Results of the research showed that the addition of 3% ESP significantly increased the ash and calcium content (p < 0.05) without changing the appearance, aroma, texture, taste profiles, and the hedonic score of gingerbread. The gingerbread samples were then stored for 2 months and were analyzed every month. The hedonic evaluation of the aroma of both gingerbread samples decreased significantly (p < 0.05) during storage. During 2 months of storage, the antioxidative activity of gingerbread fortified with 3% ESP was not significantly different compared to the control (p > 0.05), particularly in ABTS and ORACFL assay. The ABTS, DPPH, and ORACFL assays showed decreasing antioxidative activity during storage, which was also in accordance with decreasing total phenolic content of both gingerbread samples. In PCL assay, the lipid-soluble antioxidant activity in gingerbread with 3% ESP was significantly higher during 2 months of storage, compared to the control (p < 0.05). The developed product might be a potential alternative to improve the calcium  recommendation per 100 g) and antioxidant intake in order to prevent calcium deficiency. Gingerbread enriched with an organic source of calcium may become an innovative product to reduce the risk of developing osteoporosis in the elderly population, having potential health and economic significance, given the incidence of osteoporosis and the costs of treating this disease.Food waste, such as eggshell, can be an environmental problem if it is not properly managed. One of the ways to solve this is by using the eggshell as the cheap calcium source in food products. Polish gingerbread fortified with chicken eggshell powder (ESP) calcium was developed to solve the eggshell waste problem and to reduce the risk of osteoporosis. This study focused on the effect of ESP addition on basic composition, sensory evaluation, and antioxidative activity of gingerbread. Two samples of gingerbread without and with 3% ( About 200 million people in the world suffer from osteoporosis . Recent 3 . The cal piglets . An in v piglets . Not onlate (1%) ,11.3, cosmetics, and biomaterial composites) and operating supply  [Recently, the European Commission has been promoting the utilization of products’ values, resources, and materials as much as possible to support zero waste practices, which are then beneficial for the economy and environment . The wassorbent) . As an asorbent) . Among tsorbent) . TherefoCamellia sinensis) was reported to exhibit strong antioxidative activity due to its polyphenols, flavonoids including flavanols , flavonols , and phenolic acids  [Antioxidants  contribute as the anti-radicals to prevent oxidative stress, thus could reduce the risk of osteoporosis ,18. Greeic acid) . The maiic acid) . Thanks ic acid) . Severalic acid) . Tea driic acid) , men andic acid) , elderlyic acid) , Chineseic acid) ,26. Furtic acid) . In a feic acid)  indicateic acid) . Furtheric acid) . These bIn this research, Polish gingerbread (in Polish: pierniki) incorporated with chicken eggshell and green tea powder was developed to increase the calcium and antioxidant intake, as well as to solve the eggshell waste problem. Polish gingerbread is a popular bread that contains flour, honey, and various spices including cinnamon, cloves, coriander fruit, allspice, and nutmeg. Gingerbread is not only just food, but also part of the tradition and history in Poland, Europe, and world, especially during Christmas tradition ,32. The The idea to use gingerbread as the food matrix in this study was in accordance with previous studies. The application of chicken eggshell powder in bread products is recommended as it minimally changes the texture and taste of the resulting product . Bread pIn this study, it was decided to apply the eggshell in gingerbread since it is a popular snack and willingly eaten by children and the elderly, hence becoming a source of essential calcium for healthy bone growth. Moreover, the presence of vitamin D, inulin, as well as other indigestible oligosaccharides, magnesium, or short-chain fatty acids, is beneficial for calcium absorption in the body ,39,40. TThe eggshell powder was prepared according to Ray et al.  with sliThe ingredients used in the preparation of gingerbread samples were: UHT milk 3.2% fat , unrefined cane sugar , dry yeast , white flour type 650 , oat bran , salt , honey , egg , baking soda , gingerbread spice mix  , butter 82% fat , white chocolate , green tea powder , and eggshell powder.w/w of flour) and green tea powder was determined after considering prior research [w/w of flour to the cake was still acceptable in the sensory analysis [w/w of flour resulted in a more noticeable undesired typical eggshell aroma. The used concentration of green tea powder was 4% (w/w) of white chocolate topping in both control and treatment samples. The addition of green tea powder higher than 4% resulted in unacceptable taste due to high astringency and grassy aftertaste (data not shown). The resulting gingerbread with green tea-chocolate topping  using an air-acetylene flame [Prior to the determination of calcium content, the complete mineralization of 1 g of samples (powdered form) at 450 °C in a muffle furnace was conducted. It was then dissolved in 1 mol/L nitric acid . Analysis of the sample was conducted in triplicate. The samples were diluted with 0.5% LaClne flame  was condFor the evaluation of total phenolic content and antioxidative activity of gingerbread samples during storage, the samples were stored in a thermostat (20 °C) in a dark condition for 2 months. This period of storage was decided based on the approximate time that the consumer could store the product on a shelf without quality loss.2 = 0.9968) was used to calculate the TPC. The preparation of antioxidant extracts was done according to Jan et al. [Total phenolic content (TPC) was determined following the method in Shahidi and Naczk , based on et al. , with moFL, and PCL assays. The ABTS radical cation scavenging activity of gingerbread was measured according to Re et al. [2 = 0.9986) was used to evaluate the ABTS radical cation scavenging percentage. The result was expressed as mg of Trolox equivalent (TE) per 100 g of product.The antioxidative activity of gingerbread samples was determined using ABTS, DPPH, ORACe et al. , based oe et al. . The sta2 = 0.9941) was used to calculate the percentage of DPPH radical scavenging percentage and expressed as mg TE per 100 g of product.The DPPH assay was conducted following Sánchez-Moreno et al. , which iFL assay , which was described in a previous study [2 = 0.9995) and presented as mg TE per 100 g of product.The ability to scavenge the peroxyl radicals using fluorescein was conducted through ORACus study . Fluorom•−) [The PCL assay (photochemiluminescence) was performed with the use of Photochem apparatus  following the method of Gramza-Michałowska et al. . The PCL•−) . PCL evaThe sensory analysis of fresh gingerbread samples, with and without eggshell powder, with controlled green tea concentration  was condAdditionally, for storage analysis, the hedonic evaluation of the gingerbread samples was also conducted by 14 consumer panelists. During 2 months of storage, the gingerbread samples were kept in the dark condition under a controlled temperature (20 °C). The condition and period of storage were selected according to the approximate time that the consumer could store the product on the shelf without quality loss. The sensory analysis was conducted in the sensory analysis room, and all the consumer panelists were trained for the testing procedure. The mean, variance, and standard deviation of results were calculated for each attribute of each sample and session separately.T-test were used, and to determine the differences between means of samples, Tukey’s multiple range test was used (p < 0.05). Pearson’s correlation coefficient was determined between the total phenolic content of each gingerbread treatment and the antioxidative activities. SPSS 20.0  was applied for data analysis.The experiments were conducted in triplicate. One-way ANOVA and In this research, the fresh gingerbread samples (0% and 3% ESP) were compared based on basic composition and sensory analysis . Furthermore, the antioxidative activity of samples was observed during 2 months of storage and compared between treatments.p > 0.05). Alsuhaibani [3 from chicken eggshell showed lower moisture content compared to the control [p < 0.05)  . A similSalmonella or other pathogens and contaminants [The valorization of eggshell waste as calcium supplementation in food products has been conducted by many studies, including the present study. Thanks to its affordability and abundant availability, the application of eggshell waste in food products could be carried out on household scale  and induaminants . The subaminants . With thaminants .p < 0.05). A similar result was found in a study that reported a significant increase (p < 0.05) of fiber content in bread fortified with 2% eggshell powder [p < 0.05). The fortification of 4% eggshell powder also increased the crude fiber content in white bread compared to the control [The dietary fiber and its fractions contents of gingerbread samples are shown in l powder . The TDF control . Further control . It coul control . Calcium control . While t control ,67.Another concern which might affect the measurement and the increase of SDF and IDF after the eggshell powder addition to gingerbread is the mineral-binding by fibers. A study reported that lignin and cellulose, which are insoluble fibers, could bind the calcium, where the calcium binding capacity of lignin was higher than that of cellulose . As prevp < 0.05). To date, the research about fiber fraction content of chicken eggshells, especially using Van Soest assay, has not been reported yet. However, Al-awwal and Ali [p > 0.05).The NDF content, which consists of acid detergent fiber  and hemicellulose, was also determined. Both NDF and hemicellulose content of gingerbread with 3% eggshell powder were higher than the control  analysis are shown in p < 0.05). The ABTS radical cation scavenging activity was decreased significantly (p < 0.05), about 12%, after 1 month of storage in both samples. However, no significant difference in scavenging activity was found between those after 1 month and 2 months of storage. In DPPH radical scavenging activity assay, the scavenging activity decreased significantly (p < 0.05) around 4–5% every 1 month of storage, in both the control and 3% ESP samples. ORACFL assay, which is widely used to determine the antioxidant capacity written on food labels, showed similar results as a significant decrease of antioxidant was seen after 1 month (for the control sample) and after 2 months of storage (for the 3% ESP sample) (p < 0.05). The ORACFL values ranged from 996.73 to 1224.43 mg TE/100 g for the control and 1046.38 to 1194.88 mg TE/100 g for gingerbread with 3% ESP. All of those results were in accordance with the result of total phenolic content, which