Life’s Essential 8. We examined the association of total and individual CVH metrics according to Life’s Essential 8 with all-cause and cardiovascular disease (CVD)-specific mortality later in  life.The American Heart Association recently updated its construct of what constitutes cardiovascular health (CVH), called Data were from the National Health and Nutrition Examination Survey (NHANES) 2005–2018 at baseline linked to the 2019 National Death Index records. Total and individual CVH metric scores including diet, physical activity, nicotine exposure, sleep health, body mass index, blood lipids, blood glucose, and blood pressure were classified as 0–49 (low level), 50–74 (intermediate level), and 75–100 (high level) points. The total CVH metric score (the average of the 8 metrics) as a continuous variable was also used for dose–response analysis. The main outcomes included all-cause and CVD-specific mortality.A total of 19,951 US adults aged 30–79 years were included in this study. Only 19.5% of adults achieved a high total CVH score, whereas 24.1% had a low score. During a median follow-up of 7.6 years, compared with adults with a low total CVH score, those with an intermediate or high total CVH score had 40%  and 58%  reduced risk of all-cause mortality. The corresponding adjusted HRs (95%CIs) were 0.62 (0.46–0.83) and 0.36 (0.21–0.59) for CVD-specific mortality. The population-attributable fractions for high (score ≥ 75 points) vs. low or intermediate (score < 75 points) CVH scores were 33.4% for all-cause mortality and 42.9% for CVD-specific mortality. Among all 8 individual CVH metrics, physical activity, nicotine exposure, and diet accounted for a large proportion of the population-attributable risks for all-cause mortality, whereas physical activity, blood pressure, and blood glucose accounted for a large proportion of CVD-specific mortality. There were approximately linear dose–response associations of total CVH score (as a continuous variable) with all-cause and CVD-specific mortality.Life’s Essential 8 was associated with a reduced risk of all-cause and CVD-specific mortality. Public health and healthcare efforts targeting the promotion of higher CVH scores could provide considerable benefits to reduce the mortality burden later in life.Achieving a higher CVH score according to the new The online version contains supplementary material available at 10.1186/s12916-023-02824-8. Cardiovascular disease (CVD) is a leading cause of mortality worldwide . The ecoLife’s Simple 7 , no smoking, normal blood pressure, normal fasting glucose, and normal total cholesterol) to promote ideal cardiovascular health (CVH) [Life’s Simple 7 has several limitations including the confined scope of health behaviors , a crude additive scoring, and less sensitivity to interindividual differences and intraindividual change [In 2010, The American Heart Association (AHA) established th (CVH) . Althougth (CVH) . Meta-anth (CVH) . Howeverth (CVH) . In addil change .Life’s Simple 7, the AHA’s Strategic Planning Task Force and Statistical Committee recently recommended Life’s Essential 8 goals to maintain optimal CVH, which added sleep health, and updated and refined the other 7 components that were part of Life’s Simple 7  [Life’s Essential 8 in relation to mortality. From a public health perspective, it is important to validate the implementation and utility of these new CVH metrics in Life’s Essential 8 to promote awareness and adherence to the current recommendations, thereby reducing the burden of CVD and related mortality.To overcome the limitations of ntial 8) . To our Life’s Essential 8 with all-cause and CVD-specific mortality among US adults. In addition, we also calculated population-attributable fractions (PAFs) of these new CVH metrics scores in relation to mortality risk.Therefore, based on nationally representative data and using the new scoring algorithm, we examined the association of the overall and individual CVH metric scores defined in Data from the National Health and Nutrition Examination Survey (NHANES) conducted during the period of 2005–2018 were used for this study. The NHANES is an ongoing, national, cross-sectional survey with a complex, stratified, and multistage probability sampling design of the noninstitutionalized US civilian population. Information on the introduction, content, and operations of the NHANES is available elsewhere . The NHAn = 9534), those with missing data on cardiovascular metrics (n = 8208) or potential covariates (n = 1747), those who were pregnant or breastfeeding (n = 288), and those with ineligible data on death or follow-up years (n = 21), a total of 19,951 adults aged 30–79 years were included in this study. The basic characteristics between follow-up participants and those without CVH metrics are shown in Additional file As sleep health information was first collected in the 2005–2006 NHANES, we used NHANES data collected from 2005 to 2018 as baseline data. A total of 39,749 adults aged ≥ 20 years participated in the 2005 to 2018 NHANES, of which 4979, 5935, 6218, 5560, 5769, 5719, and 5569 were in the survey cycles of 2005–2006, 2007–2008, 2009–2010, 2011–2012, 2013–2014, 2015–2016, and 2017–2018, respectively. After excluding participants with the age of < 30 or > 79 years old . The AHA proposed a new method for assessing dietary quality based on quantities of adherence to the Healthy Eating Index 2015 at a population level  [Physical activity was assessed based on self-reported minutes of moderate or vigorous physical activity per week collected by a questionnaire. Physical activity time was classified as 0, 1–29, 30–59, 60–89, 90–119, 120–149, and ≥ 150 min per week, corresponding to 0, 20, 40, 60, 80, 90, and 100 points, respectively [Life’s Essential 8. There are 5 nicotine exposure categories including current smoker, former smoker (quit < 1 year) or currently using inhaled nicotine delivery systems, former smoker (quit 1 to < 5 years), former smoker (quit ≥ 5 years), and never smoker, corresponding to 0, 25, 50, 80, and 100 points, respectively). Twenty points were subtracted for adults living with active indoor smokers at home [Information on self-reported nicotine exposure collected in the questionnaire was used. In addition to combustible cigarette use, the AHA added the use of other inhaled nicotine delivery systems such as vaping devices, e-cigarettes, and secondhand tobacco smoke to the definition in  at home .Life’s Essential 8 goals based on the self-reported average hours of sleep per night collected by a questionnaire. Sleep hours per night were classified as < 4, 4 to < 5, 5 to < 6 or ≥ 10, 6 to < 7, 9 to < 10, and 7 to < 9 h, corresponding to 0, 20, 40, 70, 90, and 100 points, respectively [Sleep health was a new metric in 2, corresponding to 0, 15, 30, 70, and 100 points, respectively [Objectively measured weight and height were used to calculate BMI . BMI levels were classified as ≥ 40.0, 35.0–39.9, 30.0–34.9, 25.0–29.9, and < 25.0 kg/mBlood samples were used to measure the total and high-density lipoprotein (HDL) cholesterol. Non-HDL cholesterol was calculated as total cholesterol subtracting HDL cholesterol. Non-HDL cholesterol levels were classified as ≥ 220, 190–219, 160–189, 130–159, and < 130 mg/dL, corresponding to 0, 20, 40, 60, and 100 points, respectively [Fasting blood samples were used to measure fasting blood glucose (FBG). Both fasting and non-fasting blood samples were used to measure HbA1c. Blood glucose levels were classified as diabetes with HbA1c ≥ 10.0%, diabetes with HbA1c of 9.0–9.9%, diabetes with HbA1c of 8.0–8.9%, diabetes with HbA1c of 7.0–7.9%, diabetes with HbA1c < 7.0%, no diabetes and FBG of 100–125 mg/dL or HbA1c of 5.7–6.4%, and no history of diabetes and FBG < 100 mg/dL or HbA1c < 5.7%, corresponding to 0, 10, 20, 30, 40, 60, and 100 points, respectively [Blood pressure was measured using an appropriately sized cuff. Blood pressure levels were classified as systolic blood pressure ≥ 160 mmHg or diastolic blood pressure ≥ 100 mmHg, 140–159 or 90–99 mmHg, 130–139 or 80–89 mmHg, 120–129/ < 80 mmHg, and < 120/80 mmHg, corresponding to 0, 25, 50, 75, and 100, respectively. Twenty points were subtracted if the blood pressure levels were treated [We calculated the total score using the average of all 8 individual cardiovascular metric scores according to AHA recommendation .Baseline data from NHANES 2005–2018 were linked to mortality data from the National Death Index death certificate records until December 31, 2019, matched using a probabilistic matching algorithm to identify mortality status . The outStudy covariates included sex , age group , race/ethnicity , educational level , marital status , and the ratio of family income to poverty .P < 0.05 was considered statistically significant.We calculated the mean scores and 95% confidence intervals (CIs) and the mean levels and 95% CIs of each CVH metric by total CVH scores of 0–49 (low), 50–74 (intermediate), and 75–100 points (high), which were determined according to the subsequent dose–response relationship between CVH score and mortality. We also estimated the proportions (%) of sex, age group, race/ethnicity, education level, marital status, and the ratio of family income to poverty by low, intermediate, and high levels of total CVH scores. A direct method of standardization was used to calculate the age- and sex-standardized rates of all-cause and CVD-specific mortality per 1000 person-years  according to the three categories of the total score of CVH metrics . Kaplan–Meier survival curves were generated for the calculation of cumulative mortality using three score categories of CVH metrics , compared using the log-rank test. Competing risk analysis was used to estimate the cumulative incidence of CVD-specific mortality in consideration of non-CVD deaths as the competing events. We used Cox proportional hazards regression models to examine the associations of combined and single CVH metric scores (low level as the reference) with all-cause and CVD-specific mortality with adjustment for sex, age, race/ethnicity (model 1), and full covariates (model 2) . When the association between each CVH metric and mortality was evaluated, this metric was excluded from adjustment. The adjusted PAFs of high (score ≥ 75 points) vs. intermediate or low CVH score (score < 75 points) were estimated for the proportion of all-cause and CVD-specific mortality that could be avoided if participants with low or intermediate CVH scores achieve high CVH scores . FurtherP < 0.001). Adults with a history of heart disease or stroke were less likely to have high CVH scores .The