than 18 years, who worked in the above referred hospitality venues for more than 9 h per week and for at least 1 month at the current workplace were included in the study. Excluded were the individuals submitted to X-rays, blood transfusion or surgery between 0 and 2 months before the study and those who suffer or had suffered from cancer. Thus, from the 97 volunteers preselected, 81 were included in effect and susceptibility biomarkers analysis. Detailed contextual data can be found in Pacheco et al. .n = 62) that was subdivided according to ETS exposure on the workplace into the ETS-exposed group  including workers from Sre or mixed restaurants and bars and the non-exposed group  including workers from SFre and Cant; a group of smoking workers  containing workers from SFre and from Sre or mixed venues  and exposed to 32 mM of ethyl methanesulfonate , incubated at 37°C for 30 min, and equally processed. After gel solidification, coverslips were removed and slides were immersed in freshly prepared ice-cold lysis solution  with 10% Dimethyl Sulfoxide  and 1% Triton X-100 (Sigma), for 1 h, at 4°C, in the dark. After lysis, slides were placed on a horizontal electrophoresis tank in an ice bath, immersed in alkaline electrophoresis buffer  in the dark, for 20 min, to allow DNA unwinding. Electrophoresis was then conducted at 25 V , at 4°C for 20 min. Then, slides were rinsed with the neutralization buffer , stained with 125 μg/mL ethidium bromide (Sigma), covered with a coverslip, and kept in a dark, moist chamber. Two slides were prepared for each subject and a “blind” scorer examined 50 randomly selected cells from each slide (100 cells/subject) using a 200× magnification in a Axioplan2 imaging epifluorescence microscope  with an image analysis system . The mean percentage of DNA in the nucleoids tail  and the tail length (TL) were calculated for each worker.The alkaline version of the comet assay was used to evaluate DNA damage in PBLs from each subject and was carried out as described elsewhere , with soThe Cytokinesis-blocked micronucleus assay (CBMN) was carried out as described elsewhere  with minCBPI = (MC + 2BC + 3MTC)/Total Cells.For the SCE analysis in PBLs, cultures were established in duplicate as described previously , with miBuccal exfoliated epithelial cells were smeared onto slides, air-dried and fixed in 80% cold methanol, for 20 min . Slides GSTP1105, GSTM1, and GSTT1) and DNA repair  genes were analyzed using PCR-based assays, according to published methods with minor modifications. GSTT1 and GSTM1 genotype analysis was performed in the same reaction, in a multiplex PCR  following the manufacturer's instructions. DNA samples were stored at −20°C until analysis. Genetic polymorphisms in metabolism  and the duration of exposure to cigarette smoke (active and passive), cigarette consumption (number of cigarettes per day), cotinine concentration and age was explored by Spearman's correlation analysis. The same analysis was also used to explore correlations between the several effect biomarkers analyzed. The effect of gender on the cytogenetic parameters or in the genetic polymorphism was assessed by the Mann–Whitney U-test. Regarding the genetic polymorphisms, deviation from Hardy–Weinberg (HW) equilibrium was assessed with the Chi-Square-test. Statistical analysis using Pearson Chi-Square (2-sided), or two-sided Fisher's exact test were applied to assess differences between studied groups concerning allele distributions. To assess the influence of the genotype on each effect biomarker, non-parametric tests  were applied. Two types of comparisons were made: the influence of the gene variants on the biomarker level within each exposure group and inter-group comparison according to each allelic variant. Due to the low number of homozygous variant carriers of XRCC1194, XRCC1399, PARP1762, and XRCC3241, all subjects harboring variant alleles (homozygous and heterozygous) were pooled together.All statistical analyses were conduct using the IBM SPSS 17.0 for Windows statistical package. The level of significance considered was p = 0.008, unpaired t-test). Most employees stated to work at least 40 h per week . Mean h per week in the E group was significantly lower than in NE group  and in SW group . The average months in the current job was higher in NE group (155.6 ± 151.4) in comparison with the E group (74.9 ± 91.2) and SW group (67.6 ± 54.3). Regarding smokers (SW), the average number of cigarettes smoked per day was 16.5 ± 7.5 (range 3–30), and the mean number of years as a smoker was 22.9 ± 10.9 (range 3–49). Most workers declared not being exposed to ETS out of the work, namely at home, and have no other professional activity where they could be occupationally exposed to ETS. Biological monitoring of ETS exposure was assessed by measurement of the urinary cotinine concentration, a metabolite of nicotine, using gas chromatography–mass spectrometry, as previously reported . The cotinine values obtained for non-smokers fall in the range considered involuntary exposure (/mL) was significantly higher  than in the NE workers group .General characteristics of the studied groups are described in reported . As expeexposure , 67. Amop = 0.003, Mann–Whitney U-test) or to ETS-exposed groups , although these differences were not detected when similar comparisons were made using the mean frequencies of SCEs  , but no significant difference was detected between the group of SW and the NE group , both displaying a similar frequency of MNBC.The results of the cytogenetic and DNA damage effect biomarkers studied are presented in  of SCEs . The meare test) . Unexpecex vivo challenge assay with EMS, the data show that both parameters, tail DNA and TL, were significantly different in ETS-exposed comparatively to the NE group . The level of EMS-induced DNA damage was 26.6% lower in the E group, as compared to the NE group of workers. The individual values of tail DNA for each worker, from E or NE groups, is shown in ex vivo EMS challenge assay, the distribution of the data points from the E workers is always under a threshold of 36%, while NE individuals show a wider range of DNA damage induction, up to 57% , with the E group presenting the lowest values of tail DNA and TL in challenged lymphocytes; no difference was observed between the SW and the NE groups.As to the level of DNA damage assessed by the comet assay, no differences in the percentage of DNA in tail or in