exhibited lower total phenolic content along with longer storage time. During storage, the available antioxidant in the samples scavenged the free radicals, which are responsible for various oxidation reactions by, for example, donating the proton or electron from the hydroxyl groups of phenolics [FL did not significantly decrease during 3 months of storage.The results of total phenolic content (TPC) and antioxidative activity  are rapid, simple, and reproducible, which is useful as the biomonitoring tool, particularly in the food technology and nutrition field . In PCL The integral antioxidative capacity (IAC) was also determined as it represents the sum of antioxidative activity of water-soluble and lipid-soluble antioxidants. Generally, there was no significant difference of IAC between the control and gingerbread with 3% ESP, especially for the fresh ones and after 1 month of storage. However, after 2 months of storage, the result showed higher IAC in gingerbread with 3% ESP compared to the control. Comparing between storage times, the IAC in the control sample decreased significantly after 1 month of storage (561.90 to 481.14 mg TE/100 g), then no significant difference was found after 2 months of storage. While for gingerbread with 3% ESP, no significant changes were noticed during 2 months of storage time. To summarize, in general, gingerbread with 3% ESP could be a potential and promising functional food with good antioxidative activity and improved calcium content. Additionally, prior studies ,55 reporFL, PCL-ACW, and PCL-IAC. However, the TPC of both gingerbread samples showed a negative correlation with the antioxidative activity using PCL-ACL assay. The Folin-Ciocalteu method was used for the determination of TPC. It was reported that the Folin–Ciocalteu chromophore, which is multivalent-charged phosphor-tungsto-molybdate, has a higher affinity to water and hydrophilic compounds than the lipophilic compounds, which make it more capable of measuring the hydrophilic antioxidants [The correlation between the TPC and the antioxidative activities of gingerbread samples was also checked statistically using Pearson’s correlation coefficient . In bothoxidants . Therefooxidants  who founoxidants ,88. ConsThe appearance, aroma, texture, and taste of fresh gingerbread samples, with and without eggshell powder, with controlled green tea concentration, are presented in p > 0.05). The ingredients of both samples, especially gingerbread spice mix and green tea powder might be strong contributors to this aroma profiling. However, it is important to note that the rancidity might be more intensive along with the longer storage time due to the oxidation process of butter-containing products [In terms of aroma, the addition of 3% eggshell powder did not significantly affect the aroma of the gingerbread samples in all descriptors. In both samples, the two dominant aromas were spicy and sweet, followed by green tea, burnt, and chocolate aroma. While the rancid aroma of both samples was the least intensive aroma compared to other descriptors, and no significant difference of rancidity was found after the eggshell powder addition (products .w/w of flour) could improve the calcium content without significant grittiness changes. This result was in agreement with Ray et al., who suggested the supplementation of eggshell powder up to 6% in cake products, with consideration of calcium content, texture, and sensory properties [In texture profiling, the addition of 3% eggshell powder did not significantly affect the texture of the gingerbread samples in all descriptors. The lowest intensive descriptor in both samples was crispness (3.33–3.53). The panelists assessed that both samples were rather highly intensive in softness and porosity descriptors. Interestingly, the grittiness of gingerbread incorporated with 3% eggshell powder was not significantly different from the control. This means that the eggshell powder addition . However, no significant decrease was observed in texture and overall scores of both treatments (p > 0.05). For appearance, a significant decrease of the hedonic score was observed in the second month of storage of gingerbread with 3% ESP (p < 0.05). In terms of taste, the hedonic score decreased around 13% (p < 0.05) in gingerbread without eggshell powder after 1 month of storage, compared to 0 month, while no significant decrease was found in that with 3% ESP during 2 months of storage. Comparing between 0 and 2 months of storage, the biggest decrease (25%) was found in the aroma of the control gingerbread, followed by the appearance of gingerbread fortified with 3% ESP (17%). In the preparation of gingerbread samples, butter was added. During the storage of fat-containing products, the oxidation of fat might take place, which produces the off-flavor, such as rancid flavor [The hedonic evaluation of appearance, aroma, texture, taste, and overall of gingerbread samples, with and without eggshell powder, during 2 months of storage is presented in d flavor . Howeverd flavor . In termd flavor  explainew/w of wheat flour) eggshell powder improved the ash and calcium content of the gingerbread without significantly changing the appearance, aroma, texture, taste, and the hedonic score of gingerbread in comparison with gingerbread without eggshell powder. About 26% DV of calcium could be fulfilled by consuming 100 g of gingerbread fortified with 3% ESP . The addition of eggshell powder significantly increased the content of hemicellulose, soluble, insoluble, and total dietary fiber of gingerbread. It was also observed that the soluble dietary fiber was predominant in gingerbread fortified with 3% ESP, which might potentially have a role as a prebiotic agent and then might increase the bioavailability of microminerals, including calcium. The fortification of 3% ESP to the gingerbread did not change the antioxidative activity in ABTS and ORACFL assay. The antioxidative activities and hedonic scores of both gingerbread samples decreased during 2 months of storage. However, both samples still possessed antioxidative activity and moderate consumer acceptability after 2 months of storage. In industrial production, the sterilization and grinding process of eggshells must be conducted properly to obtain safe and sensorially acceptable eggshell powder. Although the gingerbread in this study contained increased calcium content and antioxidative activity, the sugar consumption should be reduced in further studies following the current health trend in reducing high sugar intake. The information observed in this research could be useful for scientific knowledge, especially as one of the alternative ways to solve eggshell waste problem by incorporating it into gingerbread to decrease calcium deficiency and the risk of osteoporosis, which are global health problems. Eggshell is a food waste which may cause environmental and health problems if it is not managed properly. In this study, the innovative functional food based on gingerbread enriched with antioxidants from green tea and chicken eggshell calcium was developed. The addition of 3% (


From January 26, 2020 to February 3, 2020, a total of 7 confirmed cases and 2 asymptomatic infected persons were reported in Qingdao, China. These cases had no history of sojourning in Hubei Province, contact with animals, visits to markets, or eating game meat.On January 19, 2020, patient 1, the initial case, returned to Qingdao from Kunming by air, a flight on which there were 2 persons from Wuhan City with fever. She developed fever, cough, and weakness on January 21, 2020. She went to an outpatient clinic near her community and was treated with intravenous cefazolin from January 23 to 24. Due to persistent symptoms, she was admitted to hospital on January 25, 2020, and diagnosed as a suspected case. The throat swab tested positive for coronavirus disease 2019 (COVID-19) by reverse transcriptase polymerase chain reaction (RT-PCR) on January 26, 2020.Patient 2 lived together with her mother (patient 1), and the older daughter of patient 1 (patient 4) lived in the same community. Patient 1 had gone to the house of patient 4 many times since she returned to Qingdao. On January 20, patient 1 had lunch in her father’ house, which was in another community, with her father, sister, niece , and granddaughter (patient 5). In the evening, the 2 daughters (patients 4 and 2), son-in-law , grandson, and granddaughter (patient 5) had supper with patient 1 in the house of patient 4. Patient 3 and 4 took their children back to their parents’ home for the Spring Festival on January 23. The family visited their aunt (patient 6) and uncle-in-law  on the evening of January 23. None wore surgical masks during the entire visit. Patient 7, who also did not wear a surgical mask, met patient 1 in the same clinic when patient 1 attended to the outpatient clinic  and 2. DThrough the field investigation, 115 persons were exposed during this cluster event, and the total infection rate was 6.09% (7/115). The infection rate of only this family was 41.18% (7/17). All the exposed persons were sent to designated hospitals, and they were subjected to intensive isolation and medical observation since patient 1 was confirmed. After a maximum incubation period had passed, no more cases related to this cluster were reported, and the transmission of this cluster was terminated on February 3, 2020.1 Meanwhile, Qingdao immediately entered a state of “war.” Since the first confirmed case of COVID-19 was reported, the number of cases had been increasing in Qingdao City. So far, 171 cases had been reported in Qingdao City, which included 118 confirmed cases and 53 asymptomatic infected persons. This cluster, as the first cluster in Qingdao, was of great significance for prevention and control of the epidemic. On the 1 hand, it further confirmed the importance of “4 early” . The timely detection and isolation of cases and their close contacts could effectively prevent further spread of the epidemic, which was the key point of successful control of this cluster.2 On the other hand, this cluster raised people’s awareness of effective measures to prevent and control the epidemic, that is, they should not take it lightly and especially wear masks consciously and avoid eating meals together.3 Above all, Qingdao successfully controlled several clusters of outbreak, and no community transmission occurred. As shown in our study, it was crucial to detect and isolate cases and trace and quarantine close contacts as early as possible, which was particularly important when PCR results were delayed.As the COVID-19 outbreak occurred, Shandong province launched the first-level response to this public health emergency, in accordance with the management as A class infectious disease, belonged to B class infectious disease.Our study had limitations. First, we only tested samples from patients. Samples from the environment were not obtained. Second, the isolated virus was not sequenced, and the homology analysis was not performed.