characteristics of participants across the three categories of total CVH score  are shown in Table P < 0.001 for all log-rank tests, Fig. The age- and sex-standardized mortality densities of all-cause and CVD per 1000 person-years among adults with total CVH scores at the intermediate and high levels were significantly lower than those with low level . However, the scores for BMI and blood lipids were not significantly associated with the risk of all-cause mortality. The adjusted PAF of high (score ≥ 75 points) vs. low or intermediate (score < 75 points) total CVH score with all-cause mortality was 33.4%, with the individual metric scores ranging from 4.7% for blood glucose to 16.5% for physical activity; when a score of ≥ 85 points was used to define “high score,” the adjusted PAF was 33.6%, with the individual metric scores ranging from 6.1 to 25.2% (Table During a median follow-up of 7.6 years (interquartile range: 4.3–10.9 years), compared with adults with a low total CVH score, those with intermediate or high scores had 40%  and 58%  reduced risk of all-cause mortality after adjustment for all potential covariates (Table P for trend < 0.05). The adjusted PAF of high vs. low or intermediate total CVH score with CVD-specific mortality was 42.9%, with individual CVH metrics ranging from 2.2% for sleep health to 17.8% for physical activity; when a score of ≥ 85 points was used to define “high score,” the adjusted PAF was 70.6%, with the individual metric scores ranging from 2.2 to 16.2%  and 64%  reduced risk of CVD-specific mortality after adjustment for all potential covariates. Similarly, higher scores of blood glucose were significantly associated with a reduced risk of CVD-specific mortality. Although there were non-significant associations for other individual CVH metrics including physical activity, sleep health, BMI, and blood pressure, similar trends were observed with higher scores associated with a reduced risk of CVD-specific mortality . Our findings lend support to the new CVH metrics as part of Life’s Essential 8 to have utility in the prediction of future all-cause and CVD-specific mortality.Based on a large nationally representative sample of US adults, we found that adults with a high level of total or individual CVH metrics in Life’s Simple 7 on the development of diabetes, CVD, quality of life, and mortality later in life has been shown previously [Life’s Simple 7 recommended by AHA in 2010, which may oversimplify the association between CVH metrics and mortality risk. For example, two individuals with largely different amounts of physical activity  would both be classified as having intermediate physical activity. However, the dose–response analysis showed that the associations between these two doses of physical activity and mortality risk were substantially different [Evidence to support a protective effect of ideal CVH in eviously , 13–27. eviously . Howeverifferent , 29.Life’s Essential 8, we found that a higher total CVH metric score was associated with a largely reduced risk of all-cause and CVD-specific mortality in a dose–response manner. Our findings suggest that a small improvement in total CVH score could lead to a large reduction in mortality. This is a very important finding of the present study. Particularly among those who might now have CVD or other chronic diseases , the improvements in CVH score will still improve their risk relative. So, the improvement at any level of the CVH score has a benefit based on the spline plots, and those with the lower CVH score seem to benefit more from a small improvement. In all, the linear dose–response finding of our study suggests any improvement matters and especially for those with very low CVH. That is some improvements are better than none.Based on an enhanced scoring algorithm ranging from 0 to 100 recommended in Life’s Essential 8 and underscore its key role in reducing the risk of mortality. We also found that nicotine exposure was the second important individual contributor to all-cause morality. Burnt cigarette and e-cigarette use is a serious public health issue among US adults and youth [Life’s Essential 8 by the AHA where nicotine exposure includes the use of inhaled nicotine delivery system and secondhand smoke exposure [For the individual health behaviors, we found that physical inactivity was the major individual contributor to both all-cause and CVD-specific mortality. The association of adequate physical activity substantially reducing the risk of subsequent mortality is not new , 20, butnd youth , 31, witnd youth , 20. We exposure .Life’s Essential 8 was the assessment of diet. In Life’s Simple 7, the AHA recommended the use of only 5 aspects of diet to assess the dietary score, which included intakes of fruits and vegetables, fish, fiber-rich whole grains, sodium, and sugar-sweetened beverages [Life’s Essential 8, the AHA proposed a new, more comprehensive method for the assessment of dietary quality that incorporates the use of DASH-style eating patterns or the Healthy Eating Index 2015 for population-level assessment and Mediterranean-style eating patterns for individual assessment . In this study, we used the Healthy Eating Index 2015 to assess dietary quality which was linked to mortality outcomes. In 2019, a national cohort study based on continuous NHANES surveys from 1999 to 2016 at baseline linked to 2011 mortality data showed that a diet score ≥ 69.3 using the Healthy Eating Index 2010 was associated with a reduced risk of all-cause mortality [In addition, we found that an unhealthy diet was the third important individual contributor to all-cause mortality. There is much strong evidence supporting the benefits of healthy diets in the reduction of mortality risk , 33. An Life’s Essential 8 by the AHA was the inclusion of a metric for sleep [Life’s Simple 7 [A large body of evidence has shown short (< 7 h) or long (> 8 h) habitual sleep duration associates with all-cause and CVD-specific mortality , 35. As or sleep  in additLife’s Simple 7 [Life’s Essential 8) was one of the three leading contributors to CVD-specific mortality. The difference might be explained by the change in definition for the fasting blood glucose metric in Life’s Simple 7, which likely did not comprehensively reflect the glycemic status and contribution to risk, and the various reference groups were used . Using nd HbA1c , our finLife’s Simple 7 and the risk of cardiovascular events or mortality [Life’s Essential 8, was also not associated with all-cause and CVD-specific mortality. We also found that blood lipid score was not associated with all-cause and CVD-specific mortality, which was also consistent with previous data [Life’s Essential 8. These findings suggest that the AHA should rescore the ideal level of BMI or blood lipids in future updates.Consistent with a meta-analysis of 13 prospective studies among 193,126 participants, which determined the association between the ideal BMI metric defined by ortality , we founous data . Indeed,ous data , 37, whiLife’s Essential 8 and mortality risk. In addition, we performed dose–response associations between overall CVH score and mortality risk and found that an increased CVH score significantly lowered the risk of mortality. However, several potential limitations warrant consideration. First, four lifestyle factors including dietary intake, physical activity, nicotine exposure, and sleep health were self-reported, which may lead to a recall bias. Second, despite multivariable adjustment, additional confounding by measured or unmeasured covariates might have influenced the observed associations. Third, we only used measures of 8 CVH metrics in a single visit, and we did not consider metric trajectories or changes over time that might influence the observed association. However, previous studies have shown that CVH scores seem to be relatively stable or decline [Life’s Essential 8 is calculated using equal weights for each of the 8 CVH metrics. Seventh, the generalizability of our results to the whole US population should be made with caution due to the differences in several basic characteristics between follow-up participants and those without CVH metrics.To our knowledge, this is the first large-scale, nationally representative cohort study to examine the association of total and individual CVH metrics using  decline . The potWe found that the prevalence of a high CVH score among US adults was low, and the total CVH score was inversely associated with all-cause and CVD-specific mortality. Physical inactivity, nicotine exposure, and unhealthy diet were the three leading individual contributors to all-cause mortality, whereas physical inactivity, elevated blood pressure, and elevated blood glucose were the three important ones to CVD-specific mortality. Our findings emphasize that primordial and primary prevention efforts on promoting CVH metrics, especially aiming at health factors and behaviors with high PAFs, should be strengthened to reduce early mortality risk later in life.Additional file 1: Supplementary results. Fig S1. Flow chart of inclusion/exclusion of participants. Table S1. Comparisons in differences of basic characteristic between participants with complete data and participants without CVH metrics. Table S2. Adjusted hazard ratios of all-cause and CVD-specific mortality by “Life’s Essential 8” cardiovascular health score and sex, NHANES 2005-2018. Table S3. Adjusted hazard ratios of all-cause and CVD-specific mortality by “Life’s Essential 8” cardiovascular health (CVH) score and age group, NHANES 2005-2018. Table S4. Adjusted hazard ratios of all-cause and CVD-specific mortality by “Life’s Essential 8” cardiovascular health (CVH) score and race/ethnicity, NHANES 2005-2018. Table S5. Adjusted hazard ratios of all-cause and CVD-specific mortality by “Life’s Essential 8” cardiovascular health (CVH) score and education level, NHANES 2005-2018. Table S6. Adjusted hazard ratios of all-cause and CVD-specific mortality by “Life’s Essential 8” cardiovascular health (CVH) score and marital status, NHANES 2005-2018. Table S7. Adjusted hazard ratios of all-cause and CVD-specific mortality by “Life’s Essential 8” cardiovascular health (CVH) score and ratio of family income to poverty, NHANES 2005-2018. Table S8. Adjusted hazard ratios of all-cause and CVD-specific mortality by “Life’s Essential 8” cardiovascular health (CVH) score after excluding adults with a history of CVD. Table S9. Adjusted hazard ratios of all-cause and CVD-specific mortality by “Life’s Essential 8” cardiovascular health (CVH) score after excluding death within the first 2 years of follow-up.