the tail length were observed neither between the E and NE groups nor between SW and NSW groups . In respp to 57% . When cop = 0.0004, Chi-square test). A positive correlation was found between MNBC/1000 BC in PBLs and MNC/1000 cells  or NBUD/1000 cells  in buccal cells in the E group, which was not seen in other groups.In Buccal exfoliated cells there were no significant differences between the E and NE groups, neither in respect to the frequency of MNC/1000 cells nor in NBUD/1000 cells. Considering smoking, the SW displayed the lowest NBUD frequencies and the comparison between the SW and NSW showed a significant difference in the frequency of NBUD  and a slightly lower level of DNA damage as assessed by the percentage of DNA in tail , only in the E group. None of the biomarkers was impacted by the age of the individuals. Regarding the smoking habits, there was no influence of the number of cigarettes smoked per day or the number of years of smoking in the biomarkers analyzed. Also, no relationship was found between the parameters that characterize the working time and the effect biomarkers. Furthermore, there was no correlation between the exposure biomarker (urinary cotinine concentration) and each of the effect biomarker, when considering the whole group of individuals or after their stratification according to exposure, i.e., E, NE, and SW groups.The impact of potential confounding factors was analyzed in respect to each of the effect biomarkers considering the total number of individuals studied, or after stratification by exposure. In this sense, data for each biomarker were separately analyzed according to gender, but no significant differences were observed, in spite women presented a slightly increased mean of SCEs when compared to males , except the GSTP1105genotype on the ETS-exposed group. In the study population, considering the polymorphisms in the hOGG1326, XRCC1194, and PARP1762 genes, the prevalent allele was the common allele (+/+). In addition, when considering the GSTT1 gene, the wild-type allele was prevalent in the study population; the E group presented a lower prevalence of the null genotype (17.24%) comparatively to the NE (39.39%). For the GSTM1 gene both genotypes were similarly present , where the E group presented a lower prevalence of the wild-type genotype. No significant differences were observed between SW and E or NE groups for all studied polymorphisms, although the GSTP1 Ile/Ile genotype was more prevalent in the E group (62.07%) than in the SW (31.58%).The distribution of the genotype frequencies relative to the metabolism and DNA repair genes in the studied groups is presented in  present . The disp > 0.05, Mann-Whitney) (data not shown).To ascertain the potential influence of the metabolism and DNA repair genes genotype in the genotoxic outcomes, the measurements of chromosome and DNA damage in individuals carrying the common or the variant alleles were compared both in the total study population and in each study group , 6. WhenGSTP1, GSTM1, and GSTT1 polymorphisms, no significant differences amongst the possible genotypes were observed, within SW, NE or E groups, for the frequencies of SCEs, MNBC or DNA strand breaks (% tail DNA). It may be noted that, in the SW group, the frequencies of SCEs, HFCs (data not shown) and MNBC were higher in the subjects with the GSTP1 variant allele , comparatively to those of the WT or heterozygous carriers. The E group maintained the overall trend of lower MNBC comparatively to both SW and NE group which was not influenced by the genotype. On the other hand, SW individuals with the GSTM1 allele present, showed significantly increased MNBC when compared to E group, while GSTT1 wild-type individuals from NE group presented increased MNBC when compared to E group . In respect to the EMS-induced DNA damage, irrespective of the genotype, the already observed lower levels of DNA damage in the E group comparatively with the NE or SW group is maintained either for the GSTP1, GSTM1, or GSTT1. However, workers' stratification according to their genotype, lead to a lower statistical power, due to the small samples size. This is reflected in the GSTP1 and GSTM1, where for GSTP1 the difference between the ETS-exposed and NE groups only became significant for the subset of the WT allele carriers whereas for the GSTM1 polymorphism significance was detected for the comparison between the null allele carriers. Interestingly, in the case of SW, in the absence of the GSTT1, a lower level of DNA damage was observed comparatively to GSTT1 wild-type individuals, an effect that was opposite of the observed in both E and NE groups.Considering the XRCC1194, XRCC1399 and XRCC3241, hOGG1326 and NBN185 polymorphisms, no significant influence of the genotype was observed on the frequencies of SCEs, HFCs (data not shown), MNBC or tail DNA, within NE, E, or SW groups. In respect to PARP1762 polymorphism, a significant difference in the frequency of MNBC was observed within the E group (p = 0.041), with the variant allele carriers showing a lower frequency of micronucleated cells than the WT ones. Overall, the E group maintained the trend of lower MNBC and EMS-induced DNA damage comparatively to both SW and NE group.For the Environmental tobacco smoke is a serious public health concern, recognized as one of the most common indoor pollutants worldwide. Many countries have already successfully implemented smoke-free laws for indoor public spaces and workplaces aimed at limiting exposure to ETS. In Portugal, since 2008, a partial smoke-free law is in place, allowing exceptions, as for example in the case of restaurants, bars or discotheques where smoking is allowed in smokers' designated areas if adequate reinforced ventilation systems are implemented. Thus, exposure to ETS still happens in some Portuguese restaurants and bars , 17, 19,In the present study, no effect could be ascribed to ETS exposure in relation to the basal level of DNA damage, as assessed by the comet assay in leukocytes. A similar negative result was obtained when comparing smokers to non-smokers, irrespectively of the ETS exposure. The effect of smoking on DNA damage has been thoroughly studied, mainly as a confounding factor in biomonitoring studies addressing exposure to other compounds , 69. Desex vivo with a genotoxicant  and measuring induced primary DNA lesions with the comet assay is a functional assay that allows an indirect measurement of