BCAA AUC, and Cmax values were significantly higher with HP than LP , as were insulin AUC and Cmax values . Higher glucose, urea, and triglyceride concentrations occurred in the late postprandial phase (≥180 min) with HP. In conclusion, we noted that higher milk protein intake induces increased postprandial BCAA concentrations for at least 5 h and led to higher initial insulin secretion. Gluconeogenesis due to an influx of amino acids and their degradation after HP meal might explain the late effects of protein intake on glucose and insulin.Protein intake in early life influences metabolism, weight gain, and later obesity risk. As such, a better understanding of the effects of protein intake on the postprandial metabolism and its dynamics over time may elucidate underlying mechanisms. In a randomized crossover study, we observed fasted adults who consumed two isocaloric toddler milk formulas concentrated as meals of 480 kcal with 67 g of carbohydrates 30 g (HP) or 7 g (LP) protein, and 10 g or 20 g fat, respectively. Anthropometry and body plethysmography were assessed, and blood samples collected at baseline and over five hours. Time-specific concentrations, areas under concentration curves (AUC), and maximum values of metabolites were compared by paired t-tests to examine the effects of protein content of toddler milks on postprandial plasma concentrations of insulin, glucose, branched-chain amino acids (BCAA), urea and triglycerides. Twenty-seven men and women aged 26.7 ± 5.0 years (BMI: 22.2 ± 2.5 kg/m The early protein hypothesis suggests that a higher protein supply fed to infants with conventional infant formula, as compared with breastmilk, induces an increased early weight gain and an increased risk of obesity in later life ,2,3,4. AObservational studies have suggested that the effect of protein intake on weight and BMI is not restricted to infancy but can also be seen during toddlerhood—although the available evidence is limited . The ongTherefore, in the present study, we attempt to explore potential mechanisms by analyzing the acute effects of the two TOMI milk formulas as test meals on postprandial metabolism in healthy, young adults. We hypothesize that the consumption of a higher protein test meal will be followed by larger postprandial increases in plasma concentrations of insulin and BCAA, as compared to the lower protein formula. 2, and had no known metabolic, cardiovascular, or other disorders, including lactose intolerance. Upon pre-screening, two appointments were made for each intervention, separated by 5–10 days (with a mean of 6.7 days). Of 51 screen subjects, we could include 27 subjects  diagram of the progress from recruitment through to the completion of the study is included in the subjects . ® Sarstedt) was inserted into the participants’ forearm to collect blood samples. Prior to the first intervention, the participants completed an informed consent form, as well as a health and well-being questionnaire. Data on fitness and activity level, country of origin, education, and occupation were collected through questionnaires. Upon completion, anthropometric measurements were performed, including height, weight, and waist circumference. BMI was then calculated, and body composition was determined using air displacement plethysmography  to assess body fat content and percentage of body fat. A venous, indwelling catheter , produced and provided by Nestec, Ltd., Vevey, Switzerland, now renamed to Société des Produits Nestlé S.A., Vevey, Switzerland. In order to maintain double-blinding, the milk formula was pre-packaged by the manufacturer in individual-dosed cans labeled with A or B. Each milk powder was dissolved in 500 mL of water at a temperature of 40 °C prior to consumption. The utilized quantity of milk formula in the present study was chosen to be approximately equal to the caloric content of a single adult meal and corresponds to double the concentration used for the preparation of the milk formula for children participating in the TOMI study. Thus, the higher protein, lower fat (HP) test meal contained 2010 KJ/480 kcal energy, 30 g protein, 67 g carbohydrates and 10 g fat per 500 mL. The lower protein, higher fat (LP) test meal contained 2010 KJ/480 kcal energy, 7 g protein, 67 g carbohydrates and 20 g fat per 500 mL. A randomization list and the transfer of the planned assignments to sealed and consecutively numbered envelopes was prepared by a person not involved in the conduct and lab analysis of the study. The list was produced using a randomly permuted sequence of A and B with a block length of 2 or 4 and a consecutive ID number. Group A was allocated the product sequence AB and the group B the product sequence BA. Product allocation after informed consent was performed by opening the respective sealed envelope with the id containing the product code for the first and second appointment. At each appointment, participants arrived after an overnight fast of at least 12 h to the study site. Fasted blood samples were taken (time = 0 min), followed by anthropometric measurements, which were only performed during the first appointment. The pre-packaged milk formulas A and B were chosen for each trial appointment, according to the allocation. After insertion of the venous catheter the formulas were consumed within a 5-min time period, while sitting at a table. Initially, only 450 mL of water was mixed with the test milk formula. Upon consumption of the 450 mL, an additional 50 mL of water was mixed with the leftover milk at 40 °C, in order to ensure that all formula was consumed. Trial periods for all subjects began at 8:00 a.m., and the time of meal intake for all subjects did not differ more than one hour.®, Sarstedt) to measure glucose, insulin, BCAA, triglycerides, and urea. In the fasted blood samples additionally full blood count, CRP, ALAT, and ASAT were determined to evaluate the overall health status of the subjects. The same arm was used for the venous catheter at each of the two interventions.Timing for blood samples began once the full test meal was consumed, and venous blood samples were taken at 15, 30, 60, 90, 120, 180, 240, and 300 min postprandial. With the exception of the first samples of the first subject, all blood samples were taken within a minute of each time interval. At each of the time points, including the basal fasted blood samples, serum and EDTA blood samples were taken . The subjects were encouraged not to alter their normal routines between study tests and were asked to report any major change of their lifestyle  between the tests, though none were reported. g, 4 °C) and plasma was stored at −80 °C. After collection of all the samples, plasma was analyzed via HPLC-ESI-MS/MS on an API-2000 Triple-Quadrupole-MS instrument from Sciex , as previously described in Harder et al. with slight modifications to make the method more robust and reproducible [Alanine Aminotransferase , Aspartate Aminotransferase , C-Reactive Protein , and a full blood count  and flow cytometry  were determined from the fasted serum and EDTA samples, respectively, and glucose , insulin , urea , and triglycerides  for all collected time points by the Institute of Laboratory Medicine at the University Hospital, LMU Munich. For BCAA  determination EDTA samples were centrifuged  were using trapezoidal functions for insulin, which was the primary outcome measure, as well as secondary outcomes measures including postprandial glucose, triglycerides, urea, and BCAAs. Total BCAA was calculated as the sum of valine, leucine, and isoleucine. Further exploratory measures, such as fasted baseline concentrations, concentration maximums (Cmax), and time of maximum concentration (Tmax) were also calculated and assessed.Based on studies by Hirsch et al.  and NilsComparisons between the primary and secondary outcomes, as well as exploratory outcome measures between the two milk formulas were performed with paired t-tests or Wilcoxon rank test when considered appropriate. For additional explorative analysis, linear regression with cluster options (by subject) was used to test for the effects of other factors, such as BMI, gender and age on glucose, insulin, triglycerides, urea and BCAA. Statistical significance was assumed at maximum error probability of 0.05. All statistical analyses were performed with the software SPSS v26 , and Stata 15.1 . Twenty-seven subjects  participated, as shown in One subject dropped out after the first test meal  due to reported symptoms of food intolerance. The subject denied having been aware of any lactose or food intolerance. Thus, 26 subjects were available for the main outcome analysis and 27 and 26 subjects for any HP and LP, respectively, summary statistics. No other adverse effects occurred during the entire trial. CRP and liver enzymes were all within reference ranges, and all subjects were deemed healthy. All participants had or were undergoing an education at university level, with the majority (81%) being students. Most participants (81%) were of European descent, while 3 participants were of Asian descent (11%), and 1 participant each was of African and Indian descent, respectively.Body plethysmography showed a body fat percentage of 25.4 ± 1.4% (mean ± SD) in females and 15.7 ± 1.4% in males. Most participants reported to be physically active 2–3 times per week (38.5%), followed by participants who exercised more than 6 times per week (30.8%), 4–5 times per week (19.2%), and less than once per week (11.5%). Fasted baseline concentrations, Cmax, Tmax, as well as AUC values for glucose, insulin, urea, and triglycerides are shown in p < 0.001), 240 min  and 300 min , respectively.The postprandial curves for glucose and insulin show initial steep increases, with peak concentrations reached until approximately 30 min. Subsequently, the concentration curves sharply declined until 60 min, and then more gradually for the final three hours of the observation period. In comparison to the LP meal, glucose was somewhat higher directly after HP meals, then lower and again higher from 120 min onwards, while insulin peaked early and was always higher after HP meals. Concentration maximums