Specifically, decreasing MCP content by 33% (MCP67) led to a smaller increase in non-sedimentable κ-casein and a lower decrease in αs2-casein concentrations after heating compared to other samples. Lower MCP content resulted in a moderate rise in the average particle size and turbidity, along with lower loading of β-turn structural component after heating at low pH (pH 6.3). Notably, MCP113 exhibited instability upon heating, with increased particle size, turbidity, and a significant decrease in non-sedimentable αs2-casein concentration, along with a slight increase in non-sedimentable κ-casein concentration. The FTIR results also revealed higher loading of intermolecular β-sheet, β-turn, and random coil structures, as well as lower loading of α-helix and β-sheet structures in MCP-enhanced skim milk samples. This suggests significant changes in the secondary structure of milk protein and greater formation of larger aggregates.The present study investigated the effect of micellar calcium phosphate (MCP) content and pH of skim milk on heat-induced changes in skim milk. Four MCP-adjusted samples, ranging from 67 to 113% of the original MCP content, were heated (90 °C for 10 min) at different pH values , followed by determining changes in particle size, turbidity, protein distribution, and structure. The results demonstrate a strong effect of MCP level and pH on heat-induced changes in milk, with the MCP Heat treatment is one of the most widely used unit operations in the dairy sector and includes different heat intensities, such as thermisation (~62–65 °C for 10–20 s), pasteurisation (~72–80 °C for 15–30 s), ultra-high temperature (UHT) treatment (~135–150 °C for 1–10 s), and sterilisation (~110–120 °C for 10–30 min) .Milk salts play a significant role in heat-induced changes in milk, particularly in relation to the heat-induced interactions between denatured whey proteins and casein micelles . Early iAs has been stated previously , the hea100) was ~30–31 mmol L−1 and adjustment of MCP content clearly influenced the amount of total calcium, as expected. The lowest calcium level was observed in the sample acidified to pH 6.1 prior to dialysis, i.e., sample MCP67, and the highest concentration of Ca was found in sample MCP113, which had been adjusted to pH 7.5 prior to dialysis. Adjustment of pH to 6.3, 6.6, 6.9, or 7.2 after dialysis had no major effect on the total calcium content  at pH 6.6, 6.9, or 7.2 at 90 °C for 10 min, particle size decreased by 12–22 nm  and levels of non-sedimentable whey proteins in heated samples decreased with increasing MCP content  has six features: intermolecular/aggregated β-sheets (1700–1681 cm−1), β-turns (1680–1660 cm−1), α-helix (1652–1641 cm−1), random coils (1640–1631 cm−1), intramolecular β-sheet (1630–1620 cm−1), and side chains (1618–1608 cm−1) [−1), presumably due to heat-induced aggregate formation. PCA analysis confirmed the distinction between heated and unheated samples by effectively separating them (FTIR was used to fingerprint the changes in the secondary structure of milk proteins as a result of the heat treatment . The FTI08 cm−1) . Figure ing them . Heated ing them . Furthering them .−1), as observed in FTIR analysis. The presence of β-turns in milk proteins is considered a consequence of protein unfolding from higher-order structures [113 samples, an increase in β-turn was observed (67), there were no significant changes compared to unheated milk samples. The changes in β-turns were also influenced by the pH adjustment. The intensity of the β-turn peak showed a negative correlation with pH, indicating that, at lower pH levels (pH 6.3), there was a higher presence of β-turns (−1) in heated MCP-enhanced samples (MCP113), whereas the random coils (1640–1631 cm−1) exhibited a remarkable increase after heating (−1), upon heating were expected. Interestingly, the intensity of the peak in highly MCP-reduced samples (MCP67) was close to the original unheated milk, while β-sheet rose in the control and MCP-enriched samples, which has been related to substantial aggregation [−1) was at lower pH (6.3) in heated skim milk samples, which exhibited substantial denaturation and aggregation of proteins. The rise in the loadings around 1618–1608 cm−1 has also been attributed to disruption of intramolecular hydrogen bonds within a secondary structure [Heat-induced changes in milk caused an increase in the intensity of β-turn peaks (1660–1680 cmructures . Adjustmobserved . However β-turns . In addi heating . Notably heating . Moreoveregation . In part−1) of analysis was able to detect changes in both milk carbohydrates and minerals, as shown in −1 depicts various carbohydrate vibrations, with lactose being the primary component [-1, which indicates the dissociation of phosphate from casein micelle. Conversely, higher pH demonstrated increased absorption (−1) were found to be lower for heated milk when compared to unheated milk  exhibited a lower intensity of peaks at 995–987 cm−1, while MCP-enhanced samples (MCP113) showed a higher intensity of peak around this area (995 and 987 cm−1). The pH adjustment to a low value (6.3) led to a decrease in the intensity of the peak (995 and 987 cm−1), which likely depicted the dissociation of MCP from the phosphoserine residues. Heat treatment also caused lower loading at 995 and 987 cm−1, which is indicative of the impact of heating on calcium solubility, as illustrated in Changes in the intensity of the peaks around 995 and 987 cmhoserine  and are 67). Such an adjustment of the MCP content within the casein micelle has been reported to affect the thermal stability of skim milk expressed as HCT [In addition to time and temperature, the thermal stability of milk is strongly influenced by pH and minerals . The pred as HCT  investigd as HCT . When cod as HCT ,18. κ-CNd as HCT  and in td as HCT . In the −1, suggesting the formation of intramolecular β-sheet aggregates  remained fairly consistent across most of the MCP/pH range, which changed once pH before heating was adjusted to 6.3. A notable trend was observed that the particle size and turbidity were MCP-dependent at this pH—being larger at higher MCP content , accompanied by a remarkable increase in the presence of random coils (1640–1631 cm−1) after increasing the MCP content in skim milk followed by heating  upon reducing the MCP content and heating  was added to the milk. According to a protocol described previously [67 sample thus had its MCP content reduced from the initial 100% to 67%. Conversely, in the MCP113 sample, the MCP content increased by 13% compared to the initial MCP amount of 100%. Then, the pH of the samples was adjusted to 6.3, 6.6, 6.9, or 7.2. Once the pH was stable, the samples were heated in an oil bath set at 90 °C. The time to reach 90 °C was ~3 min, after which samples were held for a further 10 min at this temperature and then cooled to 20 °C by immersion in an ice bath.The design of the experimental work is schematically presented in eviously , a predeeviously ,27. Afteg for 1 h at 20 °C in a Beckman Ultra L-70 centrifuge . After the ultracentrifugation, the clear supernatant was carefully collected from each tube using a syringe.Unheated and heated milk samples were fractionated by ultracentrifugation at 100,000× The total calcium (Ca) of the unheated MCP-adjusted pH-adjusted skim milk samples was determined using an inductively coupled plasma atomic emission spectrometer , following the method of Bijl et al. .Turbidity of the samples was measured at 860 nm using a 1 mm pathlength quartz cuvette using a UV-Visible spectrophotometer .Particle size analysis of samples was performed by dynamic light scattering  at a scattering angle of 90° and temperature was maintained at 25 °C. Samples were diluted in simulated milk ultrafiltrate (SMUF)  in a ratIndividual caseins in whole samples and ultracentrifugal supernatants were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) at room temperature using a Shimadzu HPLC system  with a Varian 9012 system controller  coupled with an RI detector  and a C4 column  using pretreatment and elution conditions as described previously by Aprianita et al. −1. At the start of measurement, the background spectrum was scanned with a blank (SMUF) and corresponding ultracentrifugal supernatant using the same instrumental conditions as for the sample spectra acquisition [FTIR measurements were conducted using an FTIR spectrometer  in the range of 4000–600 cmuisition . FTIR exuisition .p < 0.05. The design was replicated three times on three different occasions.All experiments assessed the impact of heat treatment on the selected parameters in the MCP-adjusted skim milk. SPSS software  was used to conduct a two-way analysis of variance (ANOVA) to establish differences among means, followed by Tukey’s multi-comparison of the means. The level of significance was set at The result of this study shows a strong influence of MCP adjustment on the thermal behaviour of milk proteins and, consequently, on the heat stability of milk. The key finding achieved by this study was that the sample containing the lowest MCP content appeared to have a high level of intact casein micelles and likely the greatest thermal stability among all samples. The pH level of the milk was also observed to have a direct relationship with its heat stability, with lowered pH levels resulting in reduced heat stability. MCP adjustment leads to differing behaviour of individual caseins, which dissociate to a certain extent at lower MCP content, while remaining at high levels at an MPC level above the initial. This leads to different interactions among proteins in the soluble and colloidal phases and different outcomes. Smaller particle size is observed at low pH and low MCP content; thus, greater heat stability may be expected. Notably, this work provided for the first time an insight into the effect of heat treatment on the conformational changes of MCP-adjusted and enriched skim milk heated at different pH. This can assist in greater understanding of the functional properties of MCP-adjusted skim milk in industrial-scale dairy processing to achieve skim milk with improved thermal and structural stability.