the DNA repair competence, which is critical to prevent permanent genetic instability (2O2-induced DNA damage) showed that never smokers not exposed to ETS, had the highest rates of repair of H2O2-induced breaks and that the lymphocytes of active smokers exhibited a consistent repair rate at the two administered doses (100 and 200 μM), slower that never smokers, possible due to the presence of high levels of DNA lesions, hardly or not at all repaired (6-methylguanine-DNA methyltransferase (MGMT) activity in the normal tissue of smokers compared to non-smokers although these data awaits conclusive proofs , as compared to non-exposed workers. Our finding agrees with the negatives data reported in previous studies in workers exposed to ETS in restaurants  and in aex vivo blood cells exposure to EMS. Taken together, the results from both assays may suggest that continuous and repetitive exposure to low level of ETS stimulates a cell-protective response. However, it must stressed that the long-term health consequences from this continuous stimulation cannot be foreseen. A study addressing the effect of ETS exposure on children showed a 30% increase in the frequency of MN  and former smokers displayed slightly reduced MN frequencies in comparison to non-smokers , 97. An cy of MN , althougcy of MN , 98, 99.cy of MN . This wacy of MN . Althougcy of MN , 102, thThe analysis of MN and NBUD in buccal cells, as a biomarker of local effect, did not detect differences in the number of MNC and NBUD between the E and NE groups, with both groups presenting MN frequencies similar to the average reported for healthy population  –103. A pThe remaining confounding factors, either related to smoking habits or working time did not influence the effect biomarkers analyzed. Furthermore, there was no correlation between the exposure biomarker (urinary cotinine concentration) and each of the biomarkers of effect, when considering the whole group of individuals or after their stratification according to exposure, i.e., E, NE, and SW groups.ex vivo acute genotoxic stimulus, ETS-exposed individuals displayed a higher competence to repair DNA damage than unexposed individuals, which might be related with an adaptive response triggered by the prolonged exposure to a low level of tobacco smoke components.Overall, the results of a set of biomarkers of early biological effects showed that ETS did not induce DNA or chromosome damage in blood cells from non-smoking exposed workers. Furthermore, following an PARP1762 polymorphism. PARP1 is an enzyme involved in the cellular response mechanisms to DNA damage. It participates in DNA base excision repair, single- and double-strand break repair pathways, that are active in the prevention of deletions/insertions induced by alkylating agents. PARP1 polymorphism Val762Ala (rs1136410 T>C) may be associated with prostate cancer  genotype was associated with an elevated risk of esophageal squamous cell carcinoma in smokers compared to T/T or T/C genotype (PARP1762 homozygous wild-type genotype modulated the MNBC frequency, which was lower in variant carriers, within the E group. Considering the above-mentioned studies, it would be expected that the variant allele would rather be associated with increased MNBC frequency. When looking at EMS-induced DNA damage, the PARP1762 variant allele carriers presented an increase of DNA damage in E group comparatively to the wild-type carriers, although not significant, due to small number of variant allele carriers. It can be suggested that the partial suppression of PARP1 activity in the variant carriers might have led to an overload of genetic damage in cells, triggering cell death mechanisms and removal of most damaged cell, thereby masking a genotoxic effect. Besides, a decreased MNCB frequency was observed in the variant allele carriers from E group compared to NE, similarly to the slight effect observed in E workers with normal allele, thus suggesting a possible interaction between tobacco smoke exposure and the genotype, in E group, in the genotoxic outcome observed. According to recent meta-analyses, GSTM1 and GSTT1 deletion as been associated with lung cancer in overall population (GSTP1105, in overall population (XRCC1399 and XRCC1194 polymorphism were significantly associated with lung cancer risk in caucasions (NBS1195 in Asians, but not caucasions (XRCC3241 and hOGG1326 genotype subjects (Genetic susceptibility biomarkers, such as the inherited capacity for xenobiotic biotransformation and DNA damage repair, indicate individual differences that can modulate the response to genotoxic insults , 47. Thue cancer , esophage cancer , and bree cancer . Recent ulations , 110. InPARP1762 , while Agenotype , 112. In762Ala rs6410 T>C pulation , while npulation . Concernucasions , and NBSucasions . No relasubjects –120. OveIn the present study the results of genotoxicity biomarkers used to assess the early biological effects of ETS in non-smoking workers are presented. The most relevant effect detected in restaurant workers exposed to indoor tobacco smoke was a modified response to a genotoxic challenge, compatible with an adaptive response. It remains to be determined, however, whether the induction of this kind of response may have long term consequences to the health of those workers. The other effects biomarkers characterized in lymphocytes or oral mucosa cells did not detect significant changes in ETS-exposed comparatively to unexposed workers. Although the contribution of individual susceptibility to the outcomes of this exposure did not generate conclusive results, it deserves further attention. Further investigation is also needed to better understand the mechanisms underlying the possible adaptive response reported in this work and its implication to human health. Ongoing work will try to address the relationship between ETS exposure, biomarkers of effect and alterations of the proteome.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Comissão de ética do Instituto Nacional de Saúde Doutor Ricardo Jorge. The patients/participants provided their written informed consent to participate in this study.NV, SA, and HL carried out the experiments and drafted the manuscript. MS and HL conceived the experimental strategy, supervised the work, participated in the data analysis and discussion, and in the manuscript writing and revision. TS and DP were the project coordinators and implemented the study logistics. FV was involved in workers recruitment, contextual data, and samples collection. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Low energy availability (LEA) may persist in rugby players. However, timely assessment of energy balance is important but is difficult. Therefore, a practical index that reflects energy availability (EA) is essential. A total of 19 male college rugby players participated in a 2-week pre-season summer camp. Their blood sample was collected after overnight fast prior to (Pre), in the middle (Middle), and after (Post) the camp. Their physical activity in the first half of the camp was calculated using the additive factor method in the forwards  and backs . The participants were categorized as tight five , back row , and BK for analysis. All the participants lost weight during the camp (range: from −5.9% to −0.1%). Energy balance in the first half of the camp was negative. Transferrin saturation (TSAT) and serum iron levels significantly decreased to half, or even less, compared with the Pre levels at week 1 and remained low. The changes in TSAT and serum iron levels exhibited a significant positive correlation with the changes in body weight  and with energy intake  in T5. LEA occurs in rugby summer camp but is difficult to assess using weight change. Alternately, TSAT and serum iron levels after overnight fast may be better predictors of LEA. Rugby is a contact sport that involves intermittent high-intensity anaerobic exercise followed by prolonged low-intensity aerobic exercise ,2. The aRugby is also characterized by different competitive characteristics depending on the position. Forwards  are classified into front rows , second rows , and back rows  ,8. FR anThe energy expenditure of rugby players is also affected by the game season  and the physique of the player ,18. In aProlonged LEA can reduce metabolic efficiency and increase the risk of fatigue fractures, as well as adversely affect protein synthesis, cardiovascular and psychological health, and physical growth and development . This prOne of the factors that can lead to LEA is the difficulty in assessing the state of EA, including the assessment of energy intake and expenditure. A review in 2018 noted the lack of reports that measure the dietary intakes of elite rugby players in general . A recenThis study proposes the use of transferrin saturation (TSAT) and serum iron as indicators of EA in the short term (1–2 weeks). In this study, a registered dietitian accompanied elite university rugby players during their entire pre-season training camp to assess their dietary intake. Their hematological and biochemical parameters were analyzed. In addition, coaches shared with us in real time the team schedule and the extent of practice sessions. Weight loss was observed in all the subjects before and after the training camp period. This indicates that they may have fallen into LEA. Therefore, this study aimed to find a rapid and practical indicator of EA status from the parameters obtained.A total of 30 male collegiate rugby players participated in a summer camp held from 9 August to 24 August 2017. The participants arrived at their own timing by 8 August after completing their university’s first semester exams.The first half (9 August to 14 August) of the training session mainly consisted of group training divided into forward and back units, whereas the latter half (16 August to 22 August) mainly consisted of open games with other university teams. There was a day off on 15 August.This study investigated the players’ conditions from 9 August to 22 August. Their diet was surveyed by a registered dietitian. Blood tests were conducted on 9 August , 15 , and 22 . The players’ body weights were measured Pre and Post the study period.n = 7), BR , and BK , as the training program was designed separately for FW (T5 and BR) and BK; from previous studies [Until 15 August (Middle), six players (five due to injury and one due to fever) dropped out of the training program. Another five players  dropped out by 22 August (Post). The 11 players who had dropped out were excluded from the analysis. The position and characteristics of the remaining 19 participants who completed the training program are presented in  studies ,11,12, wThis study was originally planned and conducted as a controlled trial to assess the effect of bovine lactoferrin supplementation. However, the intervention (lactoferrin or placebo) did not make any difference in body weight, hematological and biochemical parameters, and dietary intake (data not shown); therefore, the data were analyzed together without distinction by the intervention.The study protocol was approved by the Ethics Committee of Juntendo University Graduate School of Health and Sports Science  and was conducted in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The study protocol was registered to UMIN Clinical Trials Registry (UMIN-ID: UMIN000028554).To prevent LEA and protein insufficiency, the players were instructed to take at least 50 kcal/kg BW/day of energy  and 2 g/The dietary survey was conducted from 9 August to 21 August 2017, by dietary record.Meals were served in the facility’s cafeteria three times a day . The main dishes were common for each player. Cooked rice and side dishes were free for each participant to consume. Each participant weighed the amount of cooked rice and took a picture of it with the weight indicated on the scale. Side dishes were photographed prior to consumption. The photos were submitted to the dietitian for each meal. This enabled us to determine the intake of all main courses, cooked rice, and side dishes. The weights of the main dishes were measured separately. Moreover, the weights of the side dishes were calculated if they were served in portions, and otherwise estimated from photographs. The dietitian instructed the participants to consume all the food provided, and the dietitian and team managers confirmed that there was no leftover food.In addition to meals, the athletes were provided with 2 L of milk and 3 bananas daily. Protein supplement was also provided for each participant. The food supplements provided to all athletes was consumed without any leftovers. All the foods provided were reported to the dietitian along with the type and weight.There were two drink vending machines in the facility, and bottled or canned beverages could be freely purchased. Purchased beverages were photographed and submitted