and AUC for insulin were significantly higher for the HP compared to the LP milk meals . These cp < 0.05), 240 min , and 300 min .The time points of concentration maximums (Tmax) for insulin and glucose varied widely between subjects, but without significant influence by the test meals. The maximum glucose concentrations after both test milks were similar, and there were no significant differences in AUC values for glucose. However, significantly higher mean glucose concentrations after HP versus the LP test meals were observed at 180 min  and 300 min . The mean concentration curves for triglycerides for both HP and LP milk meals were characterized by similar, gradual developments until the end of the observation period with a significant difference in mean concentrations observed at 300 min .The concentration curves for urea and triglycerides showed gradual changes over the trial period for both the HP and the LP milk formulas. For urea, mean postprandial concentrations increased steadily until the end of the study (300 min) for HP, while the mean postprandial concentrations after consumption of LP showed a gradual decreasing trend. Significantly higher values after HP than LP were observed for the last two measurements at 240 min , and there was no association of BCAAs with glucose or insulin maximum concentrations. Maximum concentrations and AUCs of BCAAs were not associated with maximum concentrations or AUC values of insulin.Tmax in the HP group was reached significantly later for BCAAs than for insulin . In a linear regression model, including BMI, age, gender and the study product (HP vs. LP), 1 kg/m2 in BMI reduced the AUC insulin value by 472.5 µmol·min/L—corresponding to a 7–8% change in the AUC, and 1 year of age reduced the AUC insulin by 191.5 µmol·min/L.Age, gender, and anthropometrics  of participants were examined in relation to the AUCs of insulin, glucose, BCAAs and urea. BMI, weight, and age, but not fat mass correlated negatively and significantly with AUC insulin values , infants were randomly assigned formulas with higher and lower protein content that were consumed until twelve months of life. Fasted blood samples taken at six months indicated that infants who consumed the higher protein formula had decreased serum glucose, and increased concentrations of BCAAs, while even lower BCAA concentrations were observed in a breastfed reference group ,20. ThesAlthough studies in adults have focused on protein intake in the acute postprandial phase, there are consistencies in the data that support our hypotheses. Panahi et al. showed that postprandial glycemia and insulinemia are closely linked to meal composition, presenting lower insulin responses per kcal fat than protein . We obseMilk protein composition, primarily composed of whey and casein, is particularly relevant regarding metabolic responses. Whey protein is rapidly digested, triggering specific gastric incretins, such as glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) ,30. WheyWe noted significant differences in mean concentration of insulin, glucose and urea after HP compared to LP after 240 min. Krezowski et al.  showed tIt is unclear to which extent these acute metabolic effects observed in young, healthy adults can be extrapolated to children. While multiple observational studies have shown that early protein intake is associated with higher BMI in early childhood reflecting assumed early metabolic programming of adiposity ,41,42,43The limitations of our study include lack of a control for gastric emptying, and no measure of other endocrine factors that may have interacted with or influenced the pre- and postprandial phases. Further, we focused on the interplay of BCAAs in the postprandial metabolism, while other amino acids, particularly glucogenic, may have also played a role. Although our study included a modest sample size, we found clear group differences despite intra- and inter-individual variability in blood metabolite concentrations.The strengths of our study include the relatively long observation period of five-hours, the double-blind randomized design, and strictly controlled study conditions. Participants consumed a standard milk meal and completed no major physical activity for the duration of the observation period; therefore, we can exclude the confounding effects of exercise on the postprandial response. In an attempt to avoid any alterations in blood values, the venous catheter was always inserted into the same arm and anthropometric measurements were standardized based on the previously enacted TOMI study protocol.In conclusion, our results show that a high milk protein supply affects acute postprandial BCAA and insulin concentrations in healthy adults. The initial rise in insulin is likely due to the ingestion of glucose combined with BCAAs, while effects observed after two-hours are likely related to amino acid degradation and subsequent gluconeogenesis, due to the cumulative increase in circulating BCAAs supply over time. Though these outcomes cannot be directly compared to postprandial metabolic effects in young children, they can contribute to better understanding the mechanisms behind high milk protein supply on metabolism and growth in early life.


FL) on head, back, breast, and tail, wounds on head, back, tail, and feet, dirty birds, enlarged crop, sick birds, and dead birds. AssureWel involved scoring FL on head and back, and dirtiness of 50 random birds, and flock-level evaluation of beak trimming, antagonistic behavior, flightiness, birds needing further care, and mortality. NorWel involved scoring 8 welfare indicators on 50 random birds: FL on head, back, breast, and tail, dirtiness, and wounds on head, back, and tail. The AssureWel detected flock differences in both minor and major FL on the back (P < 0.01) as well as somewhat dirty birds (P < 0.01). The NorWel method detected flock differences in both minor and major FL on the head (P < 0.01), back (P < 0.001), breast (P < 0.001), and tail (P < 0.001) and somewhat (score 1) dirty birds (P < 0.05). The Aviary Transect method detected flock differences in FL on head, back, breast, and tail , dirty birds (P < 0.05) and enlarged crop (P < 0.001). More birds with FL on breast, and more dirty birds, were found in wall vs. central transects (P < 0.05). There was good interobserver agreement, except for dirty birds (P < 0.01), and positive correlations (P < 0.05) were identified between the Aviary Transect method and the other sampling methods for FL on head and back, and dirtiness. The three methods took similar time to complete (about 20 min/flock). In conclusion, all 3 methods detected significant differences in welfare indicator prevalence between flocks. The new Aviary Transect method provides egg producers with an efficient and sensitive whole-flock assessment of hen welfare status in multitiered aviaries.This study compared welfare assessment results in aviary flocks using 3 approaches: 1) A novel Aviary Transect method, 2) AssureWel, and 3) the Norwegian farm advisors’ NorWel method. The Aviary Transect time requirement, interobserver reliability, and within- and across-house sensitivity to detect welfare indicators were also evaluated. The study was conducted on 6 randomly chosen commercial white-strain layer flocks of similar age and flock size, kept in multitiered aviaries. The Aviary Transect method comprised standardized walks along each aisle while screening the whole flock for 12 welfare indicators: feather loss ( An integral part of ethical and sustainable egg production is to ensure acceptable hen welfare. Non-cage housing systems, such as multitiered aviaries, are increasingly used in commercial egg production. These systems offer the birds more space and opportunities to perform natural behavior compared to enriched cages . HoweverFL), dirtiness, beak trimming, antagonistic behavior, flightiness, birds needing further care, and mortality. The recommended sample size of scored birds is 50 birds for both LayWel and AssureWel, and there is good agreement in plumage scores between the methods  were randomly selected from the supplier lists of 2 different egg packing companies and were visited once between the ages of 70 to 75 wk . Produce2 to 1,000 m2 that extended around and under the tiered aviary structures. Each aisle within the aviary designated a different transect. There were 1 to 3 rows of tiered structures along the length of the house, with a wall transect along each side, and up to 2 central transects, for a total of 2 to 4 transects in the different houses  indicator descriptions  differences were adjusted for multiple comparisons using the post-hoc Tukey test. Spearman correlations were calculated using the PROC CORR script in SAS 9.3  to evaluate relationships between the 3 welfare assessment methods for all comparable welfare indicator categories. P-values < 0.05 were considered statistically significant.For the Aviary Transect data, we used the data collected by each of the 2 observers in each transect to calculate the frequency of birds with a particular welfare indicator as a proportion of the total estimated number of birds in each transect type . For the AssureWel and Norwell data, the proportion of 50 birds with scores 1 and 2 was calculated for each welfare indicator category, by the total number of birds scored in the flock (AssureWel) or by birds count per transect type and vertical level of the house (NorWel). We analyzed the data in SAS Version 9.3 (SAS Institute Inc. 2013) using 3 generalized linear models (PROC GLIMMIX) with binomial distribution, one model per welfare assessment method. Each welfare indicator evaluated within a welfare assessment method was analyzed using the method-specific model, where the response variable was a single welfare indicator. The Aviary Transect model included flock, observer and transect type  as fixed factors. The AssureWel model included only flock as a fixed factor. The NorWel model included flock, transect type , and vertical level (floor plus 3 aviary tiers) as fixed factors. The flock factor provided a measure of the sensitivity of each method across flocks, the observer factor was introduced