The Hospital Anxiety and Depression Scale (HADS) is commonly used to measure anxiety and depression, but the number of studies validating psychometric properties in older adults are limited. To our knowledge, no previous studies have utilized confirmative factor analyses in community-dwelling older adults, regardless of health conditions. Thus, this study aimed to examine the psychometric properties of HADS in older adults 70 + living at home in a large Norwegian city.In total, 1190 inhabitants ≥ 70 (range 70 – 96) years completed the HADS inventory in the population-based Trøndelag Health Study (HUNT), termed “HUNT4 70 + ” in Trondheim, Norway. Confirmatory factor analyses were performed to test the dimensionality, reliability, and construct validity.D (“I feel cheerful”) along with a correlated error term between item 2D (“I still enjoy the things I used to enjoy”) and 12D (“I look forward with enjoyment to things”) improved the fit substantially. Good to acceptable measurement reliability was demonstrated, and the construct validity was acceptable.The original two-factor-solution (Model-1) revealed only partly a good fit to the present data; however, including a cross-loading for item 6The HADS involves some items that are not reliable and valid indicators for the depression construct in this population, especially item 6 is problematic. To improve the reliability and validity of the Norwegian version of HADS, we recommend that essential aspects of depression in older adults should be included. Depression among older people, either reported as major depressive disorder (MDD) or clinically significant depressive symptoms (CSDS), is associated with decreased quality of life (QOL) , increasOlder adults aged 70 + living at home may have multiple diseases  accompanDepression is prevalent among older adults (≥ 70 years) worldwide, with a point estimate of depressive disorders of 5.4%, including both MDD and CSDS . FurtherCompared to younger age groups, older adults report a higher frequency of anxiety , 19. In Moreover, compared to younger adults, older people are less likely to report and seek help for their symptoms, have less knowledge regarding anxiety disorders and available treatment, and face barriers to treatment such as stigma, cost, transportation, and mobility . Ageism,Possibly, the lack of treatment for clinically relevant symptoms of depression and anxiety represents an alarming concern to the healthcare system and society; health politics highlights the need for health promotion interventions to keep older people in their own homes as long as possible. Undetected issues of depression and anxiety may cause reduced mental and physical functionality and increased risk of dementia and other diseases , 3–5, alIn many older people, depressive symptoms can be difficult to distinguish from age-related symptoms such as sleep disturbance, psychomotor changes, concentration disturbance, changes in nutrition and digestive functioning, and fatigue . FurtherThe Hospital Anxiety and Depression Scale (HADS) is commonly used in epidemiological research to estimate the prevalence of clinically significant anxiety symptoms and CSDS among adults  and in cThe HADS is widely used and tested with satisfactory psychometric properties , 27. HowInitially, the HADS was developed for adults receiving treatment for physical health problems in general hospitals . Still, Utilizing principal component analysis (PCA), three studies have assessed the dimensionality of the Norwegian version of HADS: one assessed older adults admitted to somatic hospitals . In comp1: The original two-factor model of HADS shows a good fit to the present data,H2: HADS demonstrates good reliability,H3: HADS shows good construct validity,H4: HADS correlates significantly and negatively with QOL and.H5: The anxiety and depression factors perform as two distinct concepts.HTo date, the dimensionality, internal consistency, construct validity, and homogeneity of the Norwegian version of the HADS have not been assessed using CFA among community-dwelling older adults ≥ 70 years. Therefore, this study aims to examine the psychometric properties of the HADS among older adults ≥ 70 living at home in Norway; dimensionality, reliability, and construct validity are interrelated measurement properties and are thus investigated in this study. The hypotheses are:1, H4, and H5 are chosen according to the theoretical assumption that HADS with anxiety and depression are two distinct concepts, distinctively different from each other and QOL, and therefore, we assume that these concepts provide good reliability and validity (H2 and H3).The hypotheses are based on theory and evidence , 23–25; During 2017–2019, persons aged ≥ 70 were recruited from one randomly selected district in Trondheim, county of Trøndelag in Central Norway, as a part of the fourth wave of The Trøndelag Health Study (HUNT). HUNT is a population-based cross-sectional study . The HUNIn total, 4667 community-dwelling inhabitants living at home in Trondheim were invited to participate, of whom a total of 1486 (response rate of 31.8%) persons 70 years or older (55.3% women and 44.7% men) participated in the regular protocol and responded to the HADS questionnaire and demographical questions (Q1 and Q2). These participants also underwent a comprehensive clinical evaluation.The inclusion criteria for the present study were: (1) living at home, (2) aged 70 years or older, (3) without dementia, and (4) having responded to all 14 HADS items since this is considered best practice utilizing CFA. In total, 126 participants were diagnosed with a dementia diagnosis, and 170 participants lacked one or more responses on HADS; all of these were excluded from the analyses, giving an effective sample N = 1190.Sociodemographic characteristics measured were age, sex , cohabiting status , and educational attainment .Medical conditions were reported using self-reported items regarding a history of asthma, diabetes, and heart attack. Functional impairment in daily life > 1 year was self-reported using one item (no or yes). Lastly, care support last year in terms of home care, in-home nursing care, and/or hospitalization in nursing homes were self-reported (yes/no).Global quality of life (QoL) was assessed with one item: “Thinking about your life at the moment, would you say that you by large are satisfied with life, or are you mostly dissatisfied?” The item was scored on a 7-point scale ranging from very satisfied to very dissatisfied.The HADS consists of 14 items, including subscales for anxiety  and depression . The items are scored on a four-point scale ranging from totally disagree to agree totally. Each item is rated from 0–3, where higher scores indicate more severe anxiety and/or depressive symptoms. The maximum score on each subscale is 21, ranging from 0–7 , 8–10 (mild disorder), 11–14 (moderate disorder), and 15–21 (severe disorder) .To increase acceptability and prevent individuals from feeling tested for mental disorders, symptoms of severe psychopathology have been excluded, which makes HADS more sensitive to milder psychopathology , 40. Acc2-statistics and conventional fit indices: χ2-statistics, the Root Mean Square Error of Approximation (RMSEA), and the Standardized Root Mean Square Residual (SRMR) with values < 0.10 are acceptable, and values ≤ 0.05 indicates a good fit [2 which is the correct asymptotic mean even under non-normality, is reported [The analyses were conducted using the IBM Statistical Package for the Social Sciences Version 28 software  and the good fit , 44. Furgood fit –46. Accogood fit , skewnesreported .CFA is well known to be sensitive to sample size ; the lar, this study investigates the dimensionality, reliability, and construct validity of the HADS among community-dwelling adults 70 years and older; dimensionality, reliability, and construct validity are interrelated measurement properties. Dimensionality concerns the homogeneity of a scale’s items [Reliability encompasses a scale’s consistency and lack of error [c) were utilized. Finally, construct validity implies various aspects, such as convergent, discriminant, and content validity. In this study, convergent and discriminant validity denote if HADS relates with other constructs as expected, while content validity embraces whether the 14 items adequately represent the theoretical content of the anxiety and depression constructs involved in HADS. Taken together, this is whether the included items cover the theoretical definition they are aimed to represent [Since we aimed to find the best fitting model, i.e., a model that represents the observed data of older home-dwelling adults in the best manner, we first tested the original model and then, after, other models based on the findings in this original model. Thus, as stated in the aim section’s items , indicatof error . To asseepresent . If the The sample of 1190 adults were aged between 70–96 years with a mean of 76.5 (SD = 5.3); 644 (54.1%) were women (mean age 76.7) while 546 (45.9%) were men (mean age 76.2) , more often female (60%), and with less education .anxiety = 0.79 =) or acceptable (αdepression = 0.66). Composite reliability (ρc) displayed values between 0.65–0.78 .The mean anxiety and depression scores were 3.4 SD = 2.9) and 3.0 (SD = 2.5), respectively , including 14 items. This solution was termed Model-1; the factor loadings (λ) ranged between 0.32 and 0.72, followed by multiple squared correlations (R2) from 0.10 to 0.52; this range of factor loadings was pretty much the same in all estimated models. Three items belonging to the depression construct; 8D (“I feel as I’m slowed down”), 10D (“I have lost interest in my appearance”) and 14D (“I can enjoy a good book or a TV program”) revealed low loadings of 0.34, 0.32 and 0.37, respectively, explaining 12%, 11% and 13% of the variance of the depression construct. The fit indices indicated misspecification: χ2 = 395.010, p = 0.00001, df = 76, χ2/df = 5.20, RMSEA = 0.059, p-close = 0.04, SRMR = 0.052, CFI = 0.91, TLI = 0.89  involved in several highly significant estimates. Hence, we scrutinized the modification indices (MI) presenting some extremely high values; item 6D exposed an extremely high MI = 77.29 with the anxiety factor and an MI = 30.374 with item 5A (“Worrying thoughts go through my mind “). Also, item 2D (“I still enjoy the things I used to enjoy”) and 12D (“I look forward with enjoyment to things”) demonstrated an exceptionally high MI = 44.383. In total, 16 MIs were ≥ 10.This study aimed to test the psychometrics of the HADS among community-dwelling people ≥ 70 years. Consequently, first, we tested the original two-factor solution  , indicating cross-loadings and significant correlations among error terms. Hence, we considered the theoretical content of the items.To further test the original model, we looked at one of the two factors at a time: anxiety and depression, both including 7 items each, demonstrated a too high χD) and feeling anxious correlate negatively. Accordingly, in Model-2 we included a path (cross-loading) from item 6D (“I feel cheerful”) to the Anxiety construct, which improved the fit considerably: χ2 = 317.951, p = 0.0001, df = 75, χ2/df = 4.24, RMSEA = 0.046, p-close = 0.262, SRMR = 0.042, CFI = 0.93, TLI = 0.92. However, the fit was still not good.It is plausible that if one is feeling cheerful, one is improbable to feel anxiety at the same time, and vice versa. Thus, it is theoretically meaningful that feeling cheerful (item 6D (“I still enjoy the things I used to enjoy”) and 12D (“I look forward with enjoyment to things”) was uncovered. It is rational that still enjoying things and looking forward to things with enjoyment correlate. Therefore, we included a correlated error term between item 2D and 12D which further improved the fit in Model-3: χ2 = 296.919, p = 0.0001, df = 74, χ2/df = 4.01, RMSEA = 0.050, p-close = 0.452, SRMR = 0.040, CFI = 0.94, TLI = 0.92  was 0.78 for the anxiety subscale and 0.65 for the depression subscale. Still, 14 normalized residuals were significant, asking for several cross-loadings and correlated errors.An extremely high MI between the items 22 = 840.194, p = 0.0001, df = 77, χ2/df = 10.91, RMSEA = 0.091, p-close = 0.0001, SRMR = 0.070, CFI = 0.78, TLI = 0.74. However, internal consistency was good ρHADS = 0.81. Hence, the misspecification was possibly caused by error covariances. Consequently, we turned to Model-3 . Therefore, we randomly split the data into two equally sized parts (N = 595) termed Sample 1 and Sample 2, representing a sample size more suitable for SEM  [2 = 225.391, p = 0.0001, df = 76, χ2/df = 2.97, RMSEA = 0.058, p-close = 0.074, SRMR = 0.057, CFI = 0.90, TLI = 0.88, ρAnxiety = 0.76 and ρDepression = 0.67. Sample2 χ2 = 257.228, p = 0.0001, df = 76, χ2/df = 3.38, RMSEA = 0.063, p-close = 0.005, SRMR = 0.053, CFI = 0.91, TLI = 0.90, ρAnxiety = 0.79 and ρDepression = 0.73. Loadings ranged between 0.31 and 0.67 for Sample1 and between 0.29 and 0.77 for Sample2.All tested models revealed a chi-square indicating misspecification. However, as already stated, it is well known that chi-square as a model fit index has limitations. First and foremost, chi-square is sensitive to sample size. The present sample is large (odeling) , 47, 52.According to the European Commission’s Green paper on mental health , depressN = 1190), revealing extraordinarily high estimates for the chi-square. When splitting the file into two parts, giving a sample size N = 595, the chi-square improved substantially, and the RMSEA was still acceptable. Hence, reflecting on the chi-square statistic in light of the large sample size, a wide variety of other indices were included to assess model adequacy. The SEM literature states that, as a minimum, RMSEA, CFI, and SRMR should be reported in combination with chi-square [The CFA approach eliminates the need to summate scales because the SEM programs such as STATA compute latent construct scores for each respondent. This process allows relationships in the model tested to be automatically corrected for error variance, a fundamental strength of CFA in construct validation. Thus, the resulting estimates are adjusted for measurement error , 47. In i-square . Using mD) along with a correlated error term between the items 2D and 12D improved these fit indices as well as the total model fit. Consequently, low reliability and content validity seemed to cause low values for CFI and TLI.Conversely, the RMSEA estimate has demonstrated lower values with large sample sizes , 57. Forfeel both cheerful and anxious at the same time is unrealistic. In contrast, people may say, “I still enjoy the things I used to enjoy” (item 2) despite occasionally feeling anxious. The same logic goes for item 4 (“I can laugh and see the funny side of things”) and 12 (“I look forward with enjoyment to things”). To feel cheerful is a feeling, an experience here and now, while being able to ‘enjoy the things that I used to enjoy’ as well as being able to ‘laugh and see the funny side of things’ are not necessarily something a person feels in the moment. These are more general future aspects, such as possibilities or attitudes. Thus, these can go together with having anxiety from time to time. Therefore, we did not allow cross-loadings to the anxiety construct for these items.Theory guided the inclusion of the cross-loading and the correlated error term. It is rational that feeling cheerful (item 6) and simultaneously feeling anxious is a contradiction. To 1. The two factors were properly correlated. However, the original two-factor solution did not reveal a good fit. Thus, H1 was only partly supported.Concerning the dimensionality of the HADS, the two-factor model undoubtedly showed the best fit to the present data; the dimensionality of the HADS questionnaire stood out to be unquestionable supporting H2) concerned the reliability of the HADS. All items were significant. Largely, the items revealed good loadings  demonstrating good reliability. Nevertheless, particularly three items belonging to the depression construct  demonstrated low factor loadings and, thus poor reliability, explaining very little of the variance in the construct. These three items caused a low reliability coefficient for depression, while anxiety displayed good reliability. Hence, H2 was not fully supported.The second hypothesis  do not correlate too strongly with measurements of a similar but distinct trait; two tests reflecting different constructs should not be strongly related to each other. If they are, we cannot be sure they are not measuring the same construct. Accordingly, discriminant validity indicates the extent of difference between two constructs. The complementary concept to divergent validity is convergent validity; both are forms of construct validity. Hence, a high correlation (higher than 0.40) [4.Testing discriminant validity, Han 0.40)  between an 0.40) . Moreovean 0.40) . The conD concerns “I feel as I’m slowed down” demonstrated low reliability and, thereby poor validity. In the present sample, with a mean age of 76.5 years, most individuals outside an active work-life have lots of time to adjust to a slower pace of life. Possibly, ‘feeling slowed down’ does not correspond well to older home-living adults’ daily experiences in relation to depression. This item did not perform to be a valid or reliable indicator of depression in this population. Moreover, about 50% of the participants reported physical or mental long-term illness, injury, or loss of function in daily life. Relevantly, a slower pace of life might seem natural and not necessarily an indicator of depression [Content validity is a central aspect of construct validity. Reliability and content validity represent interrelated measurement properties. In fact, despite good reliability, content validity might be poor. Contrariwise, validity cannot be good if reliability is low . Item 8DD, “I have lost interest in my appearance,” did not communicate well with these older adults, indicating low reliability and content validity. Losing interest in one’s appearance did not act as a valid indicator of depression in this population. Losing interest in one’s appearance may be reasoned by the inevitable age-related changes they experience rather than as a symptom of depression. Moreover, item 14D, “I can enjoy a good book or TV program,” also stood out as an unreliable indicator of depression. Plausibly, being old, enjoying a good book, or watching TV does not relate to depression. Living in your seventies-eighties-nineties, passive leisure activities are everyday activities that are useful as restoration time after active leisure activities and are related to QOL [D did not explain any variance in the depression construct and thus misbehaved as a valid indicator for the depression construct.Likewise, item 10d to QOL . ReadingD, 10D, and 14D should be carefully considered to improve reliability.These findings are consistent with previous studies among nursing home residents without cognitive impairment  and hospD to anxiety, as well as highly significant error variances between items 2D and 12D. Surprisingly, community-dwelling older adults living at home (the present study), nursing home residents  [N = 484; mean age 80.7 years) [Furthermore, the former validation study among older adults in nursing homes  also inv6 years) , and hos7 years)  respond Summarized, construct validity and reliability of anxiety were good. Conversely, the depression construct revealed low validity and reliability, which are interrelated measurement properties. Exclusively, content validity includes the extent to which elements of a measurement scale are appropriate and characteristic of the specific construct for a certain assessment purpose . In thisA notable strength of this research is the empirical examination of the HADS, which has not been tested previously in a community-dwelling older population of 70 + using CFA in Norway. Also, the large sample size is a strength, allowing the possibility to randomly split the sample into two different samples, including 595 community-dwelling older adults each.Although the older adults were selected randomly in two subsamples, we cannot state that the sample represents the community-dwelling older adults in the actual city since 3181 of 4667 declined participation. In addition, those excluded from this present study were older and had less education. Hence, in the view of representativity, we assume that the present sample may be disrupted, not representing all community-dwelling older adults.D) along with a correlated error term between item 2D and 12D, a good to acceptable measurement reliability was demonstrated, and construct validity was supported.This study showed that the two-factor structure assessing symptoms of anxiety and depression is unquestionable. In conclusion, when we included a cross-loading item , explaining the minimal variation of the depression construct in this population. Consequently, the depression factor demonstrates low reliability among older community-dwelling people aged 70 + . Therefore, to be valid indicators of depression among community-dwelling older adults, these items need to be rewritten and informed by qualitative studies exploring relevant aspects of depression among older adults living at home.However, concerning internal consistency, the original version of HADS revealed a good reliability coefficient for anxiety but a poor estimate for depression; items 8


Oxtr) has been shown to have alternative transcripts in the mouse brain which may each have different biological functions or be used in specific contexts. A popular animal model for studying oxytocin-dependent social behaviors is the prairie vole, a biparental and monogamous rodent. Alternative transcriptional capacity of Oxtr in prairie voles is unknown. We used 5′ rapid amplification of cDNA ends to identify alternative Oxtr transcription start sites in prairie vole brain tissue and uterine tissue. We then validated expression of specific transcripts in fetal brains and assessed the impact of exogenous oxytocin administration in utero on offspring brain development. We identified seven distinct Oxtr transcripts, all of which are present in both brain and uterine tissue. We then demonstrated that maternal oxytocin administration alters expression of a specific subset of Oxtr transcripts and that these different transcripts are under unique epigenetic regulation, such that in the perinatal period only one of the alternative transcripts is associated with DNA methylation in the Oxtr promoter. These data establish the existence of multiple Oxtr transcripts in prairie vole brain and uterine tissue and implicate oxytocin in the regulation of alternative transcript expression. These data have significant implications for our understanding of null mutant models in both mice and voles and translation in human birth and behavior.The neurohormone oxytocin regulates many aspects of physiology primarily by binding to its receptor, the oxytocin receptor. The oxytocin receptor gene ( Oxytocin is a neuropeptide hormone originally described for its role in promoting uterine contractions . OxytociOXTR gene, which is thought to consist of four exons and three introns . In humans, OXTR is encoded by the  introns . To some or both . Two of xtr gene . In prevhe brain . Howeverccumbens . Given tOxtr transcripts present in prairie vole nucleus accumbens and uterus. We identify seven alternative Oxtr transcripts containing the annotated fourth exon, each of which have a different transcription start site. We show that exogenous oxytocin exposure at birth differentially impacts Oxtr variant expression in the fetal brain, suggesting distinct biological functions of each of the transcripts. Finally, we provide evidence that DNA methylation in an important Oxtr regulatory region regulates only specific transcripts.In this study, we use 5′ rapid amplification of cDNA ends (5′ RACE) to identify Microtus ochrogaster) derived from wild-caught stock near Champaign, Illinois treated as previously described  from our previous study of early life experience were pooled for 5′ RACE . UterineTaq DNA Polymerase High Fidelity (Invitrogen) according to manufacturer instructions with an annealing temperature of 65°C, with the provided forward GeneRacer primer, and the reverse gene-specific primer 5′-CGTCTCTCCGGGCCTGCTGCCCT-3′. A nested PCR was then performed using 1 μL of initial PCR as input, the provided nested forward GeneRacer primer, and the nested reverse gene-specific primer 5′- CCGGGCCTGCTGCCCTTCAGGT-3′ according to manufacturer instructions. 