to the dietitian.After practice, the participants were free to shop at a convenience store outside the facility. Each time, any food purchased was photographed and submitted to the dietitian prior to consumption.The participants had no other means of obtaining food other than the aforementioned. However, players were free to consume water, including during practice, and the amounts were not reported.As described above, the type and amount of food, other than water, consumed by each participant was ascertained without omission.The energy and nutrient intake of each athlete was calculated using the nutrition calculation software Healthy Maker Pro 501 . Daily intakes were expressed per body weight (kg) for energy, protein, fat, and carbohydrates and then mg for iron.However, we were not able to determine the amount of food consumed on 14 and 21 August as dinners were served buffet style, and on 15August, which was a day off, the participants visited downtown. Hence, these 3 days were excluded from the days of assessment.Blood was collected from the cubital vein in the morning before eating breakfast.Serum albumin, creatine kinase (CK), ferritin, iron, and total iron-binding capacity (TIBC) were analyzed using a JCA-BM8060 automated analyzer . Hematologic parameters, including white blood cell, red blood cell, hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume, mean corpuscular hemoglobin (MCH), MCH concentration, and platelet (PLT), were analyzed using a Sysmex XE 2100 automated hematology analyzer . A commercial laboratory service, Hoken Kagaku Inc. , conducted the analyses and provided reference ranges of healthy Japanese male adults.Serum hepcidin was analyzed using a commercially available enzyme immunoassay kit . The sample range was provided by the manufacturer.TSAT was calculated as serum iron/TIBC. The reference range was obtained from the MDS Manual Professional Edition.The first half of the training camp (9 August to 14 August) consisted mainly of group training, whereas the second half (16 August to 22 August) consisted of open games with other university teams.The practice program for the first half was designed separately for the FW and BK players, and the duration of each activity was visually monitored by the researcher who recorded the type of practice or training and the time it took place. From these records, the average amount of energy consumed by the FW and BK players was estimated using the additive factor method ,28,29.The type of practice was selected as the equivalent from the “Revised METs (METs) Table of Physical Activity” , which was based on Ainsworth’s reports ,29. The The energy expenditure due to exercise  and the energy expenditure due to activities other than exercise  were calculated by multiplying the sum of each physical activity by 1.05 (METs × h × 1.05).The second half of the training camp was mostly open games; thus, we were unable to record and track the amount of activity for each participant.p-value adjusted via the Bonferroni correction. Dietary intakes between the positions were also compared using the generalized linear model with p-value adjusted via the Bonferroni correction.Data are expressed as median with range or estimated marginal mean with standard error. The changes in body weight were compared using Wilcoxon signed-rank test. The changes in hematological and biochemical parameters were analyzed using the generalized estimating equation with s).The changes in body weight, TSAT, and serum iron were calculated as (Post − Pre)/Pre (%), and the correlation with the intake of energy, protein, and iron was assessed using Spearman’s correlation coefficient  was used for the analysis. Statistical significance was set to r| > 0.3 .p < 0.05) and BK (p < 0.05), and the mean decrease (−1.7% and −1.9%) was similar, whereas the mean change in BR was smaller (−1.0%). The change in the BR mean was not significant due to the small sample size (n = 4) (All the participants lost weight during the study period. The weight loss was significant for T5 ( (n = 4) .For players at all positions, TSAT (reference range: 20–50%) and serum iron (reference range: 45–200 µg/dL) levels significantly decreased at Mid to half or less compared with the Pre levels and then remained low until Post. The mean TSAT levels were within the reference range at Pre but remained near or below the lower limit of the reference range at Mid .In T5 and BK, TIBC was significantly higher at Mid than Pre and lower at Post, but the changes were within the reference range (245–385 µg/dL). The means of ferritin were within the reference range (45–200 µg/dL), while they raised at Mid in BR and BK .The mean CK was above the reference range (50–250 U/L) for players at all positions throughout the camp. The other parameters were all within the reference range .During the camp (from Pre to Post), 15 (out of 19) participants, except for 4 in T5, consumed more than the goal for energy , but none of them achieved that for protein (2 g/kg/d). Meanwhile, 14 of the 19 participants consumed more than 8 g/kg/d of carbohydrates. All of them consumed more than 8 mg/d of iron.Dietary intake differed according to the positions of the players . The eneThe intake differences among positions were more apparent in the second half of the camp (Mid to Post) than the first half of the camp (Pre to Mid). BK and BR consumed significantly more energy, protein, fat and carbohydrate than T5. In the first half of the camp (Pre to Mid), BK showed greater consumptions in energy, protein, fat and carbohydrate than T5. In contract, there were no differences in iron intake between positions throughout the camp.The average physical activities were calculated for FW and BK for the first half of the summer camp, since the practice program was designed separately for FW and BK. The exercises were categorized into yoga, team practice, stretch, unit training, skill training, core exercise, weight training, strength and fitness, fitness, cap run, pool, game, and personal training .The average daily physical activity was 64.0 METs × h/kg  and 71.0 METs × h/kg  for FW and BK, respectively. Exercise accounted for 63% for FW and 68% for BK.In the second half of the summer camp, we could not calculate the physical activity as the program mainly comprised of games, and the activity of each participant was not recorded.In the first half of the camp, the energy balance was negative, i.e., energy expenditure