to test the interobserver reliability of the Aviary Transect method and the transect type factor was introduced to evaluate the within-house sensitivity of the Aviary Transect method  and NorWel method . Least Square Means (P < 0.001), dirty birds (P < 0.05) and enlarged crop  as well as somewhat (score 1) dirty birds  and major (score 2) FL on the back (both P < 0.01) and major  FL on the head. There were also differences in minor and major FL on the back (both P < 0.001), minor and major FL on the breast (P < 0.001), minor and major FL on the tail (P < 0.001) and somewhat (score 1) dirty birds , the transect scores ranged from 0.03 to 0.81% of the birds, while the corresponding ranges for AssureWel and NorWel were 0 to 28% and 0 to 22.3%, respectively. Despite these inherent differences, the results from the Aviary Transect method were highly correlated with the results from AssureWel and NorWel in the observed flocks. In fact, in 2 of 3 comparable indicators, agreement between the Aviary Transect method and each of the other 2 methods was better than that between AssureWel and NorWel. As the latter 2 methods scored FL on the head and back using identical categories  on 50 random birds, we could expect a high level of agreement between them. This was the case for FL on the head but not the back, suggesting that the whole-flock scoring done in the Aviary Transect method gives a more reliable assessment of FL in large flocks.When there is a large variation between birds in a flock, a larger sample size provides a more reliable estimate of prevalence . This maIndividual sampling is a common method for assessing laying hen welfare, including observing 50 birds from a distance  or catching 150 birds for inspection . The AviThe transect method has been shown to be an effective tool for early detection to predict welfare issues and production results in broilers ,b and tuThe Aviary Transect method scores birds in each transect, and the scores can then be combined to give an overall flock score. For most indicators, there were no differences in prevalence between transects, suggesting that birds with these issues are evenly distributed around the house. However, we found more laying hens with FL on the breast and dirtier birds in wall transects as compared to central transects. This result differs from findings in broiler flocks, where a higher prevalence of sick birds  or dead The transect method showed very good interobserver agreement when applied in aviaries, with dirty birds being the only indicator that differed significantly between observers. This is in accordance with results reported by On-farm welfare assessment is challenging because it needs to be noninvasive, adaptable to different farms, and cost-efficient . It mustIn conclusion, the present results show that all 3 methods were time efficient and detected differences between flocks for several of the assessed welfare indicators. The AssureWel and the NorWel methods gave graded scores for 8 welfare indicators, providing information about the severity of each indicator, but on relatively few animals. In contrast, the newly developed Aviary Transect method evaluated every bird in the flock for 12 indicators on a binary scale, resulting in a relatively sensitive evaluation of the prevalence of each indicator, with a focus on the more severe cases in the flock. Thus, the Aviary Transect appears to be an efficient and sensitive whole-flock assessment method for laying hens in multitiered aviary systems. The transect method has good interobserver reliability and good agreement with methods involving graded sampling of a subset of individuals, with the added benefit of including a higher number of welfare indicators. The Aviary Transect method has the potential to provide egg producers with a quantitative and specific assessment of the welfare status in the flock that is easy to use on the farm.


The World Health Organization’s global action plan on workers’ health establishes that occupational health services should carry out lifestyle interventions within the workplace, to prevent the development of non-communicable diseases. The objective of the study was to compare adherence to a healthy lifestyle six months after completion of a multi-component intervention with remotely supervised physical activity during the COVID-19 pandemic versus a multi-component intervention with in-person supervised physical exercise before the COVID-19 pandemic in university employees with unhealthy habits and predisposed to change. A prospective cohort study following the “Strengthening the Reporting of Observational studies in Epidemiology” (STROBE) guidelines was conducted, with two arms. Each multi-component intervention lasted for 18 weeks, and consisted of education on healthy habits, Mediterranean Diet (MedDiet)-based workshops, and a physical exercise program. Twenty-one middle-aged sedentary university employees with poor adherence to the MedDiet completed the study. Six months after completion of the intervention, both groups increased physical activity levels, adherence to the MedDiet, eating habits, health-promoting lifestyle, health responsibility, and health-related quality of life. There were no differences between groups in any of the variables analyzed. Therefore, remotely supervised physical exercise could be adequate to achieve long-term adherence to a healthy lifestyle in the same way as conventional face-to-face intervention, at least in a population willing to change. Recently, it has been highlighted that a healthy lifestyle is an effective strategy to improve health and reduce the incidence of non-communicable diseases (NCDs), like type 2 diabetes or cardiovascular diseases . Among tMost exercise and diet obesity interventions successfully achieve their objectives but do not maintain these changes in the long term, and after six months, generally, they lose effectiveness . Six monTo solve the frequent poor adherence to lifestyle interventions, a meta-analysis performed in 2018 highlights the importance of having psychological assistance . In thisThe COVID-19 pandemic has implied a reorganization of our society and our lifestyle, with mobility restrictions, so virtual interventions are appropriate in this context. Furthermore, sedentary behavior levels have increased during the COVID-19 pandemic ,19,20,21Another advantage of virtual interventions is that they have lower costs, greater time flexibility, and higher time savings by not having to travel, compared to face-to-face interventions, favoring adherence . HoweverHealthy lifestyles help to prevent the development of NCDs, and the work environment covers an age group with silent risk factors such as high levels of sedentary behavior . In thisVery few randomized clinical trials with Mediterranean diet interventions within the workplace managed to increase adherence to this diet pattern, although without long-term follow-up ,38. RegaThe objective of this study was to analyze whether remotely supervised physical exercise is as effective in achieving adherence to a healthy lifestyle and enhancing HrQoL as in-person supervised physical exercise in employees predisposed to change. This study hypothesized that in employees who are predisposed to change, a remotely supervised physical exercise and diet intervention achieves similar adherence to a healthy lifestyle and similar improvement in HrQoL as a physical exercise and diet intervention with in-person supervision.A prospective cohort study was conducted, with two lifestyle multi-component interventions (in-person supervised exercise cohort and remotely supervised exercise cohort), following the statement “Strengthening the Reporting of Observational studies in Epidemiology” (STROBE) .The study protocol adheres to the “Ethics Guidelines of the Declaration of Helsinki”, the last modification produced in 2011. It has the approval of the regional ethics committee of the Community of Madrid (CEIm) . All participants signed informed consent before participating in the study.The participants of both cohorts were university employees that were selected by convenience sampling from the occupational health service. At two different times, December 2018 and December 2019, informative meetings of the intervention were held. To see if they met the inclusion criteria, employees interested in participating in the intervention filled out the Mediterranean Diet Adherence Score (MEDAS), the University of Rhode Island Change Assessment Scale (URICA), and the Global Physical Activity Questionnaire (GPAQ).The inclusion criteria were the following: (1) University employees, adults; (2) Not complying with 2010 the WHO PA recommendations ; (3) HavThe exclusion criteria were having chronic diseases or musculoskeletal injuries that contraindicate physical exercise. The first cohort, the in-person supervised exercise group (ISEG) started in January 2019 a multi-component intervention. Before that, the participants received a detailed report on their lifestyle habits such as PA, sedentary behavior, or diet, and the possible long-term consequences on their health. In the first place, an educational program on healthy habits was carried out in which the participants viewed 12 weekly videos on different topics: (1) Motivation for change; (2) Nutrients, fiber and water; (3) Frequency of eating; (4) Breakfast and snacking; (5) From the food market to your dining table; (6) Circadian rhythms; (7) Physical activity recommendations; (8) False food and physical exercise myths; (9) Body composition healthy values; (10) Chronic diseases; (11) Nutritional strategies; (12) Physical exercise strategies. Three weeks after starting this first component, the diet program started in parallel, consisting of nine face-to-face weekly healthy eating workshops of 90 mins duration. In these workshops, the participants practiced diet planning following the Mediterranean diet pattern, addressed barriers to change, and reinforced the nutritional concepts of the educational program. Once the nutrition component was finished, an in-person-supervised physical exercise program was carried out, lasting for lasted six weeks, with 18 sessions