20 μL of nested PCR product was run on a 1.5% agarose gel with ethidium bromide. Single bands denoted in 5′ RACE was completed using the GeneRacer Kit with Superscript III RT (Invitrogen) according to manufacturer instructions. For uterine samples, 3 μg of RNA was used as input. For nucleus accumbens, 2.5 μg, which was pooled from four different samples, was used as input. Briefly, RNA was dephosphorylated and decapped and the GeneRacer oligo was ligated to the mRNA. mRNA was transcribed into cDNA using Superscript III RT using random primers. Touchdown PCR was performed with Platinum E. coli was transformed using the heat shock method. Transformed E. coli were spread on LB-agar plates containing 50 μg/mL ampicillin for selection and incubated at 37°C for 24 h. Three single colonies from each PCR product were picked and grown overnight in liquid LB with 50 μg/mL ampicillin at 37°C shaking at 200 rpm. Cells were pelleted by spinning at 4000 rcf for 10 min and plasmids containing 5′ end clones were isolated using the PureLink Quick Plasmid Miniprep Kit (Invitrogen). Plasmids were sent to Genewiz for sequencing. Sequences were aligned to Oxtr using NCBI’s blastn. Sequence information for each transcript is provided in Each gel-extracted band represents a PCR product that was cloned using the TOPO TA Cloning Kit (Invitrogen) according to manufacturer instructions. Briefly, 4 μL of the purified PCR product was cloned into the TOPO vector. One Shot TOP10 n = 9 dams), low dose oxytocin , medium dose oxytocin , or high dose oxytocin . An additional group of pregnant dams were not injected with anything and used as a no treatment control (n = 9 dams). 90 min following injection, pregnant dams were anesthetized with isoflurane and fetuses were removed and euthanized. Fetal brains were extracted and sectioned into thirds , then they were flash frozen on dry ice. DNA and RNA were extracted from the midbrain of one male and one female fetus per mother using AllPrep mini kits (Qiagen). All procedures for the induction labor study were approved by the IACUC at Northeastern University.Methods for generation of samples for the induction of labor study and measurement of DNA methylation in these samples were previously reported and are summarized below . PrairieOxtr-H  according to manufacturer instructions using 0.2 μM forward primer 5′-GGACCCAAGGCCTATGTCA-3′ and biotinylated reverse primer 5′-ATGAGCTTGACGCTACTGACTCG-3’. The following cycling conditions were used for amplification of the target fragment: [Step 1: (95°C/15 min)/1 cycle, Step 2: /45 cycles, Step 3: (72°C/10 min)/1 cycle, Step 4: 4°C hold. Pyrosequencing was completed using Pyromark Gold Q24 reagents (Qiagen) according to manufacturer instructions with sequencing primer 5′-TCAAGATCTGGCAGAAC-3’.Oxtr fragment, which included CpG sites −934_1, −934_2, −924, and −901: [Step 1: (95°C/15 min)/1 cycle, Step 2: /50 cycles, Step 3: (72°C/10 min)/1 cycle, Step 4: 4°C hold. Pyrosequencing was performed using two primers: 5′-GAGGGAAGGTTTTGGAGTTTTTTATAT-3′, which covers CpG sites −934_1, −934_2, and −924, and 5′-AGGGATTGAAAAGTGA-3′, which covers CpG site −901, on a Pyromark Q24 using PyroMark Gold Q24 Reagents (Qiagen) per the manufacturer protocol. Epigenotypes reported are an average of the three replicates.Methods for measurement of DNA methylation in induction of labor samples have previously been reported . Brieflyafex package in R (emmeans R package and a modified Dunnett correction (“dunnetx”) for multiple comparisons , because of the abundance of Oxtr expression in this tissue. Four of these transcripts were previously identified in mouse hippocampus  to generate schematics of the predicted protein. The predicted protein products of these transcripts are shown in Oxtr isoforms in the fetal brain are impacted by biological context using a model of labor induction  with primers that can detect distinct alternative transcripts  or groups of transcripts   .Oxtr transcripts, but post hoc tests do not indicate that any specific group differs from saline [F = 3.66, p = 0.028, Oxtr transcripts except Oxtr-H, there is again a significant group difference, with the medium group having increased expression compared to saline [F = 4.03, p = 0.020, Oxtr transcripts is altered by maternally administered oxytocin. None of the treatment group expression differences in Oxtr transcripts can be attributed to Oxtr-A, which does not change expression levels with maternal oxytocin administration [F = 1.30, p = 0.229, Oxtr-B, which does significantly differ by maternal oxytocin administration, and is elevated in the medium dose and high dose groups [F = 3.50, p = 0.033, Oxtr-H, where expression is elevated in the high dose group [F = 4.49, p = 0.008, Oxtr transcripts in the fetal brain in a dose-dependent manner and suggests the importance of clarifying expression of these specific transcripts in previous literature in the vole and the mouse. Moreover, these results underscore the importance of precise quantification of specific transcripts when examining how endogenous or exogenous factors impact Oxtr expression in the brain.Our results indicate that there are significant group differences in expression when using primers that capture all Oxtr called MT2, which is located within exon 1 and intron 1 of Oxtr, is associated with reduced transcription of OXTR in both humans and prairie voles  which we have previously shown to be sensitive to early life experience and are among the CpG sites where methylation is most highly associated with Oxtr expression  = 12.55, p < 0.001; Oxtr expression when the assay measures all transcripts except H [F = 11.28, p = 0.001; Oxtr gene expression and DNA methylation cannot be attributed to regulation of Oxtr-A, which is not associated with DNA methylation at CpG −924 [F = 1.29, p = 0.26; Oxtr-B [F = 14.15, p < 0.001; Oxtr-H [F = 2.44, p = 0.123; Oxtr-B in the fetal brain.DNA methylation is an epigenetic modification that occurs when a methyl group is covalently added to cytosine, typically in a cytosine-phosphate-guanine (CpG) context . DNA metie voles . We testpression . ResultsOxtr in prairie voles in multiple tissues. Four of these transcripts were previously identified in mouse hippocampus, indicating conservation of these transcripts across species. An additional three transcripts were identified which are novel. We confirmed the presence of five of these transcripts with RT-qPCR in the fetal brain. Putative protein product sequence-based prediction indicates that while four of these transcripts are predicted to produce full length OXTR protein, the remaining three would make partial protein products. Possible functions of these isoforms are discussed below, though we have not provided evidence that these transcripts are translated into protein. Further investigation of these transcripts is required to examine functional roles on oxytocin-dependent processes including birth and social behavior.We provide evidence of seven alternative transcripts of Oxtr-K and Oxtr-L expression, allowing the brain and/or uterus to slow Oxtr translation while keeping pools of mRNA encoding for full length Oxtr in the cell.Four of the described isoforms contain the entire protein coding sequence and could therefore be translated into the full length OXTR protein. It is possible that there is context- or tissue-dependent expression of particular transcripts which might relate to the regulation or function of OXTR and other receptors known to bind oxytocin. In other GPCRs, it has been shown that transcripts with extended 5′ untranslated regions are translated less efficiently and may allow for a quick context-dependent change in expression of a gene that is readily reversible . This meOxtr-E and Oxtr-H may regulate oxytocin binding capacity . It is well documented in other GPCRs that truncated receptors regulate both the activity and availability of full-length receptors . Crystalcapacity . OXTR hocapacity . TruncatOxtr gene. Many protein coding genes express non-coding alternative isoforms with regulatory function  the full-length OXTR, (2) other receptors that dimerize with OXTR, or (3) other proteins that interact with OXTR such as G proteins and β-arrestin. These products might also alter the transcriptional or translational ability of the canonical transcript. This has widespread implications for understanding regulation of oxytocin signaling in many tissues. Studies measuring expression of Oxtr in prairie voles should consider which of these transcripts the chosen quantification method measures for proper interpretation. Interpretation of such results should also consider that context-dependent expression of these isoforms may allow the cell to quickly and reversibly modulate oxytocin signaling in response to both internal processes such as parturition or external events such as social interaction without fully adjusting the regulation of the Oxtr gene. Further studies are necessary to examine the functional role of each of these transcripts in the cell and how the expression of each transcript is modulated by biological and environmental context. In the context of the uterus, for example, specific transcripts might be critical for contractions during birth which require oxytocin signaling, a process which might be disrupted if gene regulation goes awry. In the brain, there might be more subtle variation in transcript use which is related to specific environmental exposures, experiences, or developmental trajectories. Future studies which examine the functional characteristic of different Oxtr transcripts in the brain should also study such factors, which may explain why some transcripts are upregulated and others are not. Lastly, it has not escaped our notice that critical SNP findings in the human literature identify a prominent effect of a polymorphism in the third intron of OXTR on many behavioral and psychiatric outcomes (OXTR SNPs if present in humans.Our study does not present an exhaustive list of outcomes . Follow


Most reported a weak (53.7%) adherence to the Mediterranean diet. Almost a third (32.3%) consumed dietary supplements. Our sample presented a low adhesion to the Mediterranean dietary pattern, low consumption of fruit and vegetables, and high consumption of fast food, red and processed meat, soft drinks, and dietary supplements, including caffeine-based supplements. Dietary supplement consumption was related to a higher adhesion to the Mediterranean diet, and a higher level of professionalization and internet gaming disorder correlated with a lower adhesion. In conclusion, we infer that Portuguese and Brazilian esports players follow an unbalanced diet.As the esports industry grows, marketing campaigns for fast food, soft drinks, alcoholic and energy drinks, and dietary supplements at related events intensify. Portuguese and Brazilian esports players’ dietary patterns remain unexplored. This study aimed to characterize the dietary and gaming habits of esports players. We applied an anonymous, open online survey to a convenience sample of esports players that included the PREDIMED and the Internet Gaming Disorder Scale. The survey was shared through email and social media platforms, including Facebook, Twitter, Instagram, Discord, WhatsApp, and Twitch. The sample ( Esports involves the competitive practice of video games against other players. Some of the most well-known esports games include first-person shooters such as Counter-Strike: Global Offensive and multiplayer online battle arena games such as League of Legends . This hyThe esports industry has been expanding and gaining an audience at an extraordinarily fast pace. The worldwide overall viewership for esports is expected to reach around 600 million people around 2023 . The UniAlcohol, soft drinks, energy drinks, and fast food companies have embraced this trend and are sponsoring esports-related events, encouraging poor