was greater than energy intake, for all positions . The EAss = 607) and BK (rs = 714) for TSAT, and T5 (rs = 607) and BK (rs = 619) for serum iron  and iron (rs = 464) for T5, whereas negative correlations were observed for BR and BK  and with serum iron (rs = 0.536). However, negative correlations were observed for BR and BK  turnover daily, this turnover requires energy for the novel RBC production as well as for the degradation of old RBCs. Meanwhile, this turnover is a metabolic process that is thought to be suppressed when energy intake is low. Therefore, LEA could affect the RBC turnover. However, we could not find any literature showing that short-term LEA affects RBC turnover. Therefore, we could not discuss the theoretical mechanism by which TSAT and serum iron reflect EA status. However, the relationship between hepcidin and EA status has been discussed recently.Hepcidin, whose secretion from the liver is induced by exercise, suppresses ferroportin and inhibits the release of intracellular iron to circulation . HoweverThis study has a few limitations. First, dietary survey was conducted for each participant, but the activity level was not individually surveyed. Therefore, we were able to estimate the amount of average physical activity in the first half of the camp, when the program was organized by position, but could not assess the amount of activity of individual participant, especially in the second half of the camp. However ideally, energy expenditure should have been assessed using the doubly labeled water method. Second, body weights were not measured daily; thus, we were unable to examine the relationship between EA and weight in the first half of the camp. Furthermore, the lack of body composition measurements made it impossible to determine changes in fat mass and lean mass. In this study, we were able to demonstrate the relationship between LEA, TSAT, and serum iron in a rugby camp with 19 participants, but the number of participants at each position was limited, especially BR with only 4 participants. In addition, this study was originally designed as an intervention study. Therefore, nine players ingested bovine lactoferrin during the camp. Although significant difference was not observed in any of the parameters reported in this study, the possibility was not deniable that existence of responder/non-responder to bovine lactoferrin affected the results. Therefore, a future study with a larger number of participants will be necessary to generalize the findings of this study.LEA occurs in rugby pre-season camps. However, it is difficult to estimate the energy requirements of rugby players with large individual differences. In this study, a registered dietitian accompanied the rugby players during the pre-season camp to ascertain their intakes and encourage dietary intake; however, as a result, LEA occurred, which could not be assessed during the camp, and therefore, the condition could not be improved. On the other hand, we found that the TSAT and serum iron levels changed dramatically within a week and may be indicative of the EA status. Blood sampling post-overnight fast is a practical procedure even in rugby camps. TSAT and serum iron levels can serve as effective indicators of EA status and may be useful for managing, controlling, and improving the EA status in rugby.


There is increasing interest in identifying biological and imaging markers for the early detection of neurocognitive decline. In addition, non-pharmacological strategies, including physical exercise and cognitive interventions, may be beneficial for those developing cognitive impairment. The Feuerstein Instrumental Enrichment (FIE) Program is a cognitive intervention based on structural cognitive modifiability and the mediated learning experience (MLE) and aims to promote problem-solving strategies and metacognitive abilities. The FIE program uses a variety of instruments to enhance the cognitive capacity of the individual as a result of mediation. A specific version of the FIE program was developed for the cognitive enhancement of older adults, focusing on strengthening orientation skills, categorization skills, deductive reasoning, and memory. We performed a prospective interventional pilot observational study on older subjects with MCI who participated in 30 mediated FIE sessions (two sessions weekly for 15 weeks). Of the 23 subjects who completed the study, there was a significant improvement in memory on the NeuroTrax cognitive assessment battery. Complete sets of anatomical MRI data for voxel-based morphometry, taken at the beginning and the end of the study, were obtained from 16 participants (mean age 83.5 years). Voxel-based morphometry showed an interesting and unexpected increase in grey matter (GM) in the anterolateral occipital border and the middle cingulate cortex. These initial findings of our pilot study support the design of randomized trials to evaluate the effect of cognitive training using the FIE program on brain volumes and cognitive function. Aging is often associated with physical and cognitive comorbidity. Deterioration in cognitive function may lead to mild cognitive impairment (MCI) and subsequently to dementia. There is increasing evidence supporting the value of timely recognition and diagnosis of deteriorating cognitive function . ResearcMCI is a condition in which individuals develop cognitive impairment with minimal impairment of activities of daily living (IADL) . MCI freCognition-oriented treatments (COTs) are receiving increased interest, both from the scientific community and from the general public . StudiesSeveral MRI techniques are useful as biomarkers of aging and cognitive decline in research and clinical settings ,12. ConnThe acquisition of structural MRI is less cumbersome to older participants, since the only requirement is that they adjust to the restrictive environment of the MRI machine and refrain from moving during the scans. Anatomical connectivity can be elegantly studied using diffusion-based MRI, which may detect processes involved in brain aging and dementia ,22. Therhttps://www.fil.ion.ucl.ac.uk/spm accessed on 26 October 2021).MRI morphometry is gaining acceptance as an adjunct biomarker for differentiating normal aging from the accelerated degeneration of brain tissue associated with various dementias ,27,28. MBesides these practical considerations, there is also a major substantial incentive to include morphometry