of 60 mins each, with a frequency of three weekly sessions, combining strength and resistance exercises, and following 2010 WHO recommendations.The typical training session consisted of a 10-min warm-up of mobility exercises; a 40-min main part consisting of a strength circuit training with 2–3 sets of 12–15 repetitions of 7–8 exercises involving major muscle groups, at an intensity of 7 to 8 RPE, with a rest of 30 s between exercises and 1 min between sets, and then aerobic exercise  at an intensity of 7 to 8 on the revised category-ratio Borg scale of perceived exertion (RPE); and a 10-min cool down consisting of flexibility exercises.The second cohort, the remotely supervised exercise group (RSEG), started the same program in January 2020. The difference was that at the end of the diet component, the Spanish government ordered a strict stay-at-home lockdown to stop the expansion of the COVID-19 outbreak, so the physical exercise program was remotely supervised in real-time, as the participants were at home. In this exercise program, with the same characteristics as that of the ISEG, the participants performed self-loading strength exercises and performed aerobic exercise jogging at home since they did not have specific training material or large spaces . For each participant, the diet workshops were in-person supervised by two nutritionists for both cohorts, while the physical exercise programs were supervised by two PA professionals, in-person (ISEG) and remotely (RSEG) .The diet workshops were carried out at the university campus facilities during the employees’ workday for the ISEG and RSEG. The ISEG performed the PA program at the university campus facilities, while the RSEG performed it at home, with remote supervision, due to the COVID-19 lockdown. Both cohorts were evaluated before the multi-component intervention (T1) and six months after completion of it (T2).To analyze lifestyle, the Health-Promoting Lifestyle Profile II (HPLP-II) questionnaire was filled out. It consists of 52 items that are answered as N , S , O , and R . This questionnaire consists of six subscales: Health responsibility (9 items), Physical activity (8 items), Nutrition (9 items), Spiritual growth (9 items), Interpersonal relations (9 items), and Stress management (8 items). The score for each scale is calculated using the means of the items ,46. ConcTo analyze another of the specific components of lifestyle, the MEDAS questionnaire was filled out to assess adherence to the Mediterranean diet. This questionnaire consists of 14 items, of which each adds 0 or 1 point. The items of this questionnaire positively evaluate the consumption of vegetables, fruit, olive oil, fish, nuts, and negatively the consumption of red meat, sugary drinks, commercial pastries, or butter. It is considered high adherence ≥10 points, which is a strong protector against cardiovascular diseases, medium adherence 8 to 9 points, and poor adherence ≤7 points . PA level and sedentary behavior were analyzed using the GPAQ questionnaire ,49. ThisTo analyze HrQoL, the Short Form 36 Health Survey Questionnaire v2 (SF36) was used, which consists of 36 items scored from 0 (worst perception of HrQoL) to 100 (best perception of HrQoL) in 8 health concepts: Physical Function, Role Physical, Bodily Pain, General Health, Vitality, Social Functioning, Role Emotional, and Mental health . These eight health concepts are regrouped into the Physical Component Summary and the Mental Component Summary. A 4-point increase in any of these two components is considered clinically relevant in healthy adults .2) was calculated.Height , and weight  were measured. Then, the body mass index , the resulting sample needed to achieve the objective of the study was 22 participants, so 24 participants were sampled to compensate for a probable 10% dropout.t-test was performed for both ISEG and RSEG, to determine the time difference. Finally, to compare the difference between the results of the two interventions, the independent t-test and the Mann–Whitney U-test were used again. The level of statistical significance was set at p < 0.05. Eta partial squared (η2p) was used as a measure of effect size [All the results were analyzed by protocol and intention-to-treat analysis (ITT). The distribution and normality of the data were analyzed with the Shappiro–Wilk and Levene tests and with P-P and Q-Q plots. Data are expressed as mean ± SD. The independent T test and the Mann–Whitney U-test were used to compare the differences between both groups (RSEG and ISEG) before the multi-component intervention, with the aim of evaluating the homogeneity of the groups. Then, a paired ect size , consideect size . All staOf 15 participants initially recruited in January 2019, three (20%) did not met the inclusion criteria, so n = 12 subjects were assigned to the ISEG. Also, of 15 participants initially recruited in January 2020, three (20%) did not met the inclusion criteria, so n = 12 subjects were assigned to the RSEG. There was one dropout in the RSEG, and two dropouts in the ISEG, so the final analysis was performed on 11 RSEG and 10 ISEG subjects, as is shown in the flow diagram . This st2; while the mean age of the ISEG was 43.35 ± 7.59 years, the weight was 77.89 ± 13.88 kg, and the BMI was 27.63 ± 4.64 kg/m2. There were no significant differences in these variables between the two groups.In the RSEG, 42% of the subjects were men and 58% women, while in the ISEG, 25% were men and 75% were women. The mean age of the RSEG was 42.78 ± 6.88 years, the bodyweight was 74.98 ± 15.68 kg, and the BMI was 25.82 ± 3.70 kg/mp < 0.001; η2p = 0.68), and in the categories of Health Responsibility , Physical Activity , Nutrition , Spiritual Growth , and Stress Management , with a large effect size for these six variables. Also, the RSEG participants significantly improved their Adherence to the Mediterranean diet , with a large effect size, progressing from low adherence (MEDAS score ≤7) to medium adherence (MEDAS score 8–9). The RSEG participants also increased significantly their PA levels , progressing from low to moderate levels [p = 0.06)  .p < 0.01; η2p = 0.57), and in the categories of Health Responsibility , Physical Activity , and Nutrition , with a large effect size for these four variables. Also, the ISEG participants significantly improved their Adherence to the Mediterranean diet , with a large effect size, progressing from low adherence (MEDAS score ≤7) to medium adherence (MEDAS score 8–9). The ISEG participants also increased significantly their PA levels , progressing from low to moderate levels [p = 0.03, η2p = 0.44)  .When comparing the results in the T1-T2 analysis between the RSEG and the ISEG, there were not significant differences in any of the lifestyle variables analyzed .p < 0.01, η2p = 0.64), General Health , and the Physical Component Summary , with a large effect size for these three variables. Regarding the Physical Component Summary, the improvement was clinically significant (>4 points) [In the T1-T2 analysis, the RSEG participants significantly improved their HrQoL in the cathegories of Vitality , with a large effect size. In the case of the Mental Component Summary, there was a clinically significant improvement (>4 points) [p = 0.32)  , althoug = 0.32) .When comparing the results in the T1-T2 analysis between the RSEG and the ISEG, there were not significant differences in any of the HrQoL variables analyzed .p = 0.24), but significantly decreased in the ISEG , with a large effect size  and quantitatively (GPAQ questionnaire), with a large effect size, progressing from low PA levels to medium PA levels, with no significant differences between groups. These data contrast with a systematic review conducted in 2016 by Schoeppe et al. , in whicRegarding sedentary behavior, the ISEG decreased the daily sitting hours, with a large effect size, while the RSEG did not. These data are similar to a systematic review carried out in 2017 by Stephenson et al., in which virtual interventions failed to reduce sedentary behavior . HoweverBoth groups increased adherence to the Mediterranean diet, with large effect size, and acquired healthy habits such as following a diet low in saturated fat, limiting the consumption of sugars and sweets, eating 2–4 servings of fruit and 3–5 servings of vegetables a day, or limiting the consumption of salt, according to the HPLP-II questionnaire. Participants probably spent less time eating at restaurants and more time cooking at home. Both diet components were similar as they consisted of in-person workshops. The difference between groups was the context of the COVID-19 pandemic in which the RSEG participants were. The ISEG and the RSEG showed similar improvements, without statistically significant differences, so the context generated by the COVID-19 pandemic was not decisive in these variables to implement this change in habits, even though during this time the prevalence of snacking between meals and the consumption of alcoholic beverages and carbonated beverages have increased . Other sAs well as the participants of the RSEG and the ISEG improved their PA levels and their eating habits, their HrQoL also increased. In this regard, some observational studies have found positive correlations between HrQoL and PA levels  and adheThe participants in this study achieved long-term adherence to a healthy lifestyle, which could be partly because they were predisposed to change , and hadThe clinical relevance of the study is that it involves multi-component interventions that promote a healthy lifestyle within the workplace, from the occupational health service, as established by the WHO , in emplIn this study carried out on overweight middle-aged university employees within the workplace, both groups showed similar long-term improvements in their HrQoL and lifestyle, mainly in PA levels and eating habits. The context of the COVID-19 on which was the RSEG did not seem to have influenced the results. Therefore, remotely supervised physical exercise could be adequate to achieve these objectives in the same way as a conventional face-to-face intervention, at least in a population willing to change.