dietary habits in esports players and spectators ,8.Inadequate dietary habits are linked to lower performance in a variety of cognitive areas, which might be explained in part by ethanol neurotoxicity, increased brain inflammation caused by high fat and high sugar consumption, and increased adiposity . ContrarPlaying video games is associated with sedentarism, which increases weight gain and health risks ,14. ThusAt least six investigations have assessed some aspect of the dietary habits of esports players ,21,22,23In 2020, Rudolf and colleagues  applied n = 241) with non-players (n = 98). The esports players expressed less concern with following a balanced diet (mean difference (MD) = −0.63; p < 0.001), ingested fewer portions of fruits and vegetables , and drank fewer caffeinated beverages  than non-players.Through an online survey, Huth (2021)  comparedp = 0.02). Esports competitors also reported a statistically significant greater average consumption of fast food , and red meat  than video game players. Fast food , energy drinks , and soft drinks  were positively (poorly) correlated with video game playing time.In 2023, Soffner and colleagues  alsAlthough esports has begun to draw the attention of the scientific community, health research  and nutrThis study aimed to characterize the esports gaming behaviors, degree of internet gaming disorder, and dietary habits of Portuguese and Brazilian esports competitors. Additionally, we intended to investigate associations between sociodemographic and esports-related variables and the level of adherence to the Mediterranean diet.The finding presented in this original article may contribute to fill a knowledge gap in scientific literature and informing health professionals and individuals involved in esports on the dietary habits and gaming behaviors of Portuguese and Brazilian esports players. It may also serve as a foundation for public health institutes and esports organizations to develop strategic interventions to enhance the eating habits of these competitors.A convenience sample of esports competitors was invited to respond to an anonymous, open online survey. The inquiry was conducted between 1 December 2022 and 30 June 2023. It was disseminated via email to esports organizations and through social media platforms, including Facebook, Twitter, Instagram, Discord, WhatsApp, and Twitch.The study required participants to be between 18 and 69 years, have Portuguese or Brazilian nationality, and have played esports for at least six months. Individuals who did not fit these requirements were excluded. No rewards were offered for taking part in this investigation.A landing page was created on Google Sites, which contained two hyperlinks for two versions of the survey, generated on Google Forms: one specific for Portuguese and another for Brazilian participants. Google Analytics 4 was applied on the landing page to determine the number of unique site visitors, view rate, participation rate, and completion rate.Each Google Form contained an easy-to-read summary of the study, a permission request, an email address for additional information requests, the anticipated conclusion time, and an invitation to participate. The Google Form, available as The inquiry included:2), which was categorized based on the World Health Organization’s standards [Generic questions regarding nationality, sex, birth date, self-reported weight and height, education attainment, and professional occupation. Self-reported weight and height were used to calculate BMI , registration within an esports federation, level of professionalization , eGame that the respondent plays competitively, and their gaming rank.www.predimed.es, accessed on 2 October 2022) [The PREvención con DIeta MEDiterránea” (PREDIMED): It consists of fourteen multiple-choice questions that evaluate the typical ingestion of olive oil, spreadable fats, nuts, fruits, vegetables, legumes, fish or shellfish, meat, sugary drinks, wine, sweets, and stir-fried foods. The final score ranges from 0 to 14. We used three categories of Mediterranean diet adherence: low for values under 5, moderate for values between 6 and 9, and good for values above 10 ,33. Permer 2022) ,33 utilier 2022)  and Brazer 2022) , was reqer 2022) .Fast-food and caffeine intake: An eight-item Likert scale was applied to assess breakfast omission, ranging from “Never” to “7 days”, and fast food and caffeinated beverages with a scale ranging from “Never“ to “4 or more per day”. The Brazilian caffeine content table (BraCaffT) was used to calculate the amount of caffeine that the respondents consumed from beverages .A questionnaire consisting of five questions related to the consumption of DS, including types of DS consumed, reasons for DS consumption, sources of information, and place of acquisition.The Internet Gaming Disorder Scale—Short-Form (IGDS9) characterizes the level of engrossment concerning online video gaming . It has The anonymous electronic data gathered on Google Forms was downloaded and saved in a password-protected Excel file format (.xlsx) while complying with the EU’s General Data Protection Regulation .® SPSS® Statistics 29.0 for Windows . The significance value for all statistical tests was established at <0.05.Microsoft Excel was used to organize, evaluate, and generate descriptive statistical data for the downloaded records. All statistical analysis was performed using IBM2); “BMI—terciles” ; “federate athlete” , “No. of esports titles played” (1 to 10); “No. of years playing esports”; “No. of years playing esports—terciles” ; “No. of hours of esports play/day”; “No. of hours of esports play/day—terciles” ; “No. of competitions in the past 12 months”; “No. of competitions in the past 12 months—terciles” ; “caffeine intake” (mg); “caffeine intake—terciles” ; “DS intake” ; “No. of DS ingested” (0 to 17); “DS consumed” ; “Reasons for using DS” (17 options); “Information sources about DS” (15 options); and “Place of acquisition of DS” (8 options).The following variables were generated and added to SPSS: “PREDIMED total score” (0 to 14); “portions of fruit and vegetables/day”; “<5 portions of fruits and vegetables/day” ; “portions of red and processed meat/day”; “<1 portion of red and processed meat/day” ; “No. of soft drinks/day”; “<1 soft drink/day” ; “No. of commercial pastries or sweets/week”; “<1 commercial pastry or sweet/week” ; “breakfast consumption frequency”; “frequency of fast food intake/week”; “IGDS9 total score” (0 to 45); No. of answers “Very often”; “nationality” ; “sex” ; “age” (18 to 69); “age—quartiles” ; “years of education” (0 to 16); “university education” ; “member of an esports team” ; “level of professionalization” ; “BMI”  only for the “PREDIMED total score”, “IGDS9 total score”, “portions of red and processed meat/day”, “No. of soft drinks/day”, and “No. of commercial pastries or sweets/week” variables.t-test for “PREDIMED total score”, “IGDS9 total score”, “portions of fruit and vegetables/day”, “portions of red and processed meat/day”, “No. of soft drinks/day”, “No. of commercial pastries or sweets/week”, and Mann–Whitney’s U test for “level of professionalization”, “age”, “BMI”, “years of education”, “No. of hours of esports play/day”, “No. of competitions in the past 12 months”, “No. of years playing esports”, “No. of esports titles played”, “breakfast consumption frequency”, “frequency of fast food intake/week”, “caffeine intake”, and “No. of DS ingested”. Crosstabulations and Fisher’s exact test were used to describe the relationship between the “sex”, “<5 portions of fruits and vegetables/day”, “<1 portion of red and processed meat/day”, “<1 soft drink/day”, “<1 commercial pastry or sweet/week”, “member of an esports team”, “DS intake”, and “federate athlete” variables and “nationality”.Data from Portuguese and Brazilian players were compared using Student’s The Google website received 2039 unique visitors overall; 1566 (76.8%) of them navigated to the Google form that included the survey, and 711 (45.4%) of those completed it.A total of 132 responses were rejected: four for stating a nationality other than Portuguese or Brazilian, nine for not having played esports for at least six months, 105 for being less than 18 years old, one for reporting being older than 69, two for not reporting the esports title they played competitively, one for reporting zero hours of esports play per day, and ten for providing nonsensical answers. As a result, the information from 579 individuals was included and examined in this study.n = 305), and the remaining were of Portuguese nationality. This sample was predominantly male , aged between 18 and 29 years . The median number of years of schooling was 14 years, with about one-third of the sample  reporting having completed university education. Concerning employment status, 43.9% (n = 254) of the sample reported being an employee, whereas about one fifth  reported being a student worker, and another fifth  a student  were overweight.About half of the sample was classified as having a normal weight , fewer “years of education” (p = 0.010), and a higher “BMI” (p = 0.005). The proportion of males and females was not different among Brazilian and Portuguese players (p = 0.104).Compared to Portuguese, Brazilian players had a significantly lower “age” (n = 360), whereas the remaining self-classified as semi-professional or professional players. About a quarter  stated they were a member of an esports league or federation (The respondents reported playing esports for 8.1 (SD = 5.1) years, practicing esports 4.4 (SD = 3.2) hours a day, and participating in a total of 7.6 (SD = 10.4) competitions in the past 12 months on average. Most respondents reported being amateur esports players , had been playing esports for more years (p = 0.011), played a higher number of esports titles (p < 0.001), and reported a higher level of professionalization (p = 0.011) than the Portuguese players. Additionally, a significantly lower proportion of Brazilians reported being members of an esports team (p < 0.001) or federation (p < 0.001).The Brazilians played esports for a significantly higher number of hours per day (n = 408), with the Multiplayer Online Battle Arena genre being the second most popular , followed by racing simulators , and other sports simulators  games.Most participants played First Person Shooter games , Valorant , and FIFA 2022/23 .The specific esports titles with the most players were League of Legends , Counter-Strike: Global Offensive  accumulating a total score of 36 points or higher. About a quarter  of the total sample answered “Very often” on at least one of the eight questions included in the IGDS9  on the IGDS9 scale, with 2.6% (he IGDS9 .p < 0.001) than Portuguese players.Brazilian esports players displayed a significantly higher overall IGDS9 score (n = 311) classified as having poor adherence to the Mediterranean diet pattern (PREDIMED), and most respondents  stated eating less than five servings of fruits and vegetables per day. Over half  stated consuming fast food, 70.1% (n = 406) sweets or commercial pastries, 65.8% (n = 381) coffee, and almost a quarter  reported drinking energy drinks on one or more occasions per week. The consumption of caffeine derived from caffeinated drinks averaged 179 (SD = 208) mg per day  ate one or more servings of red/processed meat, and 40.9% (n = 237) consumed one or more soft drinks per day.Additionally, more than three-thirds of the respondents , a higher consumption of fruit and vegetables (p < 0.001), and a lower consumption of soft drinks (p < 0.001). No significant differences were observed for red and processed meat (p = 0.814) and commercial pastries and sweets (p = 0.424).When comparing the data of Portuguese players with those of Brazilian players, Portuguese players displayed a statistically significant higher PREDIMED total score (p < 0.001) and portions of red and processed meat consumed per day (p < 0.016), corresponding to a greater consumption of red and processed meat in the Portuguese and a greater intake of soft drinks in the Brazilians. No significant associations were registered between nationality and the number of portions of fruits and