in an intervention study. Gradual atrophy of brain tissue is the rule in adults, and this is even more so in the aging brain ,30,32,33This study aimed to examine the effect of the mediator-based Feuerstein Instrumental Enrichment (FIE) cognitive training program on the structure of the brain and on cognitive outcomes in older participants with MCI. Our goal was to explore whether this intervention would result in beneficial effects on brain morphometry and cognition.We performed a prospective interventional pilot observational study of older subjects with MCI who participated in a period of cognitive training using the FIE program. Measurements included cerebral volumetric MRI and functional MRI imaging to determine the association between this form of cognitive training and changes in imaging metrics. While the functional imaging data are still being processed and analyzed, we report the preliminary findings of the morphometric measurements of the brain over time as well as the cognitive outcomes based on a computerized cognitive assessment battery.www.mocatest.org accessed on 26 October 2021) was administered for initial cognitive screening, and those who received a score of 18–26 (inclusive), which is compatible with a diagnosis of MCI [All participants were residents of the same assisted living center. They were recruited following a lecture explaining the research study. A total of 55 subjects expressed their willingness to be screened for the study and provided their written informed consent. Demographic and medical background data were collected, and a diagnosis of MCI was made based on clinical criteria . A MoCA s of MCI , were inWe excluded those subjects with a medical or functional condition that would not allow them to participate in the study such as those with an unstable or symptomatic medical condition, depression, bipolar disorder, schizophrenia, or dementia. Subjects with other comorbidities, including cardiovascular disease, cerebrovascular disease, or diabetes, were included if their condition was stable. Those with a MoCA test score of 17 and below or 27 and higher were excluded. Subjects who could not perform an MRI due to the presence of metallic or electronic implants were not included. Based on the criteria outlined above, 23 subjects were considered suitable for inclusion in the study.Since all participants were recruited in a single residential facility, we divided the subjects into two groups . This was done both for administrative reasons (staggering the assessments and periods of intervention) as well as to allow for an extended baseline assessment for functional MRI (two fMRI examinations 15 weeks apart as a waiting period prior to commencement of the intervention in Group B). All participants underwent cognitive and MRI  assessments at baseline, cognitive, and functional MRI assessments post-intervention, and cognitive and MRI  assessments at one-year post-baseline .The MoCA  was deveThe CogSym instrument comprises 10 questions regarding cognitive symptoms and daily instrumental functional abilities. Subjects are requested to rate their ability on a Likert scale from 1–5  . The symDeveloped by the World Health Organization, this questionnaire comprises 5 items related to subjective well-being ,39. The https//www.neurotrax.com accessed on 26 October 2021). The NeuroTrax battery contains standard neurocognitive tests adapted for use in a computerized platform. The tests include verbal and non-verbal memory, Stroop interference, go/no-go, catch game, information processing, visual spatial and verbal functions as well as motor skills [This computerized cognitive assessment battery was designed for widespread clinical and research use in evaluating cognitive function (r skills . This bar skills . The resn = 13) was used as the experimental group (pre–post FIE intervention) and Group B (n = 10) as a control group (pre–post waiting period) to compare the cognitive changes over time. Statistical analysis was performed by investigating group-by-time interaction effects using repeated measures analysis of variance (ANOVArm) for each cognitive domain and memory component. We also included in the analysis the initial measures (T0) of the different questionnaires  as covariates. We used the IBM® SPSS® software to perform the analysis for the cognitive data.For the cognitive data analysis, Group A , adapting the optimized protocol described by Good et al. , consistt-test factorial design, adding the participant group as a covariate and selecting the ANCOVA regressors in the SPM analysis options. Each tissue segment was analyzed separately. The threshold applied to the statistical maps was p < 0.001, uncorrected. The reported GM clusters survived a threshold with p < 0.05, corrected for false detection rate.The final, MNI-space segments were fed into a paired The Feuerstein Instrumental Enrichment (FIE) Program is a cognitive training intervention based on structural cognitive modifiability and the mediated learning experience (MLE) theory . The FIEThe FIE program uses a variety of instruments to enhance the cognitive capacity of the individual as a result of mediation. Each instrument consists of a set of tasks with increasing levels of difficulty, and in a process of active learning, recommendations are provided in order to enable the learner to acquire general principles for domains and contexts beyond the original domains presented. The Feuerstein Institute developed a specific application of the classic FIE designed for cognitive enhancement of aging adults (Feuerstein Memory Program developed by the author RSF). Beyond contributing to the preservation and possible improvement in cognitive function, this program aims to promote feelings of competence and independence and benefits the emotional state.The classic FIE instruments include the following tasks: organization of dots, comparisons, spatial orientation, analytical perception, and instructions. These tasks are designed to develop skills related to systematic information gathering, reigning in impulsivity, analysis, and planning. The tasks comprising the Feuerstein Memory Program that was used in our study are memory-focused and include the following: processing and memory, the social sphere, everyday functioning, temporal orientation, and figural differences. Emphasis is placed on verbalization and outcome definition. Word fluency is addressed in every session. Strategies are