This study aimed to characterize self-reported headaches because of problems with the teeth, mouth, jaws, or dentures (HATMJD) in chronic patients with temporomandibular disorders (TMDs) in order to compare their results with those of TMD patients without such headaches and to investigate the associations of HATMJD with depression, anxiety, physical symptoms, oral behaviors, and sleep quality. We conducted a case–control study on consecutive chronic TMD patients referred to the University Medical Center of Ljubljana, Slovenia. A self-reported HATMJD was extracted from item #12 in the 49-item version of the Oral Health Impact Profile questionnaire. Axis II instruments of the Diagnostic Criteria for TMD  and the Pittsburgh Sleep Quality Index were used in this study. In total, 177 TMD patients  participated in this study; 109 (61.6%) patients were classified as TMD patients with HATMJD. TMD patients with at least mild depressive and anxiety symptoms, with at least low somatic symptom severity, and a high number of parafunctional behaviors had more HATMJD. Parafunctional behavior and sleep quality were the most prominent predictive factors of the occurrence of HATMJD. TMD patients with HATMJD have more psychosocial dysfunction, a higher frequency of oral behaviors, and poorer sleep quality than TMD patients without such headaches. Many clinical studies have confirmed associations between temporomandibular disorders (TMD) and tension-type headaches or migraine ,2,3,4. TIt is generally accepted that TMDs have a multifactorial etiology and that psychosocial components, including oral behaviors and trauma, contribute to the development, exacerbation, and progression to chronic TMD ,14. HowePsychological distress  increases the symptom burden and functional impairment in several chronic medical conditions  ,20,21,22Psychopathology may also discriminate between myogenous and arthrogenous groups of TMD patients . AccordiOral parafunctions include sleep and awake bruxism, lip biting, thumb sucking, and any other oral habit not associated with mastication, deglutition, and speech . The mosThe substantial relationship of myofascial pain on sleep quality is documented in the study, which assessed differences in sleep quality between patients with chronic daily headaches and patients with TMD . It was The diagnosis of “headache attributed to temporomandibular disorder” (HATMD) was first introduced as a secondary headache in the second edition of International Classification of Headache Disorders (ICHD-II) under the code 11.7 by the Headache Classification Subcommittee of the International Headache in 2004 and further defined in the third version  in 2018 . The HATPrimary headaches  contribute to TMD problems . ConversThis research was approved by the Faculty of Arts’ Ethics Committee, University of Ljubljana, Slovenia, on 6 June 2016, and by the National Medical Ethics Committee of the Republic of Slovenia (KME 124/05/16). All participants signed a statement regarding informed consent before beginning the study.This case–control study was performed on consecutive adult chronic TMD patients referred for TMD management to the Clinic for Orofacial Pain and Dental Sleep Medicine within the Department for Prosthetic Dentistry  between March 2016 and February 2021. Exclusion criteria included being aged less than 18, the presence of orofacial pain disorders other than TMD, or systemic rheumatic diseases. The diagnosis of TMD was based on history and physical examination findings according to DC/TMD protocol by a board-certified specialist for orofacial pain. The baseline data prior to treatment were analyzed for this study. We collected the data about demographics, such as gender, age, marital status, and level of education. The presence of the self-reported HATMJD was extracted from the Slovenian version of the psychometrically validated 49-item Oral Health Impact Profile questionnaire (OHIP), specifically from item #12  . ResponsAdditionally, Axis II instruments of the Diagnostic Criteria for TMD  and the Pittsburgh Sleep Quality Index were used in this study. All these questionnaires were translated into the Slovenian language and psychometrically validated in the Slovenian TMD population in the previous study .Depression. PHQ-9 was introduced in 2001 and is designed to screen and measure the severity of depression [pression . Patientpression .Anxiety. GAD-7 was introduced in 2006 as a self-reported questionnaire to screen and measure generalized anxiety disorder severity [severity . Patientseverity .Physical symptoms. PHQ-15 was introduced in 2002 to inquire about 15 somatic symptoms  that account for more than 90% of the physical complaints [mplaints . Subjectmplaints .Parafunctions. The Oral Behavior Checklist (OBC) is a self-report scale for identifying and quantifying the frequency of jaw overuse behaviors  [yawning) . For eacSleep quality. The Pittsburgh Sleep Quality Index (PSQI) measures the self-reported quality and patterns of sleep in adults [n adults . It diffn adults .All questionnaires were administered to our TMD patient cohort in electronic form before treatment. The questionnaires were presented in 1KA software , a free,For this study purpose, the TMD patients were subsequently divided according to the presence or absence of the self-reported HATMJD, based on item #12 from the OHIP questionnaire results. If the TMD patients responded to item #12 with “Never” or “Hardly ever”, we interpreted this as the absence of HATMJD. In contrast, if they answered to this item as “Occasionally”, “Fairly often”, or “Very often”, we interpreted this as the presence of HATMJD. Therefore, the TMD cohort was stratified into the HATMJD and non-HATMJD groups, which were our study and control groups, respectively.The variables analyzed as potential explanatory factors responsible for the variability of our observed outcome  were patient demographics, depression, anxiety, specific comorbid functional disorders, parafunctional behaviors, and sleep quality. Because measures for our constructs were continuous or ordinal in their original metric , our TMD patients were grouped according to DC/TMD recommendations as follows: depression , anxiety , specific comorbid functional disorders , and parafunctional behaviors  . Sleep qThe effect sizes with 95% confidence intervals  demonstrated the standardized differences between the two groups. Standard guidelines for interpreting effect size suggest that 0.2 is considered small, 0.5 medium, and 0.8 a large effect . The assp < 0.05. Statistical analysis was performed in SPSS for Windows software, version 25.0 .The model’s predictive power was evaluated using the Omnibus Test of Model, and the Hosmer–Lemeshow test was used to examine the goodness of the model’s fit . NagelkeOne hundred and seventy-seven adult TMD patients  participated in this study. All participants answered all items in the previously described questionnaires. Of all included TMD patients, 109 (61.6%) patients were classified as TMD patients with HATMJD, whereas 68 (38.4%) TMD patients reported no presence of HATMJD. The demographic and clinical characteristics of the studied TMD patient cohort and its subgroups  and the second subgroup  are presented in The results of the simple logistic regression analysis are presented in The complete five-step multiple logistic regression analysis is shown in 2 = 32.399, p < 0.001; Hosmer–Lemeshow test for goodness of fit: χ2 = 10.206, p = 0.251), and overall, correctly classified 70.6% of the TMD patients. The final model explained 22.7% of the variance for the presence of HATMJD (Nagelkerke’s R square was 0.227). The final model had a statistically significant predictive power  were included in multiple logistic regression analyses to identify the significant associations between the predictors (independent variables) and the outcome (dependent variable: the presence of a HATMJD). In other words, we aimed to create the best regression model from our predictor variables that most accurately explain the associations between our predictor variables and the presence of HATMJD.Our TMD patients with a high frequency of parafunctional activities had a 1.2-times higher chance of developing the self-reported HATMJD. Furthermore, in female TMD patients, the perceived HATMJD was 2.6-times more likely to occur than in male TMD patients. The female gender was the most prominent statistically significant sociodemographic characteristic in TMD patients with HATMJD. These findings are consistent with previous studies that investigated the co-morbidity of TMD and headaches ,22,56,57Medium to high somatic symptom severity was reported in 34%, moderate to severe anxiety in 19.8%, and moderate to severe depression in 19.3% of TMD patients, who reported HATMJD in our study. The association between psychological distress  and TMD pain was investigated in some previous studies ,59. In tIn the final model adjusted for age and gender, three variables were statistically significantly correlated with the presence of HATMJD in our TMD cohort. Female patients with poorer sleep quality and a higher frequency of parafunctional oral behaviors were more likely to have the presence of HATMJD. Therefore, oral behaviors and sleep quality when adjusted for gender are the statistically significant factors contributing to the perceived HATMJD. Our findings, therefore, are consistent with previous studies in patients with tension-type headaches and migraines ,32, whicInterestingly, in the recent study by Reiter et al., which retrospectively evaluated DC/TMD Axis I and Axis II data from 220 patients having pain-related TMD with HATMD, the mean depression scores measured with PHQ-9 were similar (6.88 ± 4.69) to our study (6.6 ± 5.3), mean anxiety scores, i.e., GAD-7 scores, were lower (4.35 ± 3.79) than in our study (4.9 ± 4.8), and somatic symptom severity, i.e., PHQ-15 scores, were also lower (7.18 ± 4.18) than in our study (8.4 ± 4.4) . AlthougThe study of Tchivileva et al. reported a mean PSQI summary score of 6.0 in patients with comorbid tension-type headache and HATMD and 7.6 in patients with comorbid migraine and HATMD . In contTo our knowledge, this is the first study to investigate the associations between all types of self-reported HATMJD in the TMD patient cohort and the psychosocial dysfunction, sleep quality, and parafunctional activities. Nevertheless, this study has limitations. We did not specify which TMD diagnosis is more associated with self-reported HATMJD. Our study aimed to focus on psychosocial predictors for HATMJD in TMD patients, and for this reason, we did the multiple logistic regression analysis on the complete TMD patient cohort. Additionally, the accuracy of self-perceived oral parafunctional behaviors is questionable when reported only using a questionnaire because most individuals with sleep bruxism are not aware of this oral behavior since it occurs during sleep.TMD patients with at least mild depressive and anxiety symptoms, with at least low somatic symptom severity, and a high number of parafunctional behaviors, had more HATMJD. This is additional proof that the DC/TMD Axis II constructs are crucial outcome predictors for TMD treatment purposes and HATMJD in this patient population. Furthermore, this study indicates that besides HATMD, which is a secondary headache, other types of headaches, including the primary ones, are also associated with the DC/TMD Axis II constructs and sleep quality when present in TMD patients. Therefore, assessing psychological distress, oral behaviors, and sleep quality is highly recommended in TMD patients with comorbid headache(s). Future research could also focus on how and if various TMD treatment modalities also affect different types of headaches in TMD patients.