vegetables ingested daily (p = 0.168) and the weekly consumption of commercial pastries or sweets (p = 0.413).Significant relationships were observed between nationality and the number of soft drinks (n = 133) and almost half  skipped breakfast on three or more days per week  and caffeine intake (p = 0.652) between the Brazilian and Portuguese players; however, Brazilian players did consume fast food more frequently than Portuguese players (p = 0.002).No statistically significant differences were observed regarding breakfast consumption frequency (n = 187) stated consuming DS in the past 12 months (n = 111), creatine , caffeine , and multivitamin-mineral   .p = 0.213) or the number of DS ingested (p = 0.194).No statistically significant differences were found between Brazilian and Portuguese players regarding DS consumption in the past 12 months (n = 158), prevent health problems or treat illness/injury , improve cognitive performance , gain muscle mass , and increase energy/decrease fatigue   .n = 79), medical professionals , athlete recommendations , and personal trainers  were the most frequently cited sources of DS related knowledge , as well as being a female . Not having consumed DS in the past 12 months , and a higher IGDS9 total score  were associated with a lower PREDIMED total score.Regarding adherence to the Mediterranean dietary pattern, represented by PREDIMED total score, when encompassing data relating to both nationalities, the univariate ANOVA returned statistical significance for nationality, sex, DS intake, and IGDS9 total score . Portugup = 0.004, etap2 = 0.029) and Portuguese  non-users of DS presenting a lower PREDIMED total score. The IGDS9 total score remained significant only for the Brazilian, associating with a lower PREDIMED total score . In addition, a significant relationship regarding the level of professionalization emerged for the Portuguese players , with amateurs  = 6.664) presenting a higher PREDIMED total score compared to semi-professionals (EMM = 5.886) and professionals (EMM = 5.263).When analyzing the data for Portuguese and Brazilians separately, the univariate ANOVA returned distinct results. The relationship with sex was not significant in either country, whereas DS intake remained significant in both groups, with the Brazilian , by 2019, 60.3% of Brazilian adults (≥18 years) were overweight, and 25.9% were obese . In PortIn comparison, 46.6% of the participants in our study had excess weight. More specifically, 28.0% were categorized as pre-obese and 18.7% as obese. This could have been due to our younger sample compared with that of studies conducted in reference populations ,43, as iWhen stratifying by age groups, in comparison with a reference population, our sample of Brazilian esports players presented lower pre-obesity prevalence from 18 to 24 years old (20.4% vs. 33.3%) and from 25 to 39 years (36.0% vs. 57.6%). However, they presented a higher prevalence of obesity in the same age groups: 18–24 years (18.3% vs. 10.7%) and 25–39 years (33.3% vs. 23.7%) .Our sample of Portuguese esports players presented a higher prevalence of pre-obesity in those aged between 18–24 years (24.4% vs. 19.1%) and those aged 35–44 years (48.7% vs. 37.1%), but not among the subgroup of 25–34 years (27.0% vs. 31.8%) compared to a reference population. The prevalence of obesity was also slightly higher for players aged between 18–24 years (6.5% vs. 6.3%) and 25–34 years (14.0% vs. 10.8%), but not among those aged 35–44 (15.4% vs. 20.2%) .These findings imply that esports participation may be linked to a higher prevalence of pre-obesity and obesity in particular age groups among Brazilian and Portuguese esports participants.p = 0.005), which may have been related to their significantly lower PREDIMED total score (p < 0.001), lower consumption of fruit and vegetables (p < 0.001), higher consumption of soft drinks (p < 0.001), and higher consumption frequency of fast food (p < 0.002).Furthermore, we observed significantly higher BMI values in Brazilian compared to Portuguese esports players  applied the IGDS9 questionnaire to 555 students  with a mean age of 20.3 (SD = 5.4) years who had played video games in the previous 12 months. They found that 38.2% of the volunteers had a moderate (score 16 to 20) and 13.5% had a high risk (score > 21) of gaming disorder . When apn = 15), which represented a slightly higher prevalence.In comparison, Ip et al. (2021) surveyed 526 adult video gamers in the USA  and reported that 2.3% of their total sample formally met the diagnostic criteria for IGD . Of notep < 0.001).Regarding the two nationalities covered in our study, we found significantly higher IDGS9 total score values in Brazilian esports players compared to Portuguese ones , which may have been related to differences in public food policy, food culture, and food availability between the two countries [Our Brazilian sample had a significantly lower PREDIMED total score compared to Portuguese respondents .Our study uncovered even higher values of non-compliance, with 84.5% of the total sample not reaching the recommended five portions of fruits and vegetables per day. Moreover, our sample of Portuguese players reported significantly higher average consumption of fruit and vegetables than the Brazilian players  compared to ≤2 days per week . It alsoAs the brain depends mainly on glucose as a fuel source, with glucose requirements increasing by up to 12% in the context of cognitively demanding activities , having In a 2007 study conducted in Brazil, 30% of 100 students (19 to 39 years) at a university in the city of Campinas reported skipping breakfast . In anotp = 0.809).Regarding this topic, 23% of our respondents admitted never having breakfast, whereas 47.5% reported skipping breakfast on three or more occasions per week. No statistically significant differences were observed regarding the median frequency of breakfast intake when comparing Brazilian to Portuguese esports players .Our study revealed that 39.6% of respondents ingested fast food on one, 20.4% on two to four, and 3.5% on five or more occasions per week. Despite the distinct methodologies applied in these studies, we can infer that fast food consumption was more prevalent in our sample of esports players compared to a reference population. Additionally, we registered a significantly higher consumption of fast food among players of Brazilian nationality compared to Portuguese nationality  higher relative risk for daily consumption of 100 g of red meat and 18% for 50 g of processed meat .According to the 2015-16 IAN-AF survey, about a quarter (25.5%) of Portuguese adults eat between 100 and 150 g of red meat daily . In divep = 0.814), we found that a higher proportion of Brazilian players ate less than one serving of red and processed meat per day, compared to the Portuguese (p = 0.016).Although we did not observe significant differences between the two nationalities regarding the number of servings of red and processed meat eaten per day .In comparison, we found that 40.9% of our total sample consumed one or more soft drinks daily, with a significantly higher average daily consumption of soft drinks in the Brazilian compared to the Portuguese players , who surveyed 526 adult video gamers in the USA, reported that 79.5% and 45.1% regularly consumed caffeinated and energy drinks, respectively . Additiop = 0.652), we did observe that Brazilian players’ consumption of coffee (p = 0.036) and energy drinks (p < 0.001) was significantly higher than that of Portuguese players.Although we did not observe significant differences between the two nationalities regarding caffeine consumption , protein (5%), branched amino acids (5%), nutrient-enriched foods (4%), energy drinks (4%), meal replacements for weight loss (2%), and others (5%) . AnotherThe use of DS in the previous 12 months has been reported by 29.2% of the Portuguese adult population (18 to 64 years). The most consumed DS were multivitamin complexes (33.1%), mineral salts (33.1%), and isolated vitamins (21.3%), such as folic acid and vitamin C, and other categories of DS (15.5%), including whey protein and omega 3 fatty acids .Our study unveiled a higher prevalence of DS consumption (32.3%) than those reported by the studies conducted in the Brazilian and Portuguese populations. The most consumed DS were whey protein (59.4%), creatine (47.1%), caffeine (33.2%), and multivitamin-mineral (30.5%). The differences in DS consumption may have been related to our sample being mostly of young adult age, in which the majority (84.5%) sought to improve some aspect of athletic performance or augment their muscle mass (51.9%).p = 0.213) and the number of DS ingested (p = 0.194) between our sample of Brazilian and Portuguese players.We did not observe statistically significant differences regarding the proportion of consumers of DS (n = 27) had consumed caffeine pills in the previous 12 months [Our study also uncovered a significantly higher prevalence of caffeine supplements consumption compared to a reference population, and even higher than those reported by Ip et al. (2021), who surveyed 526 adult video gamers in the USA and found that 12.1%  , developThe young adult age is a crucial period of human development in which inappropriate eating habits can have irreversible or difficult-to-reverse negative health consequences, which will extend throughout adult life . This toThis study adds to the evidence that most Portuguese and Brazilian esports players have a low level of adherence to a healthy eating pattern. It is necessary to promote healthier dietary habits in this population while involving esports organizations and public health institutions.Compared to the Portuguese, Brazilian players are more vulnerable and more in need of this type of intervention since they have a lower average age, fewer years of education, a higher level of professionalism, a higher number of hours dedicated to esports play, higher IGD score, higher unemployment, and a higher degree of dietary inadequacy, as demonstrated by lower PREDIMED scores, lower consumption of fruit and vegetables, and higher coffee, energy drinks, soft drinks, and fast-food consumption.This research may serve as a basis for the development of strategies to promote appropriate eating habits among Portuguese and Brazilian esports players.Future investigations should apply food diaries to Portuguese and Brazilian esports players to assess their eating habits more accurately, including fluid consumption, for further insight regarding possible dietary inadequacies, including micronutrient intake deficits.Additionally, one could test the hypothesis that greater adherence to a healthy dietary pattern, such as the Mediterranean diet, is associated with higher cognitive and video gaming performance in esports players.Our sample presented a low adhesion to the Mediterranean dietary pattern, low compliance to the WHO recommended amount of vegetables and fruit, and high consumption of fast food, red meat, soft drinks, and dietary supplements, including caffeine-based supplements.In comparison with the Brazilians, Portuguese esports players are older, have a lower BMI, a lower IGD score, a higher adhesion to the Mediterranean diet, higher consumption of fruit and vegetables, lower consumption of soft drinks and fast-food, a higher educational level, a higher number of members of esports teams and federate players, play esports fewer hours a day, have been playing esports for fewer years, play a lower number of esports titles, and display a lower level of esports professionalization.For both nationalities, the consumption of dietary supplements was associated with a higher adhesion to the Mediterranean diet and a higher degree of gaming disorder correlated with a lower adhesion to this dietary regimen. For the Portuguese players, a higher level of professionalization was associated with a lower adhesion to the Mediterranean diet.We infer that Portuguese and Brazilian esports players adhere to an unbalanced diet. We advocate for the implementation of initiatives to promote healthy eating habits in this community, preferably with the support of esports organizations.