taught to aid retrieval and memory, and focus is placed on strengthening the participants’ orientation skills, categorization abilities, deductive reasoning, and memory.Specifically, for this study, the Feuerstein Instrumental Enrichment (FIE) Program comprised a combination of classic IE instruments as well as novel instruments. In each session, both classic and novel IE instruments were included in the training program. In our study, FIE was administered in group settings by instructors who were specifically trained to mediate based on the needs of the older population including increased font sizes, larger spaces between the dots, and other adaptations. Subjects participated in 30 mediated FIE sessions . Attendance data were gathered throughout the intervention period.A total of 23 subjects completed the FIE program intervention (13 in Group A and 10 in Group B). The baseline characteristics of the study cohort are presented in Some of the participants found multiple imaging sessions to be challenging and, therefore, complete, two-session sets of volumetric MRI data for voxel-based morphometry were obtained from only 16 participants, eight participants from each group, five men and 11 women. The mean age was 83.5 (SD 5.0) years. Ten of the participants were widowed, five were married, and one was single.Voxel-based morphometry maps of the relative changes in brain tissues over one year in those who participated in cognitive training showed an increase in GM in two regions : (1) The3, located bilaterally in the frontal-basal regions and diffusely over the right hemisphere. Increased CSF was noticeable in the lateral ventricles as well as in the anterior longitudinal and the Sylvian fissures in both hemispheres.There was an increase in WM, abating the increased GM in the left occipital lobe. But unlike the limited increase in GM, the WM effect was broader, dispersing over the dorsal parts of both hemispheres. Expected, aging-related changes were also observed B. A decrCognitive data were gathered from a total of 23 subjects including 13 participants from Group A and 10 participants from Group B .p = 0.48), and we did not find any interaction effect  = 0.30, p = 0.59).In the global cognitive score, there was no significant effect of time  = 0.51, p = 0.05) with a much larger improvement in Group A than in Group B but without a significant interaction effect (p = 0.26).In memory, there was a significant effect of time with improvement in memory for both groups  = 4.29, p < 0.01), in delayed verbal memory  = 4.40, p < 0.05) without interaction effect in verbal memory  = 0.19, p = 0.67) and in delayed verbal memory  = 0.21, p = 0.66). In non-verbal memory, we did not find any effect of time  = 0.03, p = 0.88) and no interaction effect  = 1.44, p = 0.25).The significant effect of time was also found in verbal memory  = 9.29, p < 0.05) with a significant improvement in Group A between pre- and post-intervention  whereas no significant difference was found in Group B, apart from a decrease in the delayed non-verbal memory score .In delayed non-verbal memory, we found an interaction effect  = 5.75, p = 0.86) and no interaction effect  = 0.01, p = 0.93).In executive function, there was no main effect of time  = 0.03, p = 0.32) and no interaction effect  = 1.95, p = 0.18).In attention there was no main effect of time  = 1.02, p = 0.09) and no interaction effect  = 2.09, p = 0.16).In visual spatial abilities, there was no main effect of time  = 3.22, With regard to the MOCA, the metacognition questionnaire and the well-being questionnaire, no effects were found.Our results indicate that the FIE cognitive training program may induce a sustained, localized increase in GM and WM in older individuals with MCI. This finding is important, since research and clinical protocols that use MRI-based measurements to evaluate brain atrophy in older adults usually demonstrate an association between accelerated brain tissue degeneration and cognitive decline. However, few reports have described an increase in GM over time in adults.In a previous study, a localized increase in GM was observed, along with functional and anatomical modifications after a period of practicing simultaneous translation . GM and Interestingly, most of the previous studies involving the older population included physical activities in their interventions ,47,49,50There are few publications that describe GM increase following cognitive intervention alone. One such study applied a default-mode network-based program and found increased GM in healthy older adults  but not Our morphometric estimations also included the WM, which also increased following the cognitive training. This change was noticed near the foci of increased GM but extended to adjacent regions as well as to the right cerebral hemisphere A. Betterg) has been proposed to be engaged when predicting and monitoring the outcomes of decisions during social interactions. In particular, the MCCg processes statistical information that tracks the extent to which the outcomes of decisions meet goals when interacting with others [Behaviorally, the FIE was associated with an improvement in verbal and non-verbal memory. However, an increase in the grey matter was detected in the middle cingulate cortex and not in the retrosplenial cortex-part of the posterior cingulate cortex which is considered to be involved in episodic memory. The midcingulate cortex (MCCh others . The FIEWe must emphasize that there are a number of limitations to our study. Our imaging results were from a small treatment group of 16 participants. The statistical significance that we found in such a small sample must be regarded with caution, and it is difficult to reliably evaluate the effects of confounding variables such as age and gender. Nevertheless, other reported studies ,47,50 baIn this pilot study, we report the findings of a localized increase in both cerebral grey matter and white matter in subjects participating in a cognitive intervention program. These findings are in contrast to the expected age-related decrease in brain tissue over time. We also found a significant improvement in memory, particularly in immediate verbal memory and in delayed nonverbal memory. We believe that the results of this small pilot study support further investigation in larger randomized trials to evaluate the effect of cognitive training using the FIE program on brain volumes and cognitive function.