Gossypium hirsutum L.) planting technology. This study aims to demonstrate the effects of drip tape modes on soil hydrothermal conditions, cotton yield, and water use efficiency (WUE) of machine-harvest cotton under mulch in Xinjiang.The layout of drip tapes under mulch has changed in Xinjiang, China, with the development of machine-harvest cotton , and a conventional planting mode  as a control.Our results showed that the heat preservation and warming effects of the cotton planting model with one film, two drip tapes and six rows and the cotton planting model with one film, three drip tapes and six rows were better than that of the conventional planting mode. Soil temperature under the mulching film quickly increased and slowly decreased, which was beneficial to the early growth and development of cotton. The mean soil moisture content of the 0–60 cm soil layer in the cotton planting model with one film, three drip tapes and six rows was significantly higher than the other two treatments at the middle and late stage of cotton growth (90 days after sowing (DAS) and 135 DAS). Moreover, the water holding capacity of the middle and upper part of the tillage layer in the cotton planting model with one film, three drip tapes and six rows was the best. At the medium cotton growth stage, the main root layer in the cotton planting model with one film, three drip tapes and six rows formed a desalination zone. At the late cotton growth stage, the soil salinity content of the 0–60 cm soil layer showed that the cotton planting model with one film, three drip tapes and six rows was the lowest, the cotton planting model with one film, two drip tapes and six rows was the highest, and the conventional planting pattern was in the middle. Among these three modes, the cotton planting model with one film, three drip tapes and six rows was more efficient in controlling soil salt accumulation. The agronomic traits and cotton quality in the cotton planting model with one film, three drip tapes and six rows were better than that for the other two treatments. Compared with the other treatments, the cotton yield in the cotton planting model with one film, three drip tapes and six rows increased by 6.15% and 11.0% and 8.1% and 12.3%, in 2017 and 2018, respectively, and WUE increased by 17.4% and 22.7% and 20.9% and 22.8%, in 2017 and 2018 respectively. In conclusion, the cotton planting model with one film, three drip tapes and six rows can be recommended for machine-harvest cotton planting for arid areas in Xinjiang, considering water conservation and improving cotton yield. Gossypium hirsutum L.) planting region in China. Abundant light and heat resources provide advantages for industrial cotton growing  has been in place since the 1990s  from April 2017 to November 2018 at Shihezi University in Xinjiang, China. The region experiences a typical arid continental climate, with an average annual rainfall of 210 mm, air temperature of 7.2 °C, sunshine duration of 2,865 h and frost-free period lasting 171 days. The average accumulated temperatures above 10 °C and 15 °C are 3,463 °C and 2,960 °C, respectively. Changes in precipitation, daily reference evapotranspiration (ET0), and maximum atmospheric temperature in the cotton-growing season (from April to November) in 2017 and 2018 are presented in –1, respectively. The cotton variety ‘Nong feng 133’ is suitable for dense planting, good ventilation, light transmission among populations, early maturity, and high yield. The soil physical conditions  of 0–100 cm soil layers in the test area are shown in The experimental area was 0.06 ha. The regional ground-water table remained at a depth of 8 m. The local soil was medium loam with a pH of 7.83, a mean field water holding capacity of 19.5%, and mean permanent wilting point of 9.8%. In the surface soil (0–20 cm in depth), the amounts of soil organic matter, total nitrogen (N), phosphate (P), and available potassium (K) were 19, 0.08, 0.94, and 0.42 g kg–2. T2: the cotton planting model with one film, three drip tapes and six rows  mm. The water supply system in the study area was mainly pressurized by a water pump, and the pressure gauge and regulating valve were installed at the head of the system. The irrigation quota, irrigation volume, irrigation date, irrigation times, and fertilization times of each planting mode were the same during the experiment. The cotton was irrigated 13 times during the whole growth period. The irrigation quota was 450 mm; the N fertilizer was applied at 225 kg hm–2 and the P fertilizer at 140 kg hm–2 during the whole growth period. Irrigation was carried out by drip irrigation under the mulch. The irrigation interval was 7–10 days. The squaring period occurred four times, the flowering period occurred four times, the bolling period occurred three times, and maturity occurred two times. The fertilizer was dissolved in the water for fertilization while irrigating. The details of irrigation and fertilization management during the cotton growth period are shown in The plant spacing of cotton in the machine harvesting and traditional planting modes was 10 cm and 30 cm, respectively , and theThe soil temperature was measured at 5, 10, 15, 20, and 25 cm depths in each plot using geothermometers . Each set of geothermometers was inserted between the wide and narrow rows of cotton planting in each plot. In 2017 and 2018, soil temperature was measured at 08:00, 12:00, 14:00, 16:00, 18:00 and 20:00 on the day of seedling , squaring (50 DAS), flowering (65 DAS), bolling (90 DAS), boll opening (120 DAS), and maturity (180 DAS). The automatic weather station in the test station was used to monitor the real-time atmospheric temperature.3) and dried to measure soil moisture and bulk density. Soil samples were taken from each depth and repeated three times. A detailed description of soil sampling and soil moisture measurement was provided in In 2017 and 2018, gravimetric soil moisture was measured to a depth of 100 cm at 10 cm intervals on 45, 90, 135, and 180 DAS, respectively. On the measurement day, in T1, T2, and T3 models, three soil cores were auger drilled in each plot to collect the samples from: one below the drip tape, one in the middle of the wide row under the mulch film, and one below the bare soil between films . Soil saSoil samples were taken from wide row, narrow row and bare land between mulching film at different growth stages of cotton. The soil samples were dried, ground and passed through a 1 mm sieve. Each sample was mixed with 20 g soil according to the ratio of soil and water mass 1:5 and then placed for 2 h after shaken evenly. The conductivity of supernatant was measured by conductivity meter (DDS11-A). The absolute change (ΔS) and relative change (R) in soil salinity were calculated as:EC1:5 and EC′1:5 (ds m–1) represent soil salinity before sowing and after harvest, respectively.where, e.g., emergence, squaring, flowering, boll opening, and maturity) was recorded in all plots. The definition of each phenological stage was adopted from The aboveground dry matter was measured from 45 DAS and continued at the 45-day intervals after seeding in 2017 and 2018. On each measurement day, the aboveground parts of five cotton plants were collected. The plant samples were dried to a constant weight to measure the biomass. Moreover, the date the plants entered each phenological stage (−1). The water use efficiency  was calculated as the ratio between annual cotton yield and total evapotranspiration (ET) over the growing season in each year  in each plot and convert actual yield into total yield per hectare .where 2 (2 m × 2 m) in each plot to measure the boll weight and lint. The cotton lint sample was sent to the Cotton Quality Supervision, Inspection and Test Center of the Ministry of Agriculture and Rural Affairs to test the quality of cotton fiber with a high-volume instrument (HVI)  .P < 0.05 level.The test data were graphed and processed using SPSS 20.0  and Origin 9.0. Measurement data were analyzed using the one-way analysis of variance (ANOVA) tests. Significantly differences among various treatments are calculated through the least significant difference (LSD) at Differences in mean atmospheric temperature and mean temperature of the topsoil (5–25 cm depth) between the plots with different drip tape modes across days after sowing are shown in The wetting effects of different drip tape modes at 45 DAS, 90 DAS, 135 DAS, and 180 DAS representing different cotton-growing stages are shown in The desalinizing effects of different planting patterns at 45 DAS, 90 DAS, 135 DAS, and 180 DAS representing different cotton-growing stages are shown in i.e., 6,701 kg ha–1 in 2017 and 6,730 kg ha–1 in 2018)  , 6B. TheThe WUE in T2 was the highest , 6D, whiDifferences in the mean temperature of the topsoil (5–25 cm depth) between the plots and atmospheric temperature under the three drip tape modes under mulch are compared in Drip irrigation under mulch is small-scale irrigation, which can effectively maintain soil moisture and evenly drive out salt . In the Drip tape arrangements can affect cotton growth, which will be reflected in the cotton yield and WUE in each treatment . Deng etThe planting density in T1 and T2 was the same, but the location of drip tape was different, which can lead to soil water and salt transport differences . WUE depThis study evaluated the effects of different drip tape modes of drip irrigation on soil hydrothermal conditions, salt accumulation, cotton growth, and WUE under mulching film over 2 years in an oasis agroecosystem in Xinjiang, northwest China. We came to the following conclusions. Among the three modes, the water holding capacity of the middle and upper parts of the tillage layer in the cotton planting model with one film, three drip tapes and six rows was the best. In this mode, the main root layer of the cotton formed a desalination zone, which was more effective in controlling soil salt accumulation. The cotton plant height, leaf area index, and leaf dry matter mass were significantly higher than in the other two modes. The cotton planting model with one film, three drip tapes and six rows treatment was more effective in improving the yield and WUE. Considering water conservation and improving cotton yield, the cotton planting model with one film, three drip tapes and six rows can be recommended for machine-harvest cotton planting. This is important for promoting cotton growth and yield, improving labor productivity and the quality of machine-harvest cotton, and ensuring healthy and sustainable development of regional cotton-production in Xinjiang, China.10.7717/peerj.12004/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12004/supp-2Supplemental Information 2Click here for additional data file.